WO2012133674A1 - Procédé pour supprimer des cellules causant la cancérogenèse parmi des cellules souches - Google Patents

Procédé pour supprimer des cellules causant la cancérogenèse parmi des cellules souches Download PDF

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WO2012133674A1
WO2012133674A1 PCT/JP2012/058414 JP2012058414W WO2012133674A1 WO 2012133674 A1 WO2012133674 A1 WO 2012133674A1 JP 2012058414 W JP2012058414 W JP 2012058414W WO 2012133674 A1 WO2012133674 A1 WO 2012133674A1
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cells
cell
promoter
undifferentiated
gene
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健一郎 小戝
薫 三井
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国立大学法人 鹿児島大学
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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
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0093—Purging against cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to a method for selectively removing undifferentiated cells remaining in a cell population induced to differentiate from stem cells, a drug therefor, a differentiated cell obtained by the method, a composition for cell transplantation using the drug, and the like. .
  • Non-Patent Documents 1 and 2 The establishment of human induced pluripotent stem cells (iPS cells) (Non-Patent Documents 1 and 2) has opened the door to the practical application of cell transplantation treatment using pluripotent stem cells.
  • iPS cells human induced pluripotent stem cells
  • human embryonic stem cells can be established if iPS cells are established from the patient himself and induced to differentiate into the necessary cells and then autotransplanted into the patient.
  • Ethical problems associated with the use of (ES cells) that is, destruction of early embryos, which can be said to be the germination of life) and problems of rejection at the time of transplantation can be avoided.
  • HLA-type iPSs are used for diseases requiring early treatment such as spinal cord injury and fulminant hepatitis. It is conceivable that cells or differentiated cells derived therefrom are banked and allogeneic transplantation is performed using them. In the latter case, human ES cells may be used, but it is not easy to prepare a sufficient HLA-type repertoire due to ethical issues, and iPS cells are prepared from a small amount of cells such as skin and dental pulp. It is possible to realize more.
  • Non-patent Documents 3 to 5 Non-patent Documents 3 to 5
  • in vitro cell experiments have been conducted. It is expected that research such as the elucidation of the efficient pathophysiology of these diseases, the elucidation of genes and mechanisms related to those diseases, and the development of highly effective therapeutic drugs in high-throughput cell experiments will be advanced dramatically. .
  • pluripotent stem cells such as ES cells and iPS cells are cultured under conditions that differentiate them into cells such as cardiac muscle and nerves
  • undifferentiated cells remain in the differentiated cell population and become tumors (teratomas, carcinogenesis) (I) non-patent document 6)
  • iPS cells are artificially reprogrammed cells, and thus have peculiar safety problems (i.e., c-Myc).
  • c-Myc peculiar safety problems
  • CRA cancer-specific proliferation type adenovirus
  • CRA is a modification of the E1 gene region essential for the growth of adenovirus to differentiate the virus growth between cancer cells and normal cells.
  • Necessary for the growth of adenovirus in the E1 region A type that inhibits the inactivation of Rb and p53, which are essential for inducing a normal cellular environment, and inhibits their inactivation (ie, prevents viral growth in normal cells), and
  • E1 There is a type in which the endogenous promoter of a gene is replaced with a promoter of a gene that is highly expressed specifically in cancer, whereby the E1 gene is expressed specifically (a virus is propagated specifically in cancer).
  • Ogura et al. Can precisely control the growth of viruses with a number of cancer-specific factors such as deletions in the E1A and E1B gene regions, replacement of endogenous promoters with foreign promoters, and other cancer therapeutic genes.
  • m-CRA cancer-specific growth type adenovirus vector
  • CRA Surv.CRA
  • the virus grows depending on the activity of the gene promoter of survivin (Survivin), an apoptosis-inhibiting protein, and gastric cancer and colon cancer that specifically express survivin
  • the anticancer activity of liver cancer, cervical cancer, and osteosarcoma cell lines has been examined and reported (Patent Document 2, Non-Patent Document 7).
  • cancer-specific proliferative virus technology for other purposes, especially for the production of differentiated cells derived from pluripotent stem cells that are safe and highly reliable as drug efficacy / toxicity evaluation systems.
  • targeting to kill and remove undifferentiated cells and tumorigenic (carcinogenic) -causing cells remaining in the differentiated cell population.
  • An object of the present invention is to provide a means for selectively removing untargeted cells remaining in a cell population induced to differentiate from pluripotent stem cells and targeting and killing undifferentiated cells that cause tumorigenesis after transplantation.
  • the present inventors firstly share that pluripotent stem cells such as ES cells and iPS cells (hereinafter sometimes abbreviated as ES / iPS cells) share cancer cells in terms of pluripotency and infinite proliferation ability.
  • pluripotent stem cells such as ES cells and iPS cells
  • iPS cells hereinafter sometimes abbreviated as ES / iPS cells
  • cancer-specific genes such as TERT and Survivin were highly expressed in ES / iPS cells.
  • the present invention is as follows.
  • a promoter of a gene encoding a factor essential for replication or assembly of at least one virus is specifically expressed in cancer cells or undifferentiated cells
  • a killing agent for undifferentiated cells and / or cells that cause tumorigenesis comprising a viral vector substituted with a promoter of the gene to be treated.
  • the agent according to [1] which selectively kills undifferentiated cells remaining in a cell population induced to differentiate from stem cells and / or cells causing tumorigenesis.
  • the agent according to [2] wherein the stem cell is a pluripotent stem cell.
  • telomerase reverse transcriptase TERT
  • CEA carcinoembryonic antigen
  • HRE hypoxia responsive region
  • Grp78 L-plastin
  • hexokinase II Oct3 / 4, Nanog, Sox2, Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, miR-290 cluster or The agent according to any one of [1] to [4], which is a promoter of the miR-302 cluster.
  • the promoter of a nucleic acid encoding a factor essential for replication or assembly of at least one other virus is a promoter that can be constitutively expressed in mammals or a factor that is specifically expressed in differentiated cells.
  • the agent according to any one of [1] to [6], wherein the factor essential for at least one virus replication or assembly is a factor selected from E1A, E1A ⁇ 24, E1B, and E1B ⁇ 55K.
  • [8] Furthermore, it is under the control of a promoter of a gene that is specifically expressed in cancer cells or undifferentiated cells, a promoter that can be constitutively expressed in mammals, or a promoter of a factor that is specifically expressed in differentiated cells.
  • the cytolytic virus vector is an adenovirus vector.
  • An undifferentiated cell killing agent comprising a cytotoxic factor expression vector controlled by a cancer cell-specific promoter as an active ingredient [11] It is under the control of a promoter of a gene specifically expressed in cancer cells or undifferentiated cells A killing agent for undifferentiated cells and / or cells that cause tumorigenesis, comprising an expression vector comprising a nucleic acid encoding a cytotoxic factor. [12] The agent according to [11], wherein an undifferentiated cell remaining in a cell population induced to differentiate from a stem cell and / or a cell causing tumor formation is selectively killed. [13] The agent according to [12], wherein the stem cell is a pluripotent stem cell.
  • the pluripotent stem cells are ES cells or iPS cells.
  • the vector is a viral vector.
  • the viral vector is an adenoviral vector.
  • Promoter is telomerase reverse transcriptase (TERT), survivin, Aurora kinase, carcinoembryonic antigen (CEA), hypoxia responsive region (HRE), Grp78, L-plastin, hexokinase II, Oct3 / 4, Nanog , Sox2, Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, miR-290 cluster or miR-302 cluster promoter [11]-[ [16] The agent according to any one of [16].
  • a promoter of a nucleic acid encoding a factor essential for at least one viral replication or assembly a promoter of a gene that is specifically expressed in cancer cells or undifferentiated cells, a promoter that can be constitutively expressed in mammals, or The agent according to [15] or [16], wherein the agent is replaced with a promoter of a factor whose expression is specifically enhanced in differentiated cells.
  • the agent according to [18], wherein the factor essential for at least one virus replication or assembly is E1A, E1A ⁇ 24, E1B, or E1B ⁇ 55K.
  • (C) A method for producing differentiated cells with reduced risk of tumor formation using the agent (A) or (B) [20] A cell population induced to differentiate from a stem cell according to any one of [1] to [19] A method for producing differentiated cells with reduced risk of tumorigenesis, comprising killing undifferentiated cells remaining in the cell population and / or cells causing tumorigenesis by contacting the described agent . [21] The method according to [20], wherein the stem cell is a pluripotent stem cell. [22] The method of [21], wherein the pluripotent stem cell is an ES cell or iPS cell.
  • the agent according to any one of [1] to [19] in a cell population induced to differentiate from a stem cell And assessing the degree of killing of undifferentiated cells remaining in the cell population and / or cells that cause tumorigenesis, and assessing the risk of tumorigenesis in differentiation induction from stem cells.
  • the present invention it is possible to selectively kill and remove undifferentiated cells and / or cells that cause tumorigenesis remaining in a cell population induced to differentiate from pluripotent stem cells such as ES cells and iPS cells. It is possible to provide safe transplanted cells with reduced risk of tumorigenesis and uniform differentiated cells with a reduced mixture of cells other than target cells, enabling cell transplantation treatment using pluripotent stem cells and high throughput. It greatly contributes to the realization of in vitro efficacy and toxicity tests.
  • FIG. 1 It is a schematic diagram which shows the structure of the plasmid used for multifactor cancer cell-specific proliferation control type adenovirus (m-CRA) preparation. It is a schematic diagram which shows the structure of (a) non-growth type adenovirus and (b) multifactor growth control type adenovirus used in the Examples. It is a figure which shows the expression of survivin (Survivin) and a TERT gene in a human ES cell (KhES1) and a human iPS cell (253G1 and 201B7). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
  • Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
  • FIG. 1 It is a figure which shows the promoter activity of survivin (Surv) and a TERT gene in a human ES cell (KhES1) and a human iPS cell (253G1 and 201B7).
  • the expression of lacZ gene under the control of survivin promoter, TERT promoter, RSV promoter and CMV promoter is shown from the left of each column. No Virus indicates no virus infection.
  • m-CRA Two types of m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-propagating adenovirus vector (Ad.CA-EGFP) are applied to human iPS cells (253G1) by MOI (Multiplicity of infection) ; Number of infectious viruses per cell) 3 (left) or MOI 10 (right), 1 day after virus infection (Day 1), 2 days after (Day 2), 3 days after (Day 3), 4 days after (Day4), It is a figure which shows the ratio (Viability (%) of (non-treated (control))) of the virus-infected cell with respect to the non-infected cell (No (virus)) seven days later (Day7).
  • MOI Multiplicity of infection
  • m-CRA Two types of m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-propagating adenovirus vector (Ad.CA-EGFP) are added to human iPS cells (201B7) at MOI 3 (left) or MOI 10 (Right) Infection against uninfected cells (No Virus) 1 day after virus infection (Day 1), 2 days after (Day 2), 3 days after (Day 3), 4 days after (Day 4), 7 days after (Day 7) It is a figure which shows the ratio (Viability (non-treated) control) of the viable cell of a virus infected cell.
  • m-CRA Two types of m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-propagating adenovirus vector (Ad.CA-EGFP) are added to human ES cells (KhES1) at MOI 3 (left) or MOI 10 (Right) Infection against uninfected cells (No Virus) 1 day after virus infection (Day 1), 2 days after (Day 2), 3 days after (Day 3), 4 days after (Day 4), 7 days after (Day 7) It is a figure which shows the ratio (Viability (non-treated) control) of the viable cell of a virus infected cell.
  • KhES1 One week after induction of spontaneous differentiation in human ES cells (KhES1; FIG. 6a) and human iPS cells (201B7; FIG.
  • FIG. 6c m-CRA (TERT.m-CRA, Survivin.m -CRA) or non-proliferating adenoviral vector (Ad.dE1.3) infected with MOI 10 and alkaline phosphatase-positive cells (undifferentiated cells) that appear after 2 weeks of culture in an environment where the remaining undifferentiated cells can grow It is a figure which shows the number of colonies. No virus indicates no virus infection.
  • Nanog and Oct3 / 4 (undifferentiation marker), Nestin (ectodermal marker), GATA4 (endodermal marker), Brachyury in human ES cells (KhES1) and human iPS cells (253G1 and 201B7) after spontaneous differentiation induction (Mesodermal marker), survivin and TERT gene expression.
  • GAPDH was used as an internal standard.
  • FIG. 2 is a diagram showing expression of germ layer markers), survivin, and TERT gene. GAPDH was used as an internal standard.
  • the present invention provides a drug that selectively kills undifferentiated cells and / or cells that cause tumorigenesis, particularly undifferentiated cells and / or tumorigenic cells that remain in a cell population differentiated from stem cells.
  • the “undifferentiated cell” means an undifferentiated state (pluripotent or pluripotent) and tumorigenic after transplantation in vivo (in the present invention, both teratoma formation and carcinogenesis).
  • tumorigenic cells mean cells that have the potential to become tumors regardless of the differentiation state, but are typical. Are cells that have the ability to become cancerous. Specifically, for example, abnormalities due to reactivation of oncogenes (c-Myc, etc.) introduced at the time of iPS cell production and integration of the transgene into the chromosome ( Cells that are cancerous or have a high risk of becoming cancerous due to gene disruption or activation of oncogenes), chromosomal abnormalities caused by insufficient and / or unstable initialization associated with artificial reprogramming .
  • c-Myc oncogenes
  • undifferentiated cells also express genes that are specifically expressed in cancer cells such as TERT and survivin, while cells that have become cancerated from cells that have differentiated to some extent (The tumor-causing cell) also expresses the above-mentioned gene that serves as an undifferentiation marker (undifferentiated cell-specific gene). Therefore, cytotoxicity is controlled under the control of the promoter of the cancer-specific gene or undifferentiated cell-specific gene.
  • an expression vector in which a nucleic acid encoding the factor is placed in contact with the cell population containing the undifferentiated cells or tumorigenic cells, the cytotoxic factor is expressed only in the undifferentiated cells and / or tumorigenic cells.
  • undifferentiated cells and / or tumorigenic cells can be selectively killed and removed.
  • the undifferentiated cell / tumor-causing cell killing agent of the present invention is a gene whose nucleic acid promoter encoding a factor essential for at least one virus replication or assembly is specifically expressed in cancer cells or undifferentiated cells. It is characterized by being substituted with a promoter and / or comprising an expression cassette in which a nucleic acid encoding a cytotoxic factor is operably linked to a promoter of a cancer cell or undifferentiated cell specific gene.
  • the promoter of a gene that is specifically expressed in cancer cells has a promoter activity that can direct the expression of a sufficient amount of cytotoxic factor to specifically kill the undifferentiated cells and / or tumorigenic cells.
  • a promoter of a gene that is specifically expressed in various cancers specifically, a telomerase reverse transcriptase (TERT) promoter (Takakura, M. et al ., Cancer Res., 59: 551-557, 1999), survivin promoter, Aurora kinase promoter, hypoxia responsive region (HRE) promoter, Grp78 promoter, L-plastin promoter and hexokinase II promoter. It is done.
  • TERT telomerase reverse transcriptase
  • HRE hypoxia responsive region
  • Promoter activity that can direct the expression of a sufficient amount of cytotoxic factor to specifically kill undifferentiated cells and / or tumorigenic cells as promoters of genes that are specifically expressed in undifferentiated cells
  • promoters of genes that are specifically expressed in various stem cells specifically, Oct3 / 4, Nanog, Sox2, Cripto, Dax1, ERas
  • Examples include promoters of genes such as Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, miR-290 cluster, miR-302 cluster, and the like.
  • Preferred are Oct3 / 4 promoter, Nanog promoter, Sox2 promoter and the like, but not limited thereto.
  • the promoters of mouse and human survivin genes have been isolated and their sequence information has been disclosed (eg, Li, F. and Altieri, DC, Cancer Res., 59: 3143-3151, 1999; Li, F and Altieri, DC, Biochem. J., 344: 305-311, 1999).
  • the survivin promoter used in the expression vector of the present invention includes a human survivin gene or its ortholog in other mammals (eg, monkey, cow, horse, pig, dog, cat, sheep, goat, rabbit, mouse, rat, etc.)
  • a promoter of a gene preferably a promoter of a survivin gene derived from human or mouse, more preferably a promoter of a human survivin gene.
  • a heterologous promoter may be used as long as it can exert a promoter activity sufficient to give a sufficient killing effect on the target undifferentiated cells.
  • a vector containing a mouse survivin gene promoter can be used as a vector for killing human undifferentiated cells.
  • the nucleotide sequence length of the survivin promoter is not particularly limited as long as it is specific to the target undifferentiated cell and can activate the transcription of the downstream linked gene to such an extent that it exerts a sufficient killing effect on the cell.
  • the nucleotide sequence at positions ⁇ 173 to ⁇ 19 the nucleotide sequence from 1124 to 1278 in the nucleotide sequence shown in SEQ ID NO: 1), where the translation start point is +1
  • the human survivin gene promoter If the nucleotide sequence at positions -173 to -1 (the nucleotide sequence from 1296 to 1468 in the nucleotide sequence shown in SEQ ID NO: 2) is included with the translation start point as +1, the desired specificity and transcription activity can be obtained.
  • the survivin promoter used in the vector of the present invention has at least the 1124 to 1278th nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 1, or the at least 1296 to 1468th nucleotide in the nucleotide sequence shown in SEQ ID NO: 2.
  • the upper limit of the nucleotide sequence length of the survivin promoter is not particularly limited, but if the length of the 5 'upstream region is too large, it may adversely affect the transcription activity and specificity of the promoter.
  • the desired specificity and transcriptional activity can be obtained if the nucleotide sequence is about -6000 to -1 with the translation start point being +1, but preferably the 5 ′ end of the promoter Is downstream of the -3000 position, more preferably downstream of the -1500 position.
  • a vector in which a reporter gene is linked downstream of promoters of various lengths is prepared, introduced into target undifferentiated cells, and the expression of the promoter is used as an indicator. Can be used to determine a suitable range of sequence length for the promoter.
  • the promoter of the human TERT gene has been isolated and its sequence information has been disclosed (see, eg, Cong, YS et al., Hum. Mol. Genet., 8 (1): 137-142, 1999 ).
  • the TERT promoter used in the expression vector of the present invention includes a human TERT gene or its ortholog in other mammals (eg, monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.).
  • a promoter of a gene preferably a promoter of a TERT gene derived from human or mouse, more preferably a promoter of a human TERT gene.
  • TERT promoter Depending on the mammal to be treated, it is preferable to use the same type of TERT promoter, but a heterologous promoter may be used as long as it can exert a promoter activity that gives a sufficient killing effect on the target undifferentiated cells.
  • a vector containing a mouse TERT gene promoter can be used as a vector for killing human undifferentiated cells.
  • the nucleotide sequence length of the TERT promoter is not particularly limited as long as it can activate the transcription of the gene linked downstream so that it is specific to the target undifferentiated cell and exhibits a sufficient killing effect on the cell.
  • the TERT promoter used in the vector of the present invention comprises at least the nucleotide sequence from 3852 to 3996 in the nucleotide sequence shown in SEQ ID NO: 3.
  • the upper limit of the nucleotide sequence length of the TERT promoter is not particularly limited, but if the length of the 5 'upstream region becomes too large, it may adversely affect the transcriptional activity and specificity of the promoter.
  • the desired specificity and transcriptional activity can be obtained as long as the nucleotide sequence is about -4000 to -1 with the translation start point being +1, but preferably the 5 ′ end of the promoter Is downstream of -3000 position, more preferably downstream of -2000 position.
  • a vector in which a reporter gene is linked downstream of a promoter of various lengths is prepared, introduced into target undifferentiated cells, and the expression of the promoter is used as an indicator. Can be used to determine a suitable range of sequence length for the promoter.
  • the Aurora kinase promoter used in the expression vector of the present invention is not particularly limited as long as it is a promoter derived from a gene belonging to the Aurora kinase family.
  • a Drosophila Aurora-A, -B and -C gene mammal for example, , Human, monkey, cow, horse, pig, dog, cat, sheep, goat, rabbit, mouse, rat, etc.
  • a promoter of Aurora kinase A gene or Aurora kinase B gene derived from human or other mammal is preferable, and a promoter of human Aurora kinase A gene or human Aurora kinase B gene is more preferable.
  • a heterologous promoter can be used as long as it can exert a promoter activity sufficient to give a sufficient killing effect on the target undifferentiated cells.
  • a vector containing a mouse Aurora kinase gene promoter can be used as a vector for killing human undifferentiated cells.
  • the nucleotide sequence length of the Aurora kinase promoter is not particularly limited as long as it is specific to the target undifferentiated cell and can activate the transcription of the downstream linked gene to such an extent that it exerts a sufficient killing effect on the cell.
  • the nucleotide sequence at positions ⁇ 124 to +354 the nucleotide sequence from 1363 to 1840 in the nucleotide sequence shown in SEQ ID NO: 4
  • the transcription start point is +1
  • human Aurora kinase B In the case of a gene promoter, if it contains a nucleotide sequence at positions -185 to +361 (the nucleotide sequence from 1595 to 2140 in the nucleotide sequence shown in SEQ ID NO: 5), where the transcription start point is +1, the target specificity and Transcriptional activity can be obtained.
  • the human Aurora kinase promoter used in the expression vector of the present invention is at least nucleotides 1363 to 1840 in the nucleotide sequence shown in SEQ ID NO: 4, or at least 1595 to 1 in the nucleotide sequence shown in SEQ ID NO: 5.
  • the upper limit of the nucleotide sequence length of the Aurora kinase promoter is not particularly limited, but if the length of the 5 'upstream region is too large, it may adversely affect the transcription activity and specificity of the promoter.
  • nucleotide sequence from position -1486 to +354 (nucleotide sequence shown in SEQ ID NO: 4) with the transcription start point being +1
  • the transcription start point in the case of the human Aurora kinase B gene promoter, the transcription start point If the nucleotide sequence is from -1779 to +361 (nucleotide sequence shown in SEQ ID NO: 5) with +1 as +1, the desired specificity and transcriptional activity can be obtained.
  • the 5 ′ end of the human Aurora kinase promoter used in the expression vector of the present invention is the nucleotides 1 to 1363 in the nucleotide sequence shown in SEQ ID NO: 4, or the nucleotide sequence shown in SEQ ID NO: 5.
  • Examples include nucleotides 1 to 1595 in the nucleotide sequence.
  • a preferable region can be selected in the same manner.
  • the promoter of a gene specifically expressed in cancer cells or undifferentiated cells in the present invention is also a nucleic acid capable of hybridizing under stringent conditions with a promoter of a natural mammal-derived cancer cell or undifferentiated cell-specific gene.
  • nucleic acids having substantially the same properties as the native promoter are also a nucleic acid capable of hybridizing under stringent conditions with a promoter of a natural mammal-derived cancer cell or undifferentiated cell-specific gene.
  • nucleic acids having substantially the same properties as the native promoter are substantially identical characteristics” a property that drives gene expression specific to the target undifferentiated cells and / or tumorigenic cells, and the degree of transcriptional activity is equivalent (eg, about 0.5 to about 2), but the quantitative factors may be different as long as they can drive gene expression to such an extent that they can exert a sufficient killing effect on the undifferentiated cells and / or tumorigenic cells. .
  • a nucleic acid capable of hybridizing under stringent conditions with the complementary strand sequence of the nucleotide sequence shown in SEQ ID NO: 4 or 5 is exemplified.
  • examples of such a nucleic acid include, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more, particularly preferably about 97% or more, most preferably the nucleotide sequence shown in SEQ ID NO: 4 or 5. Include nucleic acids containing nucleotide sequences having about 98% or more homology.
  • Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to stringent conditions.
  • Stringent conditions include: (1) low ionic strength and high temperature for washing, for example, using 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C, and (2) such as formamide A denaturant such as 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinyl pyrrolidone / 750 mM sodium chloride, 75 mM mM sodium phosphate buffer (pH 6.5) containing 50% alkaline (pH 6.5) v) Reaction conditions characterized by using formamide at 42 ° C are exemplified.
  • stringent conditions are: 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhart solution, sonicated sperm Use DNA (50 mg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, wash with 0.2xSSC and 50% formaldehyde at 55 ° C, followed by 0.1x SSC containing EDTA at 55 ° C High stringency washing may be performed.
  • a person skilled in the art can easily achieve a desired stringency by appropriately adjusting the temperature during the hybridization reaction and / or washing, the ionic strength of the buffer, and the like according to factors such as the probe length.
  • Cancer cell or undifferentiated cell-specific gene promoters are derived from cells or tissues derived from humans or other mammals (eg monkeys, cows, horses, pigs, dogs, cats, sheep, goats, rabbits, mice, rats, etc.) From the extracted genomic DNA, a known gene promoter sequence (for example, Li, F. and Altieri, DC, Cancer Res., 59: 3143-3151, 1999; Li, F. and Altieri, DC, in the case of survivin promoter) Biochem. J., 344: 305-311, 1999; for TERT promoter, see Cong, YS et al., Hum. Mol. Genet., 8 (1): 137-142, 1999; for Aurora kinase promoter Tanaka, M.
  • a known gene promoter sequence for example, Li, F. and Altieri, DC, Cancer Res., 59: 3143-3151, 1999; Li, F. and Altieri, DC, in the case of survivin promoter
  • the genomic DNA containing the promoter region is cloned using a nucleic acid consisting of a nucleic acid consisting of a DNA-decomposing enzyme, for example, an appropriate restriction enzyme as desired. Cut DNA fragment containing the partial promoter sequences, after separation by gel electrophoresis, it can be prepared by purifying the DNA to recover the desired band.
  • the promoter partial sequence is obtained by PCR using a primer synthesized based on a promoter sequence specific to a known cancer cell or undifferentiated cell using the crude cell extract or genomic DNA isolated therefrom as a template. Amplification and isolation are also possible.
  • a BLAST search should be performed on the genomic DNA of the animal using the cDNA sequence of the cancer cell or undifferentiated cell-specific gene as a query. By this, the nucleotide sequence of the cancer cell or undifferentiated cell-specific gene promoter region of the animal can be obtained.
  • the cancer cell or undifferentiated cell-specific gene promoter is based on the known promoter sequence (for example, the nucleotide sequence represented by SEQ ID NO: 4 or 5 in the case of the Aurora kinase promoter) or all of the nucleotide sequence.
  • the nucleic acid containing a part can also be obtained by chemically synthesizing using a commercially available DNA / RNA automatic synthesizer.
  • the undifferentiated cell / tumor causing cell killing agent of the present invention comprises, as an active ingredient, an expression vector comprising a nucleic acid encoding a cytotoxic factor under the control of a cancer cell or an undifferentiated cell-specific gene promoter. contains.
  • the nucleic acid encoding a cytotoxic factor under the control of a cancer cell or undifferentiated cell-specific gene promoter used in the expression vector of the present invention can be directly expressed, for example, when the nucleic acid is transcribed (and translated). Alternatively, it may encode any protein or RNA as long as it indirectly causes cell death or at least growth inhibition.
  • cytotoxic factors include apoptosis-inducing genes (such as Fas), genes that code for constituent proteins of ion channels (such as sodium channels), and proteins that can damage cells by converting prodrugs into poisons (suicide genes) ) (HSV-thymidine kinase, cytosine deaminase, etc.), antisense nucleic acids for reprogramming genes (antisense nucleic acids for Oct3 / 4, antisense nucleic acids for Nanog, etc.), miRNAs that promote apoptosis or suppress cell proliferation, or mimics thereof Or antisense nucleic acids, aptamers, ribozymes, etc. of miRNA that have an apoptosis-inhibiting action or a cell growth-promoting action.
  • apoptosis-inducing genes such as Fas
  • genes that code for constituent proteins of ion channels such as sodium channels
  • proteins that can damage cells by converting prodrugs into poisons suicide genes
  • Nucleic acid encoding a cytotoxic factor can be isolated as cDNA from a cell or tissue producing the nucleic acid by a method known per se, and is functionally linked downstream of a cancer cell or undifferentiated cell-specific gene promoter. Can do.
  • An expression cassette comprising a nucleic acid encoding a cytotoxic factor under the control of a cancer cell or undifferentiated cell specific gene promoter preferably comprises an appropriate polyadenylation sequence downstream of the nucleic acid or gene.
  • the vector having an expression cassette containing a gene promoter specific for a cancer cell or an undifferentiated cell operably linked to a nucleic acid encoding a cytotoxic factor of the present invention contains a nucleic acid encoding a protein required for viral replication or assembly. It may be placed under the control of an exogenous promoter different from the promoter of a cancer cell or undifferentiated cell-specific gene, or a cancer cell-specific gene and an undifferentiated cell-specific gene.
  • a promoter derived from cytomegalovirus e.g., CMV (Early early promoter), promoter from human immunodeficiency virus (HIV) (eg, HIV LTR), promoter from Rous sarcoma virus (RSV) (eg, RSV LTR), promoter from mouse mammary tumor virus (MMTV) (eg, MMTV LTR) , Moloney murine leukemia virus (MoMLV) -derived promoter (eg, MoMLV LTR), herpes simplex virus (HSV) -derived promoter (eg, HSV thymidine kinase (TK) promoter), SV40-derived promoter (eg, SV40 early promoter), Epstein Bar Virus (EBV) promoter, Adeno-associated virus (CMV) (e.g., CMV (Early early promoter), promoter from human immunodeficiency virus (HIV) (eg, HIV LTR), promoter from Rous sarcom
  • a AV p5 promoter adenovirus (AdV) -derived promoter (Ad2 or Ad5 major late promoter), and other constructs such as gene promoters of mammalian constituent proteins such as ⁇ -actin gene promoter, PGK gene promoter, transferrin gene promoter, etc.
  • a promoter can be used.
  • a foreign promoter different from the promoters of cancer cell-specific genes and undifferentiated cell-specific genes is a factor whose expression is specifically increased in differentiated cells in which target undifferentiated cells and / or tumorigenic cells coexist
  • These promoters and inducible promoters can also be used.
  • Undifferentiated cells that cause tumorigenesis do not always maintain a completely undifferentiated state, and often retain undifferentiated characteristics in a state where differentiation has progressed to some extent.
  • both the differentiation marker and the undifferentiation marker are expressed, and therefore the promoter of any of these marker genes can be used.
  • promoters of factors whose expression is specifically increased in differentiated cells include albumin and ⁇ -fetoprotein promoters specific to the liver, prostate specific antigen (PSA) promoter specific to the prostate, muscle and Mitochondrial creatine kinase (MCK) promoter specific to various organs such as the brain, as well as myelin basic protein (MB), glial fibrillary acidic protein (GFAP) and neuron specific enolase specific to the nervous system such as the brain (NSE) promoter and the like.
  • a metallothionein-1 gene promoter etc. can be used, for example.
  • inducers such as heavy metals such as gold, zinc, and cadmium, steroids such as dexamethasone, alkylating agents, chelating agents, and cytokines at desired times and / or tumors.
  • inducers such as heavy metals such as gold, zinc, and cadmium
  • steroids such as dexamethasone
  • alkylating agents such as gold, zinc, and cadmium
  • chelating agents such as amethasone
  • cytokines By locally administering to the location (transplantation site) where the morphogenic cells are present, the expression of a cytotoxic factor such as a viral protein can be induced in the target undifferentiated cells and / or tumorigenic cells at any time.
  • the present invention is characterized in that the promoter of a nucleic acid encoding a factor essential for at least one viral replication or assembly is replaced with a cancer cell or undifferentiated cell specific gene promoter.
  • the present invention relates to a target undifferentiated cell and / or a tumorigenic cell-specific propagation virus vector (conditionally replicating virus: CRV) (hereinafter also referred to as “cancer cell or undifferentiated cell-specific gene promoter-dependent CRV”). That is, the present invention relates to a vector characterized by specifically growing in a target undifferentiated cell and / or a tumorigenic cause cell (superior to a differentiated cell or a non-cancerous cell).
  • CRV tumorigenic cell-specific propagation virus vector
  • viral vectors not only cause the proliferation of the virus specifically in the target undifferentiated cells and / or tumorigenic cells, but also result in killing the target undifferentiated cells and / or tumorigenic cells ( Dissolve).
  • the virus released from the lysed cells infects neighboring vector-untransfected undifferentiated cells and / or tumorigenic cells, and this step is repeated so that eventually all unresolved cells in the cell population are present.
  • the vector of the present invention is introduced into differentiated cells and / or tumorigenic cells, and an effect of killing and removing undifferentiated cells and / or tumorigenic cells can be obtained.
  • the cancer cell or undifferentiated cell-specific gene promoter-dependent CRV of the present invention places a nucleic acid encoding a protein required for replication or assembly of at least one virus under the control of a cancer cell or undifferentiated cell-specific gene promoter.
  • Nucleic acid encoding a protein necessary for viral replication or assembly means a nucleic acid encoding one of the proteins essential for the virus to self-replicate, such as a structural protein of the virus, or the virus performs the assembly. Means a nucleic acid encoding any of the essential proteins. More specifically, the nucleic acid encoding a protein required for virus replication or assembly varies depending on the virus species used.
  • E1A, E1B, E2 and E4 which are early genes that act on E. coli, or Rb-binding region-deficient E1A (E1A ⁇ 24) and p53-binding region-deficient E1B (E1B ⁇ 55K) described later.
  • E1A is transcribed first after infection with adenovirus, and if there is no expression of E1A, subsequent viral replication does not occur, and thus target undifferentiated cells and / or tumorigenicity with cancer cell or undifferentiated cell-specific gene promoters.
  • the late genes (Late gene) L1, L2, L3, L4, and L5 of the nucleic acid encoding the structural gene of adenovirus are transcribed at a later stage of cell division after infection, and constitute the virus structure.
  • the expression of these late genes is controlled by a cancer cell or undifferentiated cell-specific gene promoter, the virus growth can be controlled specifically for the target undifferentiated cells and / or tumorigenic cells.
  • a gene encoding a viral protein whose expression is controlled by a cancer cell or undifferentiated cell-specific gene promoter is a viral replication.
  • any viral gene that is essential for assembly may be used.
  • viral vectors other than adenovirus for example, in the case of herpes simplex virus, such as Rep78 and Rep68 under the control of the p5 promoter, Rep52 and Rep40 under the control of the p19 promoter in the case of an adeno-associated virus.
  • initial gene products such as ICP0, ICP4, ICP22, and ICP27, thymidine kinase, and the like, in the case of Sendai virus, include N protein, P protein, and L protein.
  • Such a cancer cell or undifferentiated cell-specific gene promoter-dependent propagation type viral vector replaces an endogenous promoter of a nucleic acid encoding a protein necessary for viral replication or assembly with a cancer cell or undifferentiated cell-specific gene promoter. Can be obtained.
  • the propagating viral vector of the present invention is an adenoviral vector
  • the nucleic acid encoding E1A and / or E1B more preferably the nucleic acid encoding at least E1A is under the control of a cancer cell or undifferentiated cell-specific gene promoter. Smelled.
  • the cancer cell or undifferentiated cell-specific gene promoter-dependent CRV of the present invention introduced into the cell is in an environment where the cancer cell or undifferentiated cell-specific gene promoter is not activated (differentiated cells or non-cancerous cells). The cells are not damaged because they cannot proliferate.
  • the cancer cell or undifferentiated cell-specific gene promoter-dependent CRV of the present invention is within the environment (target undifferentiated cell and / or tumorigenic cause cell) in which the cancer cell or undifferentiated cell-specific gene promoter is activated. Enter the cell, where the virus grows and the cells are damaged by the cytotoxicity of the viral protein.
  • the virus released from the lysed cells successively infects undifferentiated cells and / or tumorigenic cells without surrounding vector introduction, and the same steps are repeated.
  • the proliferative virus vector of the present invention can be finally introduced into all undifferentiated cells and / or tumorigenic cells in the cell population.
  • nucleic acid encoding a protein required for viral replication or assembly is under the control of a cancer cell or undifferentiated cell-specific gene promoter, viral growth or assembly is specific for cancer cells or undifferentiated cells. Any nucleic acid encoding a protein required for replication or assembly of other viruses is different from the promoters of cancer cell-specific genes and undifferentiated cell-specific genes. May be placed under the control of a foreign promoter.
  • a promoter derived from cytomegalovirus e.g., CMV (Early early promoter), promoter from human immunodeficiency virus (HIV) (eg, HIV LTR), promoter from Rous sarcoma virus (RSV) (eg, RSV LTR), promoter from mouse mammary tumor virus (MMTV) (eg, MMTV LTR) , Moloney murine leukemia virus (MoMLV) derived promoter (eg MoMLV LTR), herpes simplex virus (HSV) derived promoter (eg HSV thymidine kinase (TK) promoter), SV40 derived promoter (eg SV40 early promoter), Epstein Bar Virus (EBV) promoter, Adeno-associated virus (AAV) promoter
  • CMV cytomegalovirus
  • HSV human immunodeficiency virus
  • RSV Rous sarcoma virus
  • MMV mammary tumor virus
  • MoMLV LTR her
  • AAV p5 promoter adenovirus (AdV) -derived promoter (Ad2 or Ad5 major late promoter), and constitutive genes such as ⁇ -actin gene promoter, PGK gene promoter, transferrin gene promoter, etc.
  • a promoter can be used.
  • promoters of factors whose expression is specifically enhanced in differentiated cells include, for example, albumin and ⁇ -fetoprotein promoters specific to liver, prostate specific antigen (PSA) promoter specific to prostate, muscle and Mitochondrial creatine kinase (MCK) promoter specific to various organs such as the brain, as well as myelin basic protein (MB), glial fibrillary acidic protein (GFAP) and neuron specific enolase specific to the nervous system such as the brain (NSE) promoter and the like.
  • PSA prostate specific antigen
  • MCK Mitochondrial creatine kinase
  • MB myelin basic protein
  • GFAP glial fibrillary acidic protein
  • NSE neuron specific enolase specific to the nervous system
  • the inducible promoter for example, a metallothionein-1 gene promoter can be used.
  • target undifferentiated cells and / or tumors such as heavy metals such as gold, zinc and cadmium, steroids such as dexamethasone, alkylating agents, chelating agents, and cytokines are targeted at a desired time.
  • steroids such as dexamethasone
  • alkylating agents such as gold, zinc and cadmium
  • chelating agents such as chelating agents
  • cytokines are targeted at a desired time.
  • the promoters used may be the same or different.
  • the TERT promoter and survivin promoter can be used together in one vector.
  • the cancer cell or undifferentiated cell-specific gene promoter-dependent CRV may further comprise an expression cassette containing a cancer cell or undifferentiated cell-specific gene promoter functionally linked to a nucleic acid encoding a cytotoxic factor. Good.
  • the viral vector of the present invention is essential for inducing the cellular environment necessary for viral growth of viral proteins in differentiated cells and non-cancerous cells, but in target undifferentiated cells and / or tumorigenic cells. Regions that are not necessary for the growth of the cells may be deleted. For example, in order to propagate adenovirus in differentiated cells and non-cancerous cells, it is necessary to inactivate Rb and p53 in order to rotate the cell cycle, but already in undifferentiated cells and tumorigenic cells. Since the cell cycle is rotating, the Rb binding region of E1A and the p53 binding region of E1B are not necessary for the growth of adenovirus in undifferentiated cells and tumorigenic cells.
  • the E1A24KDa region is deleted (E1A ⁇ 24), the E1B55KDa region is deleted (E1B ⁇ 55), or the E1B19KDa region is deleted (E1B ⁇ 19). Allows cell-specific virus propagation.
  • this type of viral vector an undifferentiated cell and / or a tumorigenic cell even if a nucleic acid encoding a protein necessary for viral replication is not under the control of a cancer cell or an undifferentiated cell-specific gene promoter Specific growth can occur.
  • the present invention provides for the propagation of viruses in differentiated or non-cancerous cells, where none of the nucleic acids encoding proteins required for viral replication or assembly are under the control of a cancer cell or undifferentiated cell specific gene promoter.
  • a region that is essential to induce the required cellular environment but is not required for virus growth in target undifferentiated cells and / or tumorigenic cells eg, E1A24KDa region, E1B55KDa region, and / or E1B19KDa region
  • a vector in which a nucleic acid encoding a cytotoxic factor is operably linked under the control of a cancer cell-specific gene promoter.
  • areas that are essential for inducing the cellular environment necessary for virus growth in differentiated and non-cancerous cells, but are not necessary for virus growth in target undifferentiated cells and / or tumorigenic cells may be under the control of a cancer cell or undifferentiated cell-specific gene promoter, Any nucleic acid encoding a protein required for viral replication other than a defective viral protein may be placed under the control of a cancer cell or undifferentiated cell specific gene promoter.
  • the vector of the present invention provides an origin of replication for autonomous amplification in host cells and a selection marker gene for selection of transformed cells (tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, etc.) Or a gene that complements an auxotrophic mutation, etc.).
  • a selection marker gene for selection of transformed cells (tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, etc.) Or a gene that complements an auxotrophic mutation, etc.).
  • the vector of the present invention may be a viral vector or a non-viral vector, but is preferably an adenovirus, a retrovirus, an adeno-associated virus, a herpes virus, a herpes simplex virus, a lentivirus, a vaccinia virus, a poxvirus, Virus vectors such as poliovirus, Sindbis virus, Sendai virus.
  • Adenoviruses have such advantages that gene transfer efficiency is extremely high, they can be introduced into non-dividing cells, and integration of the transgene into the host chromosome is extremely rare.
  • a gutted (gutless) vector that replaces almost the entire length of the adenovirus genome other than the packaging signal ( ⁇ ) ⁇ with the transgene has solved the problem of immunogenicity in the first generation vector, and as a result transgene expression Long-term sustainability was realized.
  • adeno-associated virus has relatively high gene transfer efficiency, can be introduced into non-dividing cells, and it is known that transgene expression persists for a long time by in vivo administration in animal experiments.
  • the multifactor cancer-specific growth-regulated recombinant adenovirus system developed by the present inventors (m-CRA; JP 2005-046101 and WO 2005/012536)
  • a cancer cell or undifferentiated cell specific gene promoter is used.
  • An example of a plasmid vector suitably used for construction of m-CRA is presented in FIG.
  • a cancer cell-specific gene promoter is used as promoter A and / or promoter B in plasmid vector P1
  • cancer cell or undifferentiated is used as promoter C (controlling the expression of cytotoxic factor) of plasmid vector P2.
  • any other promoter can be used, such as a cell-specific gene promoter or another target undifferentiated cell and / or tumorigenic cell-specific promoter or a constitutive promoter.
  • the target undifferentiated cell and / or tumorigenic cause cell-specific promoter include the above-mentioned various promoters that are specifically expressed only in undifferentiated cells and / or tumorigenic cause cells.
  • the cytotoxic factor controlled by the promoter C include the various cytotoxic factors described above.
  • the E1A gene (which may be deficient in the 24 KDa region) operably linked to the TERT or survivin promoter, and a constitutive promoter (such as a CMV promoter) and functional
  • a plasmid vector P1 containing an E1B gene (which may be deficient in the 19 KDa or 55 KDa region), a reporter gene operably linked to a constitutive promoter (such as a CMV promoter) (as a model system for cytotoxic factors)
  • a backbone plasmid P3 containing an adenoviral genome lacking the E1 region (which may have a target cell specific mutation in the fiber gene).
  • CRA cell-specific proliferating adenovirus
  • the growth of the vector is controlled by two factors, the promoter (TERT or survivin) that controls E1A expression and the E1B gene (E1B ⁇ 55K).
  • the promoter By substituting the promoter to be controlled, the promoter to control the expression of the cytotoxic factor, the cytotoxic factor, and the fiber gene of the backbone with other elements, it becomes possible to further control the growth and expression by multiple factors.
  • the non-viral vector of the present invention comprises an expression cassette comprising a nucleic acid encoding a cytotoxic factor under the control of a cancer cell or undifferentiated cell specific gene promoter.
  • vectors plasmids derived from E.
  • coli e.g., pBR322, pBR325, pUC12, pUC13
  • plasmids derived from Bacillus subtilis e.g., pUB110, pTP5, pC194
  • yeast-derived plasmids e.g., pSH19, pSH15
  • animal cell expression plasmids e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • cytotoxic factor has the same meaning as described above.
  • non-viral vector of the present invention in addition to the above, those containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin, and the like can be used as desired.
  • the selection marker include dihydrofolate reductase (dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance (Amp r ) gene, neomycin resistance (Neo r ) gene (G418 resistance) and the like.
  • a non-viral vector the vector can be introduced using a polymer carrier such as a poly L-lysine-nucleic acid complex or encapsulated in liposomes.
  • Liposomes are phospholipid capsules with a particle size of several tens to several hundreds of nanometers, and vectors such as plasmids containing nucleic acids encoding cytotoxic factors under the control of cancer cell or undifferentiated cell-specific gene promoters. Can be enclosed. Alternatively, the vector can be directly introduced into the target cell using the particle gun method.
  • a target undifferentiated cell and / or tumor characterized in that the promoter of a nucleic acid encoding a factor essential for replication or assembly of at least one virus of the present invention is replaced with a cancer cell or undifferentiated cell-specific gene promoter Cell-specific growth type viral vector, or a vector containing a cytotoxic factor under the control of a cancer cell or undifferentiated cell-specific gene promoter, specifically propagates in target undifferentiated cells and / or oncogenic cells Or the cytotoxic factor can be expressed, and if necessary mixed with a pharmacologically acceptable carrier to form various preparations such as injections, then undifferentiated cells / tumor-causing cells Undifferentiated remaining in a cell population derived from a killing agent, preferably stem cells, especially pluripotent stem cells It can be used as agents to selectively kill cells and / or tumorigenic causes cells.
  • a cancer cell or undifferentiated cell-specific gene promoter Cell-specific growth type viral vector, or a vector containing a
  • the pharmacologically acceptable carrier various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, It is blended as a solubilizer, suspending agent, isotonic agent, buffer, soothing agent and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used.
  • excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light
  • Anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate and the like can be mentioned.
  • Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
  • Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
  • Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
  • the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
  • solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
  • suspending agent examples include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, such as polyvinyl alcohol, polyvinyl Examples include pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and other hydrophilic polymers, polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
  • isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
  • Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
  • Preferable examples of the soothing agent include benzyl alcohol.
  • Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • Preferable examples of the antioxidant include sulfite and ascorbate.
  • Suitable examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, and edible blue Nos.
  • water-insoluble lake dyes (Example: Aluminum salt of water-soluble edible tar pigment, etc.), natural pigment (eg, ⁇ -carotene, chlorophyll, Bengala, etc.).
  • natural pigment eg, ⁇ -carotene, chlorophyll, Bengala, etc.
  • sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
  • Administration of the undifferentiated cell / tumor-causing cell killing agent containing the vector of the present invention involves introducing the vector into a cell population induced to differentiate from stem cells, and culturing for a certain period of time to remain undifferentiated cells and / or tumorigenicity.
  • the ex-vivo method in which the resulting differentiated cells are transplanted into the patient after killing and removing the causative cells, and the in-vivo method in which the cells are induced to differentiate from the stem cells are performed separately.
  • administration of the preparation may be performed by, for example, local injection into a cell transplant site, cell transplant site of an implant incorporating the vector of the present invention It is desirable to do so by transplanting to the other.
  • the vector can be introduced into the target cells by a microinjection method, a calcium phosphate coprecipitation method, a PEG method, an electroporation method, or the like.
  • the administration of the undifferentiated cell / tumor-causing cell killing agent of the present invention may be performed prior to the cell transplant, or may be performed simultaneously with or after the cell transplant.
  • the differentiated cell population in which undifferentiated cells and / or tumorigenic cause cells may remain, for which the undifferentiated cell / tumor causing cell killing agent of the present invention can be used, for example, is provided as follows. .
  • the stem cell as a source for inducing a differentiated cell is not particularly limited as long as it has pluripotency or multipotency and a self-replicating ability capable of proliferating while maintaining an undifferentiated state.
  • IPS cells embryonic germ cells derived from primordial germ cells (EG) cells, multipotent germline stem (mGS) cells isolated in the process of establishing GS cells from testicular tissue, multipotent adult progenitor cells isolated from bone marrow ( MAPC) and other tissue stem cells such as hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, neural stem cells, vascular endothelial progenitor cells, and the like are preferable, and pluripotent stem cells are more preferable.
  • the ES cell may be an ES cell produced by nuclear reprogramming from a somatic cell.
  • the animal species from which the stem cells are derived may be any mammal in which any of the stem cells has been established or can be established, and examples thereof include humans, mice, monkeys, pigs, rats, dogs, etc. Preferably it is a human or a mouse.
  • a “somatic cell” may be any cell other than a germ cell derived from a mammal (eg, human, mouse, monkey, pig, rat, etc.), for example, keratinized epithelial cell (Eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (Eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells (eg, vascular endothelial cells) in the inner chain, and ciliated cells that are capable
  • undifferentiated progenitor cells including somatic stem cells
  • terminally differentiated mature cells It can be used as the source of somatic cells in the invention.
  • undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • the nuclear reprogramming substance is not particularly limited as long as it is a gene that is specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof. 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28 Lin28b, Nanog, Esrrb or Esrrg. These nuclear reprogramming substances may be used in combination when iPS cells are established.
  • a combination including at least one, two, or three of the nuclear reprogramming substances preferably a combination including four.
  • nucleotide sequences of mouse and human cDNAs of each of the above nuclear reprogramming substances refer to NCBI accession numbers described in WO 2007/069666, and mouse and human cDNAs of L-Myc, Lin28, Lin28b, Esrrb and Esrrg
  • sequence information can be obtained by referring to the following NCBI accession numbers.
  • a person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.
  • expression vectors When these nuclear reprogramming substances are introduced into somatic cells in the form of nucleic acids, expression vectors may be used.
  • expression vectors in the present invention include plasmids, artificial chromosome vectors, and viral vectors.
  • Artificial chromosome vectors include, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC) and the like.
  • a viral vector retrovirus vector, lentiviral vector (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920 , 2007), adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) .
  • a plasmid a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008 and WO 2009/032456).
  • an expression vector such as a plasmid or an artificial chromosome vector
  • a method such as lipofection, liposome, microinjection, or gene gun method.
  • the expression vector can contain regulatory sequences such as a promoter, an enhancer, an internal ribosome entry site (IRES), a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed.
  • regulatory sequences such as a promoter, an enhancer, an internal ribosome entry site (IRES), a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed.
  • promoter used examples include EF1 ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV- A TK (herpes simplex virus thymidine kinase) promoter or the like is used.
  • EF1 ⁇ promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR ⁇ promoter and the like can be mentioned.
  • selectable marker sequences such as drug resistance genes (for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, diphtheria toxin gene, green fluorescent protein (GFP), red fluorescent protein , ⁇ glucuronidase (GUS), reporter gene sequences such as FLAG, and the like.
  • drug resistance genes for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.
  • thymidine kinase gene diphtheria toxin gene, green fluorescent protein (GFP), red fluorescent protein , ⁇ glucuronidase (GUS), reporter gene sequences such as FLAG, and the like.
  • GFP green fluorescent protein
  • GUS red fluorescent protein
  • GUS ⁇ glucuronidase
  • transposons include, for example, piggyBac, a transposon derived from lepidopterous insects (Kaji, K. et al., Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766 -770 (2009), WO 2010/012077).
  • the above expression vectors are replicated without chromosomal integration and are present episomally with the origins of lymphotropic herpesvirus, BK virus and Bovine papillomavirus. It may contain a sequence related to the replication. Examples include EBNA-1 and oriP or Large T and SV40ori sequences (WO 2009/115295, WO 2009/157201 and WO 2009/149233). Moreover, in order to simultaneously introduce a plurality of nuclear reprogramming substances, an expression vector for polycistronic expression may be used.
  • the gene coding sequence is derived from IRES or picornavirus (foot-and-mouth disease virus (FMDV), equine rhinitis virus B (ERAV), Thosea asigna virus (TaV), etc.) It may be linked by the 2A sequence (Science, 322: 949-953, 2008; BMC Biology, 6:40, 2008; WO 2009/092042 and WO 2009/152529).
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis virus B
  • TaV Thosea asigna virus
  • the nuclear reprogramming substance When the nuclear reprogramming substance is introduced in the form of a protein, it may be introduced into a somatic cell by a technique such as lipofection, binding with a cell membrane permeable peptide, or microinjection.
  • HDAC histone deacetylase
  • VPA valproic acid
  • MC 1293 sodium butyrate
  • M344 small molecule inhibitors
  • siRNA and shRNA against HDAC eg, HDAC1 siRNA Smartpool TM (Millipore), HuSH 29mer shRNA Constructs against HDAC1 Nucleic acid expression inhibitors such as (OriGene) etc.
  • DNA methyltransferase inhibitors eg 5'-azacytidine
  • G9a histone methyltransferase Inhibitors eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.), etc.
  • Nucleic acid expression inhibitors, etc.] L-channel calcium agonist (eg Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)) P53 inhibitors (eg siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt signaling activator (eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), Cytokines such as LIF or bFGF, ALK5 inhibitors (eg SB431542) (Nat Methods, 6: 805-8 (2009)), mitogen-activated protein kinase signaling inhibitors, glycogen synthase kinase-3 inhibitors (PloS Biology, 6 (10), 2237-2247 (2008)), miRNAs such as miR-291-3p, miR-294, miR-295 (RL Judson et al., Nat.
  • L-channel calcium agonist eg Bayk8644
  • P53 inhibitors eg siRNA and shRNA against p53
  • Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10 to 15% FBS or knockout serum replacement (KSR) (these medium further include LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.), (2) ES cell culture medium containing bFGF or SCF, for example, mouse ES cell culture medium ( For example, TX-WES medium, Thrombo X) or primate ES cell culture medium (for example, primate (human & monkey) ES cell culture medium, Reprocell, Kyoto, Japan).
  • a low protein medium or a cell cycle arrester-containing medium may be used (WO 2010/004989).
  • culture methods include, for example, contacting somatic cells with a nuclear reprogramming substance (nucleic acid or protein) on DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C. Incubate for ⁇ 7 days, then re-spread cells onto feeder cells (eg, mitomycin C-treated mouse embryonic fibroblasts (MEF), STO cells, SNL cells, etc.) After 10 days, the cells are cultured in a culture medium for primate ES cell culture containing bFGF, and iPS-like colonies can be generated about 30 to about 45 days or more after the contact. Moreover, in order to increase the induction efficiency of iPS cells, the cells may be cultured under conditions of an oxygen concentration as low as 5-10% (WO 2010/013845).
  • a nuclear reprogramming substance nucleic acid or protein
  • feeder cells eg, mitomycin C-treated mouse embryonic fibroblasts (MEF), STO cells, S
  • DMEM medium containing 10% FBS on feeder cells eg, mitomycin C-treated MEF, STO cells, SNL cells, etc.
  • feeder cells eg, mitomycin C-treated MEF, STO cells, SNL cells, etc.
  • LIF penicillin / streptomycin
  • puromycin e.g., puromycin
  • L- Glutamine e.g., non-essential amino acids, ⁇ -mercaptoethanol, etc.
  • an ES-like colony can be formed after about 25 to about 30 days or more.
  • the medium is replaced with a fresh medium once a day from the second day after the start of the culture.
  • the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
  • a marker gene-expressing cell When a gene containing a drug resistance gene is used as a marker gene, a marker gene-expressing cell can be selected by culturing in a medium (selective medium) containing the corresponding drug.
  • the marker gene is a fluorescent protein gene
  • the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, and by adding a chromogenic substrate in the case of a chromogenic enzyme gene. Can be detected.
  • Examples of ES cell production methods include culturing an inner cell mass at the blastocyst stage of a mammal (see, for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)) , A method of culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Akira Iriya et al., Protein nucleic acid enzyme , 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999) Wakayama et al., Proc.
  • ES cells can be obtained from a predetermined institution, and further commercially available products can be purchased.
  • human ES cells KhES-1, KhES-2, KhES-3, KhES-4 and KhES-5 are available from the Institute of Regenerative Medicine, Kyoto University.
  • somatic cell nuclear transfer the type of somatic cells and the source from which the somatic cells are collected are the same as in the case of the iPS cells.
  • EG cells can be induced by isolating primordial germ cells according to conventional methods and culturing them in the presence of LIF, bFGF and SCF.
  • mGS cells can be prepared from testis cells according to the method described in WO 2005/100548.
  • Pluripotent adult progenitor cells can be isolated from bone marrow according to the method described in J. Clin. Invest. 109: 337-346 (2002).
  • tissue stem cells can be isolated and cultured by a technique known per se.
  • ES cells can be differentiated into hematopoietic progenitor cells by coculturing with irradiated C3H10T1 / 2 cell line to induce sac-like structures (ES-sac) (Blood, 111: 5298-306 , 2008).
  • ES-sac sac-like structures
  • Neural stem cell / nerve cell differentiation induction methods from ES cells include embryoid body formation method (Mech Div 59 (1) 89-102, 1996), retinoic acid method (Dev Biol 168 (2) 342-57, 1995).
  • the SDIA method Neuron 28 (1) 31-40, 2000
  • the NSS method Neurosci Res 46 (2) 241-9, 2003
  • factors such as retinoic acid, TGF ⁇ 1, FGF, dynorphin B, ascorbic acid, nitric oxide, FGF2 and BMP2, Wnt11, PP2, Wnt3a / Wnt inhibitors have been used in the medium
  • a method for inducing myocardial differentiation by Noggin (Nat Biotechnol 23 (5) 611, 2005) have been reported.
  • the dose of the undifferentiated / tumor-causing cell killing agent of the present invention in the in vivo method is as follows: vector type, promoter activity in the target cell, cytotoxic factor type, administration route, disease severity, administration
  • vector type for example, when using an undifferentiated cell or a tumor-causing cell-specific proliferating adenovirus as a viral vector, conventional cancer gene therapy, depending on the target animal species, drug acceptability, body weight, age, etc.
  • 1x10 10 to 10 12 particles / tumor of virus particles have been confirmed to be safe, so the same dose is an indication for administration (Molecular Therapy, 18: 429-434, 2010 ).
  • a single adult dose is about 2 to about 10 mg, preferably about 5 to about 8 mg.
  • Uniform differentiated cells obtained by killing and removing undifferentiated cells and / or tumorigenic cells obtained as described above are mixed with a pharmaceutically acceptable carrier according to a conventional method, for example, by injection, suspension.
  • a pharmaceutically acceptable carrier for example, by injection, suspension.
  • Manufactured as a parenteral preparation such as a suspension or infusion.
  • pharmaceutically acceptable carriers include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants.
  • An aqueous liquid for injection can be mentioned.
  • the transplantation therapeutic agent of the present invention includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine, etc.), a stabilizer (for example, human serum). Albumin, polyethylene glycol, etc.), preservatives, antioxidants and the like.
  • a buffer for example, phosphate buffer, sodium acetate buffer
  • a soothing agent for example, benzalkonium chloride, procaine, etc.
  • a stabilizer for example, human serum
  • Albumin polyethylene glycol, etc.
  • preservatives antioxidants and the like.
  • the transplantation therapeutic agent of the present invention is provided in a state of being cryopreserved under conditions normally used for cryopreservation of cells, and can be used after thawing at the time of use.
  • serum or an alternative thereof an organic solvent (eg, DMSO) and the like may further be included.
  • the concentration of serum or an alternative thereof may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v), although not particularly limited.
  • the concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).
  • the present invention also makes the undifferentiated cell / tumor-causing cell killing agent of the present invention contact a cell population induced to differentiate from stem cells, and kills the undifferentiated cells and / or tumorigenic-causing cells remaining in the cell population.
  • a method for evaluating the risk of tumorigenesis in inducing differentiation from stem cells is provided. As shown in the Examples below, there are many cells that still show expression of undifferentiated markers according to RT-PCR analysis even in cells that have morphologically differentiated morphology.
  • the undifferentiated cell / tumor-causing cell killing agent of the present invention is capable of killing cells that are determined to be differentiated cells in such a morphological form but retain an undifferentiated state to some extent. By examining the degree, it is possible to evaluate whether or not the cell population induced to differentiate by a certain method has a high risk of becoming a tumor after transplantation without analyzing the expression of differentiation / undifferentiation markers.
  • the present invention also reduces the remaining undifferentiated cells and / or tumorigenic cells obtained by bringing the cell population induced to differentiate from stem cells into contact with the undifferentiated cells / tumorigenic cell killing agent of the present invention.
  • the present invention relates to utilization of differentiated cells (target cells) for various cell experiments for basic research and drug discovery research and development, such as elucidation of the mechanisms of diseases that express pathologies in the target cells and development of therapeutic drugs.
  • the stem cell for inducing the target cell or the somatic cell derived therefrom is preferably a cell derived from a patient suffering from the target disease.
  • the present invention is also obtained by bringing the cell population induced to differentiate from stem cells into contact with the undifferentiated cell / tumor-causing cell killing agent of the present invention.
  • a biological research reagent comprising undifferentiated cells and / or differentiated cells with reduced tumorigenic cells is provided.
  • the reagent can be produced by blending various kinds of pharmacologically acceptable additives as necessary with the differentiated cells, as with the transplantation therapeutic agent.
  • a compound library is obtained by inducing differentiation from human iPS cells and contacting the undifferentiated cells / tumor-causing cell killing agent of the present invention to reduce the remaining undifferentiated cells and / or tumorigenic cells.
  • a compound having therapeutic activity against the disease can be selected as a drug candidate compound by contacting and measuring a change in cell function as an index of the therapeutic effect on the target disease.
  • neuronal cells are induced to differentiate from human iPS cells (see, for example, Proc. Natl. Acad. Sci. USA, 105: 5856, 2008), and undifferentiated using the undifferentiated / tumor-causing cell killing agent of the present invention. After the cells and / or tumorigenic cells are killed and removed, the test substance is brought into contact with the nerve cells, and a functional assay relating to nerve cell death, neurite outgrowth, electrophysiology, etc. is performed, thereby improving the nerve function. Can be selected as a therapeutic drug candidate for neurodegenerative diseases and the like.
  • an assay system relating to a neurotransmitter can be constructed using various mature nerves (dopamine nerve, acetylcholine nerve, etc.) obtained by selective differentiation culture.
  • disease-specific iPS cells are established from ALS patients and AD patients having inherited mutations, and pathological conditions (for example, abnormal A ⁇ production, tau Differentiation into neuronal cells reflecting abnormal accumulation, neurodegeneration, etc.), and killing and removing undifferentiated cells and / or tumorigenic cause cells using the undifferentiated cells / tumorizing cause cell killing agent of the present invention.
  • a test substance can be brought into contact with a nerve cell, and a candidate compound for a therapeutic agent for sporadic ALS or AD can be selected using as an index the presence or absence or improvement of the disease state.
  • cardiotoxicity and hepatotoxicity using a cell evaluation system have been predicted from a relatively early stage of drug discovery in a drug toxicity evaluation test.
  • One of the serious side effects caused by drugs is ventricular arrhythmia associated with QT prolongation. Inhibition of HERG channel is known as the main mechanism of QT prolongation.
  • Electrophysiological methods for evaluating cardiotoxicity of drugs are widely used (Biophys. J., 74: 230, 1998). However, if cardiomyocytes derived from iPS cells that reflect more physiological conditions are used, an efficient and more accurate cardiotoxicity evaluation can be performed.
  • cardiomyocytes are differentiated from human iPS cells (see, for example, Circulation, 118: 498, 2008), and undifferentiated cells and / or tumorigenic cause cells using the undifferentiated / tumor-causing cell killing agent of the present invention After killing and removing the pulsatile region from the cardiomyocyte colony and placing it on the electrode of the multi-electrode measuring device, measuring the extracellular potential in the presence of the test substance, By testing the degree, it is possible to exclude a test substance having a QT prolonging action as a cardiotoxic substance from the drug candidates.
  • Reference Example 1 Expression of cancer cell-specific genes in ES / iPS cells (1) Sorting of undifferentiated cells by FACS Human ES cells treated overnight with Rock inhibitor (10 ⁇ M Y27632) (KhES1: transferred from Kyoto University) Human iPS cells (201B7, 253G1: above, RIKEN Cell Bank) were trypsinized and dispersed into single cells. After counting the number of cells, the required amount of cells was centrifuged (1,000 rpm, 5 min) and resuspended in PBS containing 10 ⁇ M Y27632.
  • the aqueous phase (upper phase) was transferred to another sample, isopropanol was added and mixed, and the mixture was allowed to stand at room temperature for 10 minutes. Centrifuge again at 4 ° C, 12,000 g for 15 minutes, add 1-2 ml of 75% ethanol to the remaining precipitate, stir, fully suspend the precipitate, and centrifuge again at 4 ° C, 12,000 g for 15 minutes. The supernatant was removed and the resulting precipitate was naturally dried for about 5 minutes. DEPC water was added and heated at 60 ° C. for 15 minutes, and then allowed to stand at 4 ° C.
  • Example 1 Promoter assay and expression of foreign gene specific to undifferentiated cells
  • Human ES cells (KhES1) and human iPS cells (201B7, 253G1) treated overnight with a Rock inhibitor were seeded at 3 ⁇ 10 5 in a 6-well plate. .
  • the next day the number of cells per well was counted, and the non-proliferating adenovirus carrying the lacZ gene under the control of survivin, TERT, RSV and CMV promoters (Ad.Survivn-LacZ, Ad.TERT-LacZ, Ad. RSV-LacZ, Ad.CMV-LacZ; FIG. 2a) was infected with MOI 10 and a promoter assay was performed.
  • cytotoxic genes such as HSV-tk (Herpes simplex virus thymidine kinase) gene are more than necessary and sufficient (if RSV promoter activity is sufficient). It has been shown that a promoter activity considerably lower than the activity intensity is sufficient) to induce cytotoxic effects (Hepatology. 2003 37 (1): 155-63.). Therefore, from the above results, if a cytotoxic gene such as HSV-tk gene is linked to a cancer cell-specific promoter such as Survivin or TERT, undifferentiated cells remaining in the cell population induced to differentiate from ES cells or iPS cells. It is clear that the cells can be killed sufficiently.
  • HSV-tk Herpes simplex virus thymidine kinase
  • Example 2 In vitro m-CRA killing effect of undifferentiated cells
  • human ES cells KhES1
  • human iPS cells 201B7, 253G1 treated with a Rock inhibitor were placed on a 96-well plate at 4-6 ⁇ 10 4 per well. Sowing. The next day, after counting the number of cells per well, two types of m-CRA (TERT.m-CRA, Survivin.m-CRA; FIG. 2b) or non-proliferating adenovirus vector (Ad.CA-EGFP; FIG.
  • Survivin.m-CRA showed a stronger cytotoxic effect. This result reflected the promoter assay result of Reference Example 2 in which the survivin promoter was more active than the TERT promoter. Comparing the cytotoxic effect of hES cells (FIG. 5-3) and hiPS cells (FIGS. 5-1, 5-2), the cytotoxic effect of m-CRA was stronger in hiPS cells.
  • Example 3 Removal of undifferentiated cells using m-CRA controlled for growth by survivin or TERT According to a maintenance culture protocol for human pluripotent stem cells (RIKEN), treatment was performed overnight with Rock inhibitor on matrigel. Spontaneous differentiation was promoted by culturing KhES1, 201B7 and 253G1 in a dispersed culture and culturing in DMEM (10% FCS).
  • m-CRA TERT.m-CRA, Survivin.m-CRA; Fig. 2b
  • MOI 10 mitomycin C-treated MEF feeder cells.
  • Example 4 Effect of m-CRA on ES / iPS cells after induction of spontaneous differentiation
  • IPS cells 201B7 strain
  • ES cells KhES-1 strain
  • KhES-1 strains induced to differentiate for 15 days
  • KhES-1 strains induced to differentiate for 20 days
  • Total RNA was extracted from 5 types of cells, reverse transcribed to cDNA, Oct3 / 4 and Nanog (undifferentiation marker), Nestin (ectodermal marker), Brachyury (mesoderm marker), and GATA4 (endoderm marker) RT-PCR was performed. GAPDH was used as an endogenous control. Unlike the undifferentiated cells, these cells spread on the dish and showed a differentiated form as confirmed under a microscope. RT-PCR confirmed the expression of differentiation markers in almost all cell types.
  • KhES1 which was induced to differentiate for 25 days, was infected with TERT.m-CRA, Survivin.m-CRA, and Ad.CApr-EGFP.
  • TERT.m-CRA TERT.m-CRA
  • Survivin.m-CRA Survivin.m-CRA
  • Ad.CApr-EGFP Ad.CApr-EGFP
  • Example 5 Differentiation state after embryoid body (EB) formation From the results of Example 4, differentiation is not sufficient in spontaneous differentiation induction in which pluripotent stem cells are cultured in the absence of bFGF. It was insufficient to study the effect. Therefore, differentiation induction by EB formation, which is closer to a more natural differentiation method, was performed and the effect of m-CRA was examined. Each pluripotent stem cell treated with a ROCK inhibitor was cultured at 1 ⁇ 10 4 / well on a low-adhesion 96 well plate to form an embryoid body (EB). On the 7th day after differentiation (Day 7), one EB was seeded per well on a gelatin-coated 24-well plate, and differentiation was further promoted.
  • EB embryoid body
  • TERT.m-CRA On the 15th day after differentiation (Day 15), TERT.m-CRA, Survivin.m-CRA, and Ad.CA-EGFP were infected with MOI 10 for 1 hour, and the cells after infection were observed for 2 weeks. Total RNA was collected on Day 0, 7, 15, and 22, and the gene expression status was confirmed by RT-PCR.
  • TP63 As an ectoderm marker
  • GATA4 as an endoderm marker
  • GAPDH as an endogenous control.
  • the following primers were used for Nanog and Oct4, which are markers for TERT, survivin, and undifferentiated cells not included in the kit.
  • KhES1-derived differentiated cells were confirmed to be beating one after another.
  • pulsating cells from day 22 can be confirmed in differentiated cells derived from the 253G1 strain. These pulsating cells were observed regardless of the presence or absence of virus infection, but no pulsating cells were observed in the 201B7 strain by day 31 when observation was continued. Differentiated cells were found to be infected with m-CRA because EGFP expression was observed, but the number of EGFP-positive cells was hardly increased, indicating that m-CRA was not proliferating (Fig. 9-1 to 9-3).
  • the undifferentiated cell / tumor causing cell killing agent of the present invention selects undifferentiated cells and / or tumorigenic cause cells remaining in a cell population derived from pluripotent stem cells such as ES cells and iPS cells. Therefore, it is possible to provide highly reliable and uniform differentiated cells as a system for evaluating safe transplanted cells, drug efficacy, and toxicity with a reduced risk of tumor formation, and therefore, many of them. It is extremely useful for realizing cell transplantation treatment using potent stem cells and efficient and highly accurate screening of drug candidate compounds.

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Abstract

La présente invention concerne un agent létal pour des cellules non différenciées et/ou des cellules susceptibles de causer la cancérogenèse, l'agent létal contenant un vecteur viral dans lequel un promoteur pour un acide nucléique pour coder pour un facteur essentiel pour répliquer ou assembler au moins un virus a été remplacé par un promoteur pour un gène qui est spécifiquement exprimé dans des cellules cancéreuses ou des cellules indifférenciées. La présente invention concerne en outre un agent létal pour des cellules indifférenciées et/ou des cellules susceptibles de causer la cancérogenèse, l'agent létal contenant un vecteur d'expression comprenant un acide nucléique pour coder pour un facteur cytotoxique sous le contrôle d'un promoteur pour un gène qui est spécifiquement exprimé dans des cellules cancéreuses ou des cellules indifférenciées. La présente invention concerne en outre un procédé pour produire des cellules différenciées ayant un risque réduit de cancérogenèse, le procédé étant caractérisé en ce qu'un desdits médicaments mentionnés ci-dessus est mis en contact avec une population de cellules induite pour se différencier à partir de cellules souches, de manière à tuer les cellules non différenciés ou les cellules susceptibles de causer une cancérogenèse restantes dans la population de cellules.
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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
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