WO2012133674A1 - Method for removing tumorigenesis-causing cells among stem cells - Google Patents

Method for removing tumorigenesis-causing cells among stem cells Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
promoter
undifferentiated
gene
Prior art date
Application number
PCT/JP2012/058414
Other languages
French (fr)
Japanese (ja)
Inventor
健一郎 小戝
薫 三井
Original Assignee
国立大学法人 鹿児島大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人 鹿児島大学 filed Critical 国立大学法人 鹿児島大学
Priority to JP2013507734A priority Critical patent/JP6037393B2/en
Publication of WO2012133674A1 publication Critical patent/WO2012133674A1/en

Links

Images

Classifications

    • 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.

Abstract

The present invention provides a lethal agent for undifferentiated cells and/or cells that would cause tumorigenesis, the lethal agent containing a virus vector in which a promoter for a nucleic acid for encoding an essential factor for replicating or assembling at least one virus has been replaced with a promoter for a gene which is specifically expressed in cancer cells or undifferentiated cells. The present invention further provides a lethal agent for undifferentiated cells and/or cells that would cause tumorigenesis, the lethal agent containing an expression vector including a nucleic acid for encoding a cytotoxic factor under the control of a promoter for a gene which is specifically expressed in cancer cells or undifferentiated cells. The present invention additionally provides a method for producing differentiated cells having a reduced risk of tumorigenesis, the method being characterized in that an aforesaid drug is brought into contact with a cell population induced to differentiate from stem cells, to thereby kill the undifferentiated cells or cells that would cause tumorigenesis remaining within the cell population.

Description

幹細胞における腫瘍化原因細胞の除去方法Methods for removing tumorigenic cells in stem cells
　本発明は、幹細胞から分化誘導された細胞集団に残存する未分化細胞の選択的除去方法、そのための薬剤、並びに当該方法により得られる分化細胞または当該薬剤を用いた細胞移植治療用組成物などに関する。 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. .
　ヒト人工多能性幹細胞（iPS細胞）の樹立により（非特許文献１、２）、多能性幹細胞を用いた細胞移植治療の実用化への扉が開かれた。例えば、パーキンソン病やI型糖尿病のような慢性疾患の場合、患者本人からiPS細胞を樹立し、必要な細胞に分化誘導した後に該患者に自家移植することが可能になれば、ヒト胚性幹細胞（ES細胞）の使用に伴う倫理的問題（即ち、生命の萌芽ともいえる初期胚の破壊）や移植時の拒絶反応の問題を回避することができる。一方で、iPS細胞の樹立から目的細胞への分化誘導まで最短でも2-3ヶ月を要することから、脊髄損傷や劇症肝炎などの早期治療を必要とする疾患については、様々なHLAタイプのiPS細胞またはそれら由来の分化細胞をバンキングしておき、それらを用いて同種移植を行うことが考えられる。後者の場合、ヒトES細胞の利用も想定されるが、倫理的問題から十分なHLAタイプのレパートリーを準備するのは容易ではなく、また、iPS細胞は皮膚や歯髄などの少量の細胞から作製することができるので、より実現の可能性が高い。そのため、将来の臨床応用に向けて、ヒトiPS細胞から種々の体細胞への分化誘導やiPS細胞を用いた疾患モデルマウスの治療といった研究が急速に進展している。
　また患者由来の細胞から疾患特異的iPS細胞が樹立できたことが多数報告されており（非特許文献３～５）、疾患特異的iPS細胞を使えば、体外（in vitro）での細胞実験によりそれらの疾患の効率的な病態解明、それらの疾患に関連する遺伝子やメカニズムの解明、ハイスループットの細胞実験での極めて効率的な治療薬の開発などの研究が飛躍的に進むと期待されている。
　また一方、一つの医薬品開発には10年以上の歳月と約900億円の費用がかかるといわれているが、実際に薬として上市される確率は約2万分の1であることから、より効率的な研究開発投資を行う上でも、創薬開発のできるだけ早期の段階でヒト細胞を用いて薬効試験や毒性試験を行うシステムの開発が切望されている。現在は各種のヒト細胞が入手できないことが障害となり、ヒト細胞での早期の薬効・毒性試験が実現していない。しかし、もしヒトES細胞やヒトiPS細胞から各種の目的細胞を培養皿上（in vitro）で分化誘導して、その目的細胞を単離し活用できれば、この問題が解決でき、創薬事業が大きく進展することになる。
　このように再生医療だけでなく、創薬開発における薬効・毒性試験事業においても、ヒトES細胞、iPS細胞研究は大きな期待が寄せられている。 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. For example, in the case of chronic diseases such as Parkinson's disease and type I diabetes, 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. On the other hand, since it takes at least 2-3 months from the establishment of iPS cells to the induction of differentiation into target cells, various 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. Therefore, researches such as differentiation induction from human iPS cells into various somatic cells and treatment of disease model mice using iPS cells are rapidly progressing for future clinical application.
In addition, it has been reported that many disease-specific iPS cells have been established from patient-derived cells (Non-patent Documents 3 to 5). If disease-specific iPS cells are used, 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. .
On the other hand, it is said that it takes more than 10 years and costs of about 90 billion yen to develop one drug, but the probability of actually being marketed as a drug is about 1 / 20,000, so more In order to efficiently invest in research and development, there is a strong demand for the development of a system for performing drug efficacy tests and toxicity tests using human cells at the earliest possible stage of drug development. Currently, the availability of various human cells has become an obstacle, and early drug efficacy / toxicity tests on human cells have not been realized. However, if various target cells can be induced to differentiate from human ES cells or human iPS cells on a culture dish (in vitro), and the target cells can be isolated and used, this problem can be solved, and the drug discovery business has greatly advanced. Will do.
Thus, not only in regenerative medicine, but also in the drug efficacy and toxicity test business in drug development, there are great expectations for research on human ES cells and iPS cells.
　しかし、ES細胞やiPS細胞などの多能性幹細胞を心筋や神経などの細胞に分化させる条件下で培養すると、分化した細胞集団の中に未分化細胞が残存し、腫瘍化（奇形腫、発癌）の原因となっていることが報告されており（非特許文献６）、さらにiPS細胞は、人工的に初期化された細胞であるがゆえの特有の安全性の問題（即ち、c-Myc等の原癌遺伝子の導入やウイルスベクターの使用による腫瘍化リスク、由来となる体細胞の種類に依存した分化抵抗性による腫瘍化リスク等）をも抱えている。
　このように、多能性幹細胞を用いた再生移植治療の実用化には、腫瘍化の問題を克服することが不可欠である。これまでにiPS細胞由来の発癌の抑制に対しては、癌遺伝子を含まない初期化因子の組合せの探索、非ウイルスベクターの使用、タンパク質導入によるiPS細胞の樹立といったより安全なiPS細胞樹立という観点からは様々な試みがなされてはいる。しかしこれらは、iPS作製時の工夫による「発癌リスクの多少の抑制」という、腫瘍化抑制に関しては「間接的」なアプローチに過ぎず、完全に発癌を阻止できるレベルのものではない。iPS細胞からの発癌細胞を「直接的」に阻止・除去、あるいはiPS細胞由来の腫瘍化細胞（発癌細胞）を直接標的治療する技術の開発という観点からは未だ取り組まれていないため、何ら有効な技術は開発されていないのが現状である。またiPS細胞由来の発癌細胞に限らず、ES細胞にも共通する多能性幹細胞であるがゆえの未分化細胞の残存による腫瘍化（目的細胞以外の多種細胞が混在して腫瘤を形成する奇形腫）のリスクに対しても、何ら有効な解決策は提供されていない。
　また患者由来の疾患特異的iPS細胞を使用した病態解明やその疾患への創薬開発においても、目的の分化細胞以外の未分化細胞の混入は他の細胞種を生じさせるために、目的細胞以外の細胞の混在により、正確な実験を行うことができない。
　また同様に創薬における薬効・毒性試験への正常ヒト細胞の供給という点でのヒトES細胞、iPS細胞の応用においても、目的の分化細胞以外の未分化細胞の混入は他の細胞種を生じさせるために、目的細胞以外の細胞の混在により、測定の精度が低下し信頼性が低下することが考えられる。 However, when 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), and iPS cells are artificially reprogrammed cells, and thus have peculiar safety problems (i.e., c-Myc). The risk of tumorigenesis due to the introduction of proto-oncogenes such as the above and the use of viral vectors, and the risk of tumorigenesis due to differentiation resistance depending on the type of somatic cells from which they are derived.
Thus, overcoming the problem of tumorigenesis is essential for the practical application of regenerative transplantation treatment using pluripotent stem cells. So far, for the suppression of iPS cell-derived carcinogenesis, the viewpoint of safer iPS cell establishment, such as searching for combinations of reprogramming factors that do not contain oncogenes, using non-viral vectors, and establishing iPS cells by introducing proteins Since then, various attempts have been made. However, these are only “indirect” approaches for suppressing tumorigenesis, which is “a slight suppression of the risk of carcinogenesis” by means of iPS production, and are not at a level that can completely prevent carcinogenesis. It is not yet effective from the viewpoint of developing a technology that directly blocks and removes carcinogenic cells from iPS cells or directly targets iPS cell-derived tumorigenic cells (carcinogenic cells). Currently, no technology has been developed. In addition, it is not limited to carcinogenic cells derived from iPS cells, but it is a pluripotent stem cell that is common to ES cells. No effective solution has been provided for the risk of tumors.
In elucidation of pathology using patient-specific disease-specific iPS cells and drug development for the disease, contamination of undifferentiated cells other than the desired differentiated cells causes other cell types. Due to the mixture of cells, accurate experiments cannot be performed.
Similarly, in the application of human ES cells and iPS cells in terms of supplying normal human cells for drug efficacy and toxicity testing in drug discovery, contamination of undifferentiated cells other than the desired differentiated cells will produce other cell types. Therefore, it is conceivable that the measurement accuracy decreases and the reliability decreases due to the mixture of cells other than the target cells.
　ところで、近年、癌治療には癌特異的増殖型アデノウイルス（conditionally replicating adenovirus：CRA）ベクターが有望視されている。CRAはアデノウイルスの増殖に必須なE1遺伝子領域を改変して癌細胞と正常細胞とでウイルス増殖に違いを持たせたものであり、(1) E1領域内にある、アデノウイルスの増殖に必要な細胞環境を誘導するのに必須なRbやp53との結合領域を欠損させて、それらの不活性化を阻害する（即ち、正常細胞でのウイルス増殖を阻止する）タイプと、(2) E1遺伝子の内因性プロモーターを、癌特異的に高発現している遺伝子のプロモーターに置換することにより、癌特異的にE1遺伝子を発現させる（癌特異的にウイルスを増殖させる）タイプのものがある。
　小戝らは、E1AおよびE1B遺伝子領域内の欠損、当該遺伝子の内因性プロモーターの外来プロモーターによる置換、さらには他の癌治療遺伝子といった多数の癌特異化因子で、精緻なウイルス増殖制御が可能な、次世代CRAというべき多因子制御による癌特異的増殖型アデノウィルスベクター（m-CRA）を効率的に作製できる技術を開発した（特許文献１）。さらに、この技術を用いて、アポトーシス抑制タンパク質であるサバイビン（Survivin）の遺伝子プロモーターの活性依存的にウイルスが増殖するCRA（Surv.CRA）を作製し、サバイビンを特異的に発現する胃癌、大腸癌、肝癌、子宮頸癌、骨肉腫細胞株におけるその抗癌作用を検討し、報告している（特許文献２、非特許文献７）。 By the way, in recent years, cancer-specific proliferation type adenovirus (CRA) vectors are promising for cancer treatment. 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. (1) 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 (2) 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. Developed a technology that can efficiently produce a cancer-specific growth type adenovirus vector (m-CRA) based on multi-factor control that should be the next generation CRA (Patent Document 1). Furthermore, using this technology, we produced CRA (Surv.CRA) in which 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).
　しかしながら、癌特異的増殖型ウイルス技術を他の用途、特に安全で、かつ薬効・毒性評価系としての信頼性の高い多能性幹細胞由来の分化細胞の作製のために利用した例は皆無であり、そもそも分化細胞集団の中に残存する未分化細胞や腫瘍化（発癌）原因細胞を標的化して殺傷除去しようという発想自体が存在しないのが現状である。 However, there are no examples of using 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. In the first place, there is no idea of targeting to kill and remove undifferentiated cells and tumorigenic (carcinogenic) -causing cells remaining in the differentiated cell population.
特開2005-46101号公報JP 2005-46101 A 国際公開第2005/115476号International Publication No. 2005/115476
　本発明の目的は、多能性幹細胞から分化誘導された細胞集団内に残存し、移植後の腫瘍化の原因となる未分化細胞を標的化して殺傷することにより選択除去する手段を提供することであり、それによって腫瘍化リスクのない安全な移植細胞・薬効、毒性評価系としての信頼性の高い分化細胞を提供し、幹細胞を用いた細胞移植治療の実用化、創薬開発の進展への途を開くことである。 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. As a result, we provide safe transplanted cells and drug efficacy without risk of tumorigenesis, and highly reliable differentiated cells as a toxicity evaluation system, and practical application of stem cell-based cell transplantation treatment and progress in drug development Open the way.
　前述のとおり、従来の、ヒトiPS細胞をはじめとする多能性幹細胞を用いた細胞移植治療の実用化へ向けた安全面での取り組みは、いずれも腫瘍発生の抑制を主眼とするものしかなかった。しかしながら、導入遺伝子の組合せや導入、あるいは由来となる体細胞の選択といった予防的措置では、せいぜいES細胞と同等レベルまで腫瘍化リスクを低減させることができるのみであり、未分化細胞の混在という根本的な問題を解決することは不可能である。
　そこで、本発明者らは、従来の発想を大きく転換し、「そもそも腫瘍化を完全に阻止することはできない」との前提に立って、多能性幹細胞由来の分化細胞集団内に不可避的に残存する未分化細胞を標的化して殺傷することにより、腫瘍化原因細胞を完全に排除できる方法の開発を模索した。本発明者らはまず、ES細胞やiPS細胞（以下、ES/iPS細胞と略記する場合がある）などの多能性幹細胞は、多能性と無限増殖能という点において癌細胞と共通することに着目し、癌細胞特異的に増殖制御されるウイルスを利用すれば分化細胞集団内に残存する未分化細胞を選択的に殺傷除去できるのではないかと考え、ES/iPS細胞における種々の癌特異的遺伝子の発現を調べたところ、TERT、サバイビン（Survivin）などの癌特異的遺伝子がES/iPS細胞において高発現していることが明らかとなった。そこで、これらの遺伝子のプロモーターによって増殖制御されるm-CRAを用い、ES/iPS細胞を分化条件下で一定期間培養した後に該m-CRAを感染させ、さらに未分化状態を維持できる条件で培養を続けたところ、m-CRA感染群では非感染群と比較して未分化コロニー数が有意に減少したことから、癌細胞特異的増殖型ウイルスを用いて未分化細胞を選択的に殺傷除去できることが確認された。
　本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。 As mentioned above, all of the conventional safety efforts toward the practical application of cell transplantation treatment using pluripotent stem cells such as human iPS cells mainly focus on suppression of tumor development. It was. However, preventive measures such as transgene combinations, introduction, or selection of somatic cells from which they are derived can only reduce the risk of tumorigenesis to the same level as ES cells, and it is fundamentally a mixture of undifferentiated cells. It is impossible to solve a general problem.
Therefore, the present inventors greatly changed the conventional idea, inevitably in the differentiated cell population derived from pluripotent stem cells on the premise that "cannot completely prevent tumor formation in the first place". We sought to develop a method that could completely eliminate tumorigenic cells by targeting and killing the remaining undifferentiated cells. 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. In view of the above, it is thought that the use of a virus whose growth is specifically controlled for cancer cells can selectively kill and remove the undifferentiated cells remaining in the differentiated cell population. When the expression of genetic genes was examined, it was revealed that cancer-specific genes such as TERT and Survivin were highly expressed in ES / iPS cells. Therefore, using m-CRA whose growth is controlled by the promoters of these genes, culturing ES / iPS cells for a period of time under differentiation conditions, infecting the m-CRA, and further culturing under conditions that can maintain the undifferentiated state As a result, the number of undifferentiated colonies in the m-CRA-infected group was significantly reduced compared to the non-infected group, so that undifferentiated cells can be selectively killed and removed using a cancer cell-specific proliferating virus. Was confirmed.
As a result of further studies based on these findings, the present inventors have completed the present invention.
　即ち、本発明は以下の通りである。
（Ａ）増殖型ウイルスを有効成分とする未分化細胞殺傷剤
［１］少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする遺伝子のプロモーターが、癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターで置換されているウイルスベクターを含有してなる、未分化細胞および／または腫瘍化の原因となる細胞の殺傷剤。
［２］幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を選択的に殺傷することを特徴とする、［１］記載の剤。
［３］幹細胞が多能性幹細胞である、［２］記載の剤。
［４］多能性幹細胞がES細胞またはiPS細胞である、［３］記載の剤。
［５］癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターがテロメラーゼ逆転写酵素（TERT）、サバイビン、Auroraキナーゼ、がん胎児性抗原（CEA）、低酸素応答性領域（HRE）、Grp78、L-プラスチン、ヘキソキナーゼII、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3B、miR-290クラスターまたはmiR-302クラスターのプロモーターである、［１］～［４］のいずれかに記載の剤。
［６］更に、少なくとも１つの他のウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターで置換されていることを特徴とする、［１］～［５］のいずれかに記載の剤。
［７］少なくとも１つのウイルスの複製またはアッセンブリに必須の因子が、Ｅ１Ａ、Ｅ１ＡΔ２４、Ｅ１Ｂ、およびＥ１ＢΔ５５Ｋから選択される因子である、［１］～［６］のいずれかに記載の剤。
［８］更に、癌細胞もしくは未分化細胞で特異的に発現する遺伝子のプロモーター、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターの制御下にある、細胞毒性因子をコードする核酸を含む発現カセットを含有する、［１］～［７］のいずれかに記載の剤。
［９］ウイルスベクターが細胞溶解性ウイルスベクターである、［１］～［８］のいずれかに記載の剤。
［１０］細胞溶解性ウイルスベクターがアデノウイルスベクターである、［９］記載の剤。 That is, the present invention is as follows.
(A) Undifferentiated cell killing agent containing a proliferating virus as an active ingredient [1] 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.
[2] 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.
[3] The agent according to [2], wherein the stem cell is a pluripotent stem cell.
[4] The agent according to [3], wherein the pluripotent stem cells are ES cells or iPS cells.
[5] Promoters of genes specifically expressed in cancer cells or undifferentiated cells are 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 The agent according to any one of [1] to [4], which is a promoter of the miR-302 cluster.
[6] Further, 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 [5], which is substituted with a promoter.
[7] 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, E1AΔ24, E1B, and E1BΔ55K.
[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 agent according to any one of [1] to [7], which contains an expression cassette containing a nucleic acid encoding a cytotoxic factor.
[9] The agent according to any one of [1] to [8], wherein the viral vector is a cytolytic viral vector.
[10] The agent according to [9], wherein the cytolytic virus vector is an adenovirus vector.
（Ｂ）癌細胞特異的プロモーターにより制御される細胞毒性因子発現ベクターを有効成分とする未分化細胞殺傷剤
［１１］癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターの制御下にある細胞毒性因子をコードする核酸を含む発現ベクターを含有してなる、未分化細胞および／または腫瘍化の原因となる細胞の殺傷剤。
［１２］幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を選択的に殺傷することを特徴とする、［１１］記載の剤。
［１３］幹細胞が多能性幹細胞である、［１２］記載の剤。
［１４］多能性幹細胞がES細胞またはiPS細胞である、［１３］記載の剤。
［１５］ベクターがウイルスベクターである、［１１］～［１４］のいずれかに記載の剤。
［１６］ウイルスベクターがアデノウイルスベクターである、［１５］記載の剤。
［１７］プロモーターがテロメラーゼ逆転写酵素（TERT）、サバイビン、Auroraキナーゼ、がん胎児性抗原（CEA）、低酸素応答性領域（HRE）、Grp78、L-プラスチン、ヘキソキナーゼII、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3B、miR-290クラスターまたはmiR-302クラスターのプロモーターである、［１１］～［１６］のいずれかに記載の剤。
［１８］更に、少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが癌細胞もしくは未分化細胞で特異的に発現する遺伝子のプロモーター、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターで置換されていることを特徴とする、［１５］または［１６］記載の剤。
［１９］少なくとも１つのウイルスの複製またはアッセンブリに必須の因子が、Ｅ１Ａ、Ｅ１ＡΔ２４、Ｅ１Ｂ、またはＥ１ＢΔ５５Ｋである、［１８］記載の剤。 (B) 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.
[14] The agent according to [13], wherein the pluripotent stem cells are ES cells or iPS cells.
[15] The agent according to any one of [11] to [14], wherein the vector is a viral vector.
[16] The agent according to [15], wherein the viral vector is an adenoviral vector.
[17] 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].
[18] Furthermore, 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.
[19] The agent according to [18], wherein the factor essential for at least one virus replication or assembly is E1A, E1AΔ24, E1B, or E1BΔ55K.
（Ｃ）（Ａ）または（Ｂ）の剤を用いた腫瘍化リスクの低減された分化細胞の製造方法
［２０］幹細胞から分化誘導された細胞集団に［１］～［１９］のいずれかに記載の剤を接触させることにより、該細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を殺傷することを特徴とする、腫瘍化リスクの低減された分化細胞の製造方法。
［２１］幹細胞が多能性幹細胞である、［２０］記載の方法。
［２２］多能性幹細胞がES細胞またはiPS細胞である、［２１］記載の方法。 (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.
（Ｄ）（Ｃ）で得られた分化細胞を用いたex vivo細胞移植療法（剤）／（Ａ）または（Ｂ）の剤を用いたin vivo細胞移植療法（剤）
［２３］［２０］～［２２］のいずれかに記載の方法により得られる有効量の分化細胞を、該分化細胞の移植を必要とする哺乳動物に移植すること、あるいは、幹細胞から誘導された分化細胞集団を移植されたか、もしくは移植される哺乳動物に、有効量の［１］～［１９］のいずれかに記載の剤を投与することを特徴とする、腫瘍化リスクが低減された細胞移植療法。
［２４］［２０］～［２２］のいずれかに記載の方法により得られる分化細胞を含有してなる、細胞移植療法剤。
［２５］幹細胞から誘導された分化細胞集団と組み合わせて投与される、細胞移植療法のための［１］～［１９］のいずれかに記載の剤。 (D) ex vivo cell transplantation therapy (agent) using differentiated cells obtained in (C) / in vivo cell transplantation therapy (agent) using agent (A) or (B)
[23] Transplanting an effective amount of differentiated cells obtained by the method according to any one of [20] to [22] into a mammal in need of transplantation of the differentiated cells, or derived from stem cells A cell with reduced risk of tumor formation, characterized by administering an effective amount of the agent according to any one of [1] to [19] to a mammal transplanted with a differentiated cell population or transplanted mammal Transplantation therapy.
[24] A cell transplantation therapeutic agent comprising a differentiated cell obtained by the method according to any one of [20] to [22].
[25] The agent according to any one of [1] to [19] for cell transplantation therapy, which is administered in combination with a differentiated cell population derived from stem cells.
（Ｅ）（Ａ）または（Ｂ）の剤を用いた分化誘導における腫瘍化リスクの評価方法
［２６］幹細胞から分化誘導された細胞集団に［１］～［１９］のいずれかに記載の剤を接触させ、該細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞の殺傷の程度を検定することを特徴とする、幹細胞からの分化誘導における腫瘍化リスクの評価方法。 (E) Tumorization risk evaluation method in differentiation induction using the agent (A) or (B) [26] 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.
（Ｆ）（Ａ）または（Ｂ）の剤を用いて未分化細胞の混在が低減された目的細胞の薬効・毒性試験あるいはその他の細胞機能の評価実験への用途
［２７］［２０］～［２２］のいずれかに記載の方法により得られる分化細胞と、被検物質とを接触させる工程、および該細胞における目的の薬効または毒性の発現を検定する工程を含む、被検物質の薬効または毒性試験方法。
［２８］分化細胞が患者由来の幹細胞から誘導されたものである、［２７］記載の方法。
［２９］［２０］～［２２］のいずれかに記載の方法により得られる分化細胞を含有してなる、生物学的研究用試薬。
［３０］分化細胞が患者由来の幹細胞から誘導されたものである、［２９］記載の試薬。 (F) Use of the agent of (A) or (B) for a medicinal effect / toxicity test of a target cell in which the mixture of undifferentiated cells is reduced or other cell function evaluation experiment [27] [20]-[ 22], comprising the step of contacting the differentiated cell obtained by the method according to any one of the above and the test substance, and the step of assaying the expression of the target drug effect or toxicity in the cell. Test method.
[28] The method according to [27], wherein the differentiated cell is derived from a stem cell derived from a patient.
[29] A reagent for biological research comprising a differentiated cell obtained by the method according to any one of [20] to [22].
[30] The reagent according to [29], wherein the differentiated cell is derived from a stem cell derived from a patient.
　本発明によれば、ES細胞やiPS細胞などの多能性幹細胞から分化誘導された細胞集団の中に残存する未分化細胞および／または腫瘍化の原因となる細胞を選択的に殺傷除去することができ、腫瘍化リスクの低減された安全な移植細胞・目的細胞以外の細胞の混在の低減された均一な分化細胞の提供が可能となり、多能性幹細胞を用いた細胞移植治療・ハイスループットなin vitro薬効・毒性試験の実現に大いに寄与する。 According to 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.
多因子癌細胞特異的増殖制御型アデノウイルス（m-CRA）作製に用いるプラスミドの構造を示す模式図である。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. 実施例で用いた（ａ）非増殖型アデノウイルスおよび（ｂ）多因子増殖制御型アデノウイルスの構造を示す模式図である。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. ヒトES細胞（KhES1）およびヒトiPS細胞（253G1および201B7）におけるサバイビン（Survivin）およびTERT遺伝子の発現を示す図である。内部標準としてグリセルアルデヒド3リン酸脱水素酵素（GAPDH）を用いた。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. ヒトES細胞（KhES1）およびヒトiPS細胞（253G1および201B7）におけるサバイビン（Surv）およびTERT遺伝子のプロモーター活性を示す図である。各カラム左からサバイビンプロモーター、TERTプロモーター、RSVプロモーターおよびCMVプロモーターの制御下にあるlacZ遺伝子の発現を示す。No Virusはウイルス非感染を示す。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. ヒトiPS細胞（253G1）に二種類のm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター(Ad.CA-EGFP)をMOI（Multiplicity of infection; 感染多重度；細胞一個当たりの感染性ウイルス数） 3（左）、またはMOI 10（右）で感染させた場合の、ウイルス感染1日後（Day1）、2日後（Day2）、3日後（Day3）、4日後（Day4）、7日後（Day7）における非感染細胞（No Virus）に対するウイルス感染細胞の生細胞の割合（Viability (% of non-treated control)）を示す図である。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). ヒトiPS細胞（201B7）に二種類のm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター(Ad.CA-EGFP)をMOI 3（左）、またはMOI 10（右）で感染させた場合の、ウイルス感染1日後（Day1）、2日後（Day2）、3日後（Day3）、4日後（Day4）、7日後（Day7）における非感染細胞（No Virus）に対するウイルス感染細胞の生細胞の割合（Viability (% of non-treated control)）を示す図である。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. ヒトES細胞（KhES1）に二種類のm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター(Ad.CA-EGFP)をMOI 3（左）、またはMOI 10（右）で感染させた場合の、ウイルス感染1日後（Day1）、2日後（Day2）、3日後（Day3）、4日後（Day4）、7日後（Day7）における非感染細胞（No Virus）に対するウイルス感染細胞の生細胞の割合（Viability (% of non-treated control)）を示す図である。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. ヒトES細胞（KhES1; 図６ａ）、ヒトiPS細胞（201B7; 図６ｂおよび253G1; 図６ｃ）に自発的な分化を誘導して1週間後に、m-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.dE1.3）をMOI 10で感染させ、残存未分化細胞が増殖できる環境下で2週間培養した後に出現するアルカリフォスファターゼ陽性細胞（未分化細胞）のコロニー数を示す図である。No virusはウイルス非感染を示す。One week after induction of spontaneous differentiation in human ES cells (KhES1; FIG. 6a) and human iPS cells (201B7; FIG. 6b and 253G1; 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. 自発的な分化誘導後のヒトES細胞（KhES1）およびヒトiPS細胞（253G1および201B7）における、NanogおよびOct3/4（未分化マーカー）、Nestin（外胚葉マーカー）、GATA4（内胚葉マーカー）、Brachyury（中胚葉マーカー）、サバイビンおよびTERT遺伝子の発現を示す図である。内部標準としてGAPDHを用いた。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. 25日間分化誘導を行った後m-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.CA-EGFP）を感染させたヒトES細胞（KhES1）における、感染2日後（day2）（図８ａ）および3日後（day3）（図８ｂ）の細胞の様子とEGFP発現の観察結果を示す図である。In human ES cells (KhES1) infected with m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-proliferating adenovirus vector (Ad.CA-EGFP) after induction of differentiation for 25 days, It is a figure which shows the observation result of the mode of a cell and EGFP expression 2 days after infection (day2) (FIG. 8a) and 3 days after (day3) (FIG. 8b). ヒトiPS細胞（201B7）から胚様体（EB）形成を誘導後15日目（Day15）にm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.CA-EGFP）をMOI 10で感染させ、感染翌日（day16）から6日目（day21）までの細胞の様子とEGFP発現の観察結果を示す図である。On day 15 after induction of embryoid body (EB) formation from human iPS cells (201B7), m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-proliferating adenovirus vector (Ad. It is a figure which shows the observation result of the mode of a cell from the day following infection (day16) to the 6th day (day21) by infecting CA-EGFP) with MOI | 10. ヒトiPS細胞（253G1）から胚様体（EB）形成を誘導後15日目（Day15）にm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.CA-EGFP）をMOI 10で感染させ、感染翌日（day16）から6日目（day21）までの細胞の様子とEGFP発現の観察結果を示す図である。On day 15 after induction of embryoid body (EB) formation from human iPS cells (253G1), m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-proliferating adenovirus vector (Ad. It is a figure which shows the observation result of the mode of a cell from the day following infection (day16) to the 6th day (day21) by infecting CA-EGFP) with MOI | 10. ヒトES細胞（KhES1）から胚様体（EB）形成を誘導後15日目（Day15）にm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.CA-EGFP）をMOI 10で感染させ、感染翌日（day16）から6日目（day21）までの細胞の様子とEGFP発現の観察結果を示す図である。On day 15 after induction of embryoid body (EB) formation from human ES cells (KhES1), m-CRA (TERT.m-CRA, Survivin.m-CRA) or non-proliferating adenovirus vector (Ad. It is a figure which shows the observation result of the mode of a cell from the day following infection (day16) to the 6th day (day21) by infecting CA-EGFP) with MOI | 10. 胚様体形成法による分化誘導後0（day0）、7（day7）、15（day15）および22日目（day22：15日目にm-CRA（TERT.m-CRA、Survivin.m-CRA）または非増殖型アデノウイルスベクター（Ad.CA-EGFP）をMOI 10で感染）のヒトES細胞（KhES1）における、NanogおよびOct4（未分化マーカー）、TP63（外胚葉系のマーカー）、GATA4（内胚葉系のマーカー）、サバイビンおよびTERT遺伝子の発現を示す図である。内部標準としてGAPDHを用いた。Day 0 (day 0), 7 (day 7), 15 (day 15) and 22 (day 22: day 15: m-CRA (TERT.m-CRA, Survivin.m-CRA) after differentiation induction by embryoid body formation method Or Nanog and Oct4 (undifferentiation marker), TP63 (ectodermal marker), GATA4 (internal) in human ES cells (KhES1) infected with non-propagating adenovirus vector (Ad.CA-EGFP) with MOI 10 FIG. 2 is a diagram showing expression of germ layer markers), survivin, and TERT gene. GAPDH was used as an internal standard.
　本発明は、未分化細胞および／または腫瘍化の原因となる細胞、特に幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化原因細胞を選択的に殺傷する薬剤を提供する。ここで「未分化細胞」とは、未分化状態（多能性もしくは多分化能）を保持し、かつ生体内に移植された後に腫瘍化（本発明においては、奇形腫形成および発癌の両方を含む概念として用いる）する潜在的能力を有する細胞であれば特に制限されないが、典型的には、移植された場合にその未分化性（多分化能）のために無秩序に分化して目的細胞以外の細胞種が腫瘤を形成するような細胞であり、具体的には、アルカリフォスファターゼ染色陽性、SSEA3染色陽性、SSEA4染色陽性、Tra-1-60染色陽性、Tra-1-81染色陽性、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3Bの遺伝子発現、miR-290クラスターのmiRNA、miR-302クラスターのmiRNAの発現等の未分化マーカーの発現によって特徴付けられる。形態学的には分化した形態を呈する細胞であっても、未分化マーカーの発現を認める細胞は、本発明においては未分化細胞に包含される。
　一方、「腫瘍化の原因となる細胞」（以下、腫瘍化原因細胞と略記する場合がある）とは、分化状態に拘泥されず腫瘍化する潜在的能力を有する細胞を意味するが、典型的には、癌化する能力を有する細胞であり、具体的には、例えばiPS細胞作製時に導入されたがん遺伝子（c-Myc等）の再活性化や導入遺伝子の染色体への組込みによる異常（遺伝子破壊やがん遺伝子の活性化）、人工的なリプログラミングに伴う不十分および／または不安定な初期化により生じる染色体異常などの原因により癌化するか、癌化するリスクの高い細胞である。 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. To do. Here, 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). It is not particularly limited as long as it has a potential ability to be used as a concept to include, but typically, when transplanted, other than the target cell that differentiates randomly due to its undifferentiation (multipotency) Cell types that form a tumor, specifically, alkaline phosphatase staining positive, SSEA3 staining positive, SSEA4 staining positive, Tra-1-60 staining positive, Tra-1-81 staining positive, Oct3 / 4, Nanog, Sox2, Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B gene expression, miR-290 cluster miRNA, miR-302 cluster miRNA Characterized by the expression of undifferentiated markers such as Even cells that are morphologically differentiated are recognized as undifferentiated cells in the present invention.
On the other hand, “cells that cause tumorigenesis” (hereinafter sometimes abbreviated as 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 .
　これらの未分化マーカーを発現する細胞（未分化細胞）では、TERTやサバイビンをはじめとする癌細胞で特異的に発現する遺伝子も発現しており、一方、ある程度分化した細胞から癌化した細胞（腫瘍化原因細胞）では、上記の未分化マーカーとなる遺伝子（未分化細胞特異的遺伝子）も発現しているので、該癌特異的遺伝子もしくは未分化細胞特異的遺伝子のプロモーターの制御下に細胞毒性因子をコードする核酸を配置した発現ベクターを、当該未分化細胞や腫瘍化原因細胞を含む細胞集団に接触させれば、未分化細胞および／または腫瘍化原因細胞のみで該細胞毒性因子が発現して未分化細胞および／または腫瘍化原因細胞を選択的に殺傷除去することができる。 Cells expressing these undifferentiated markers (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. When 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. Thus, undifferentiated cells and / or tumorigenic cells can be selectively killed and removed.
　即ち、本発明の未分化細胞／腫瘍化原因細胞殺傷剤は、少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターで置換されていること、ならびに／あるいは、細胞毒性因子をコードする核酸が癌細胞または未分化細胞特異的遺伝子のプロモーターに機能的に連結した発現カセットを含むことを特徴とする。
　癌細胞で特異的に発現する遺伝子のプロモーターとしては、未分化細胞および／または腫瘍化原因細胞において特異的に該細胞を殺傷するのに十分な量の細胞毒性因子の発現を指示できるプロモーター活性を発揮し得るものであれば特に制限はないが、例えば、種々の癌で特異的に発現が認められる遺伝子のプロモーター、具体的には、テロメラーゼ逆転写酵素（TERT）プロモーター（Takakura, M. et al., Cancer Res., 59: 551-557, 1999）、サバイビン（survivin）プロモーター、Auroraキナーゼプロモーター、低酸素応答性領域（HRE）プロモーター、Grp78プロモーター、L-プラスチンプロモーターおよびヘキソキナーゼIIプロモーターなどが挙げられる。好ましくはTERTプロモーター、サバイビンプロモーター、Auroraキナーゼプロモーターである。
　未分化細胞で特異的に発現する遺伝子のプロモーターとしては、未分化細胞および／または腫瘍化原因細胞において特異的に該細胞を殺傷するのに十分な量の細胞毒性因子の発現を指示できるプロモーター活性を発揮し得るものであれば特に制限はないが、例えば、種々の幹細胞で特異的に発現が認められる遺伝子のプロモーター、具体的には、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3B、miR-290クラスター、miR-302クラスター等の遺伝子のプロモーターなどが挙げられる。好ましくはOct3/4プロモーター、Nanogプロモーター、Sox2プロモーター等であるが、それらに限定されない。 That is, 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. Although it is not particularly limited as long as it can be exerted, for example, 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. Preferred are TERT promoter, survivin promoter, and Aurora kinase promoter.
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 Is not particularly limited, for example, 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.
　マウスおよびヒトのサバイビン遺伝子のプロモーターは単離されており、その配列情報は開示されている（例えば、Li, F. and Altieri, D.C., Cancer Res., 59: 3143-3151, 1999; Li, F. and Altieri, D.C., Biochem. J., 344: 305-311, 1999を参照）。
　本発明の発現ベクターに用いられるサバイビンプロモーターとしては、ヒトサバイビン遺伝子または他の哺乳動物（例えば、サル、ウシ、ウマ、ブタ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、マウス、ラット等）におけるそのオルソログ遺伝子のプロモーター、好ましくはヒトまたはマウス由来のサバイビン遺伝子のプロモーター、より好ましくはヒトサバイビン遺伝子のプロモーターである。処置対象である哺乳動物に応じて、それと同種のサバイビンプロモーターを用いることが好ましいが、標的未分化細胞に対する十分な殺傷効果を与える程度のプロモーター活性を発揮し得る限り、異種プロモーターを用いてもよい。例えば、ヒト未分化細胞の殺傷用ベクターとして、マウスサバイビン遺伝子のプロモーターを含むベクターを用いることができる。 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. Depending on the mammal to be treated, it is preferable to use the same type of survivin promoter, but 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. . For example, a vector containing a mouse survivin gene promoter can be used as a vector for killing human undifferentiated cells.
　サバイビンプロモーターのヌクレオチド配列長は、標的未分化細胞特異的で、かつ当該細胞に対して十分な殺傷効果を発揮する程度に、下流に連結された遺伝子の転写を活性化し得る限り特に制限されない。例えば、マウスサバイビン遺伝子プロモーターの場合、翻訳開始点を+1として-173～-19位のヌクレオチド配列（配列番号１に示されるヌクレオチド配列中1124～1278番目のヌクレオチド配列）、ヒトサバイビン遺伝子プロモーターの場合、翻訳開始点を+1として-173～-1位のヌクレオチド配列（配列番号２に示されるヌクレオチド配列中1296～1468番目のヌクレオチド配列）を含んでいれば、目的の特異性および転写活性が得られうる。従って、好ましくは、本発明のベクターに用いられるサバイビンプロモーターは、配列番号１に示されるヌクレオチド配列中少なくとも1124～1278番目のヌクレオチド配列、または配列番号２に示されるヌクレオチド配列中少なくとも1296～1468番目のヌクレオチド配列を含む。サバイビンプロモーターのヌクレオチド配列長の上限も特に制限はないが、5’上流域の長さが大きくなりすぎると却ってプロモーターの転写活性や特異性に好ましくない影響を与える場合がある。例えば、ヒトサバイビン遺伝子プロモーターの場合、翻訳開始点を+1として約-6000～-1位のヌクレオチド配列であれば、目的の特異性および転写活性が得られうるが、好ましくはプロモーターの5’末端は-3000位より下流、より好ましくは-1500位より下流である。他の哺乳動物由来のサバイビンプロモーターを用いる場合も、種々の長さのプロモーターの下流にレポーター遺伝子を繋いだベクターを作製し、標的未分化細胞に導入してレポーターの発現を指標にして該プロモーター活性を評価することにより、該プロモーターの好適な配列長の範囲を決定することができる。 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. For example, in the case of the mouse survivin gene promoter, 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, and 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. Can be. Therefore, preferably, 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. Contains nucleotide sequence. 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. For example, in the case of a human survivin gene 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. When using survivin promoters derived from other mammals, 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.
　ヒトのTERT遺伝子のプロモーターは単離されており、その配列情報は開示されている（例えば、Cong, Y.S. et al., Hum. Mol. Genet., 8(1): 137-142, 1999を参照）。
　本発明の発現ベクターに用いられるTERTプロモーターとしては、ヒトTERT遺伝子または他の哺乳動物（例えば、サル、ウシ、ウマ、ブタ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、マウス、ラット等）におけるそのオルソログ遺伝子のプロモーター、好ましくはヒトまたはマウス由来のTERT遺伝子のプロモーター、より好ましくはヒトTERT遺伝子のプロモーターである。処置対象である哺乳動物に応じて、それと同種のTERTプロモーターを用いることが好ましいが、標的未分化細胞に対する十分な殺傷効果を与える程度のプロモーター活性を発揮し得る限り、異種プロモーターを用いてもよい。例えば、ヒト未分化細胞の殺傷用ベクターとして、マウスTERT遺伝子のプロモーターを含むベクターを用いることができる。 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. 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. . For example, a vector containing a mouse TERT gene promoter can be used as a vector for killing human undifferentiated cells.
　TERTプロモーターのヌクレオチド配列長は、標的未分化細胞特異的で、かつ当該細胞に対して十分な殺傷効果を発揮する程度に、下流に連結された遺伝子の転写を活性化し得る限り特に制限されない。例えば、ヒトTERT遺伝子プロモーターの場合、翻訳開始点を+1として-145～-1位のヌクレオチド配列（配列番号３に示されるヌクレオチド配列中3852～3996番目のヌクレオチド配列）を含んでいれば、目的の特異性および転写活性が得られうる。従って、好ましくは、本発明のベクターに用いられるTERTプロモーターは、配列番号３に示されるヌクレオチド配列中少なくとも3852～3996番目のヌクレオチド配列を含む。TERTプロモーターのヌクレオチド配列長の上限も特に制限はないが、5’上流域の長さが大きくなりすぎると却ってプロモーターの転写活性や特異性に好ましくない影響を与える場合がある。例えば、ヒトTERT遺伝子プロモーターの場合、翻訳開始点を+1として約-4000～-1位のヌクレオチド配列であれば、目的の特異性および転写活性が得られうるが、好ましくはプロモーターの5’末端は-3000位より下流、より好ましくは-2000位より下流である。他の哺乳動物由来のTERTプロモーターを用いる場合も、種々の長さのプロモーターの下流にレポーター遺伝子を繋いだベクターを作製し、標的未分化細胞に導入してレポーターの発現を指標にして該プロモーター活性を評価することにより、該プロモーターの好適な配列長の範囲を決定することができる。 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. For example, in the case of the human TERT gene promoter, if the translation start point is +1 and the nucleotide sequence at positions −145 to −1 (the nucleotide sequence from 3852 to 3996 in the nucleotide sequence shown in SEQ ID NO: 3) is included, Specificity and transcriptional activity can be obtained. Therefore, preferably, 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. For example, in the case of a human TERT gene 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. When using a TERT promoter derived from other mammals, 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.
　本発明の発現ベクターに用いられるAuroraキナーゼプロモーターとしては、Auroraキナーゼファミリーに属する遺伝子由来のプロモーターであれば特に制限はないが、例えば、ショウジョウバエAurora-A、-Bおよび-C遺伝子の哺乳動物（例えば、ヒト、サル、ウシ、ウマ、ブタ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、マウス、ラット等）オルソログが挙げられる。好ましくはヒトまたは他の哺乳動物由来のAuroraキナーゼA遺伝子またはAuroraキナーゼB遺伝子のプロモーター、より好ましくはヒトAuroraキナーゼA遺伝子またはヒトAuroraキナーゼB遺伝子のプロモーターである。処置対象である哺乳動物に応じて、それと同種のAuroraキナーゼプロモーターを用いることが好ましいが、標的未分化細胞に対する十分な殺傷効果を与える程度のプロモーター活性を発揮し得る限り、異種プロモーターを用いてもよい。例えば、ヒト未分化細胞の殺傷用ベクターとして、マウスAuroraキナーゼ遺伝子のプロモーターを含むベクターを用いることができる。 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. For example, 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. Depending on the mammal to be treated, it is preferable to use the same type of Aurora kinase promoter, but 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. Good. For example, a vector containing a mouse Aurora kinase gene promoter can be used as a vector for killing human undifferentiated cells.
　Auroraキナーゼプロモーターのヌクレオチド配列長は、標的未分化細胞特異的で、かつ当該細胞に対して十分な殺傷効果を発揮する程度に、下流に連結された遺伝子の転写を活性化し得る限り特に制限されない。例えば、ヒトAuroraキナーゼA遺伝子プロモーターの場合、転写開始点を+1として-124～+354位のヌクレオチド配列（配列番号４に示されるヌクレオチド配列中1363～1840番目のヌクレオチド配列）、ヒトAuroraキナーゼB遺伝子プロモーターの場合、転写開始点を+1として-185～+361位のヌクレオチド配列（配列番号５に示されるヌクレオチド配列中1595～2140番目のヌクレオチド配列）を含んでいれば、目的の特異性および転写活性が得られうる。従って、好ましくは、本発明の発現ベクターに用いられるヒトAuroraキナーゼプロモーターは、配列番号４に示されるヌクレオチド配列中少なくとも1363～1840番目のヌクレオチド配列、または配列番号５に示されるヌクレオチド配列中少なくとも1595～2140番目のヌクレオチド配列を含む。Auroraキナーゼプロモーターのヌクレオチド配列長の上限も特に制限はないが、5’上流域の長さが大きくなりすぎると却ってプロモーターの転写活性や特異性に好ましくない影響を与える場合がある。例えば、ヒトAuroraキナーゼA遺伝子プロモーターの場合、転写開始点を+1として-1486～+354位のヌクレオチド配列（配列番号４に示されるヌクレオチド配列）、ヒトAuroraキナーゼB遺伝子プロモーターの場合、転写開始点を+1として-1779～+361位のヌクレオチド配列（配列番号５に示されるヌクレオチド配列）であれば、目的の特異性および転写活性が得られうる。従って、好ましい一実施態様においては、本発明の発現ベクターに用いられるヒトAuroraキナーゼプロモーターの5’末端として、配列番号４に示されるヌクレオチド配列中1～1363番目のヌクレオチド、または配列番号５に示されるヌクレオチド配列中1～1595番目のヌクレオチドが挙げられる。他の哺乳動物由来のAuroraキナーゼプロモーターを用いる場合も、同様にして好ましい領域を選択することができる。 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. For example, in the case of the human Aurora kinase A gene promoter, 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), where 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. Therefore, preferably, 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. Contains the 2140th nucleotide sequence. 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. For example, in the case of the human Aurora kinase A gene promoter, the nucleotide sequence from position -1486 to +354 (nucleotide sequence shown in SEQ ID NO: 4) with the transcription start point being +1, and 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. Accordingly, in a preferred embodiment, 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. In the case of using an Aurora kinase promoter derived from another mammal, a preferable region can be selected in the same manner.
　本発明における癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターはまた、天然の哺乳動物由来の癌細胞または未分化細胞特異的遺伝子のプロモーターとストリンジェントな条件下でハイブリダイズし得る核酸であって、該天然プロモーターと実質的に同一の特性を有する核酸を包含する。「実質的に同一の特性」とは、標的の未分化細胞および／または腫瘍化原因細胞に特異的な遺伝子発現を駆動する性質を意味し、転写活性の程度は同等（例えば、約0.5～約2倍）であることが好ましいが、当該未分化細胞および／または腫瘍化原因細胞に対して十分な殺傷効果を発揮できる程度の遺伝子発現を駆動し得る限り、量的要素は異なっていてもよい。例えば、ヒトAuroraキナーゼAまたはBプロモーターの場合、配列番号４または５に示されるヌクレオチド配列の相補鎖配列とストリンジェントな条件下でハイブリダイズし得る核酸が挙げられる。このような核酸としては、例えば、配列番号４または５に示されるヌクレオチド配列と約80%以上、好ましくは約90%以上、より好ましくは約95%以上、特に好ましくは約97%以上、最も好ましくは約98%以上の相同性を有するヌクレオチド配列を含有する核酸などが挙げられる。本明細書におけるヌクレオチド配列の相同性は、例えば、相同性計算アルゴリズムNCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) を用い、以下の条件 (期待値=10; ギャップを許す; フィルタリング=ON; マッチスコア=1; ミスマッチスコア=-3) にて計算することができる。 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. And nucleic acids having substantially the same properties as the native promoter. By “substantially identical characteristics” is meant 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. . For example, in the case of the human Aurora kinase A or B promoter, 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. The homology of the nucleotide sequence in this specification is, for example, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) 、, and the following condition (expected value = 10; allow gap; filtering = ON ; Match score = 1; mismatch score = -3)
　ハイブリダイゼーションは、自体公知の方法あるいはそれに準じる方法、例えば、Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、添付の使用説明書に記載の方法に従って行なうことができる。ハイブリダイゼーションは、好ましくは、ストリンジェントな条件に従って行なうことができる。ストリンジェントな条件としては、(1) 洗浄に低イオン強度および高温、例えば、50℃で0.015 M 塩化ナトリウム/0.0015 M クエン酸ナトリウム/0.1% 硫酸ドデシルナトリウムを使用し、(2) ホルムアミドのような変性剤、例えば、0.1% ウシ血清アルブミン/0.1% フィコール/0.1% ポリビニルピロリドン/750 mM 塩化ナトリウム、75 mM クエン酸ナトリウムを含む50 mM リン酸ナトリウム緩衝液 (pH 6.5) とともに、50% (v/v) ホルムアミドを42℃で使用することを特徴とする反応条件が例示される。あるいは、ストリンジェントな条件は、50% ホルムアミド、5xSSC (0.75 M NaCl、0.075 M クエン酸ナトリウム)、50 mM リン酸ナトリウム (pH 6.8)、0.1% ピロ燐酸ナトリウム、5xデンハート溶液、超音波処理鮭***DNA (50 mg/ml)、0.1% SDS、及び10% 硫酸デキストランを42℃で使用し、0.2xSSC及び50% ホルムアルデヒドで55℃で洗浄し、続いて55℃でEDTAを含有する0.1xSSCからなる高ストリンジェント洗浄を行うものであってもよい。当業者は、プローブ長等のファクターに応じて、ハイブリダイゼーション反応および/または洗浄時の温度、緩衝液のイオン強度等を適宜調節することにより、容易に所望のストリンジェンシーを実現することができる。 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. Alternatively, 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.
　癌細胞または未分化細胞特異的遺伝子プロモーターは、ヒトまたは他の哺乳動物（例：サル、ウシ、ウマ、ブタ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、マウス、ラット等）由来の細胞・組織から抽出したゲノムＤＮＡより、公知の当該遺伝子プロモーター配列（例えば、サバイビンプロモーターの場合、Li, F. and Altieri, D.C., Cancer Res., 59: 3143-3151, 1999; Li, F. and Altieri, D.C., Biochem. J., 344: 305-311, 1999参照; TERTプロモーターの場合、Cong, Y.S. et al., Hum. Mol. Genet., 8(1): 137-142, 1999参照; Auroraキナーゼプロモーターの場合、Tanaka, M. et al., J. Biol. Chem., 277(12): 10719-26, 2002; Kimura, M. et al., Biochem. Biophys. Res. Commun., 316: 930-6, 2004参照）からなる核酸をプローブとして該プロモーター領域を含むゲノムDNAをクローニングし、DNA分解酵素、例えば、適当な制限酵素を用いて所望の部分プロモーター配列を含むDNA断片に切断、ゲル電気泳動で分離後、所望のバンドを回収してDNAを精製することにより調製することができる。あるいは、上記細胞の粗抽出液もしくはそこから単離したゲノムDNAを鋳型として、公知の癌細胞または未分化細胞特異的遺伝子プロモーター配列を基に合成したプライマーを用いたPCRにより、当該プロモーター部分配列を増幅、単離することもできる。癌細胞または未分化細胞特異的遺伝子プロモーターのヌクレオチド配列が未知の哺乳動物については、該癌細胞または未分化細胞特異的遺伝子のcDNA配列をクエリーとして該動物のゲノムDNAに対してBLAST検索を行うことにより、該動物の癌細胞または未分化細胞特異的遺伝子プロモーター領域のヌクレオチド配列を入手することができる。
　また、癌細胞または未分化細胞特異的遺伝子プロモーターは、公知の当該プロモーター配列（例えば、Auroraキナーゼプロモーターの場合、配列番号４または５で表されるヌクレオチド配列）を基に、そのヌクレオチド配列の全部または一部を含む核酸を、市販のDNA/RNA自動合成装置を用いて化学合成することによっても得ることができる。 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. et al., J. Biol. Chem., 277 (12): 10719-26, 2002; Kimura, M. et al., Biochem. Biophys. Res. Commun., 316: 930-6, 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. Alternatively, 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. For a mammal whose nucleotide sequence of a cancer cell or undifferentiated cell-specific gene promoter is unknown, 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.
In addition, 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.
　一実施態様において、本発明の未分化細胞／腫瘍化原因細胞殺傷剤は、癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子をコードする核酸を含む発現ベクターを有効成分として含有する。本発明の発現ベクターに用いられる、癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子をコードする核酸は、例えば、該核酸が転写（および翻訳）された場合に、直接的もしくは間接的に、細胞に対して死、もしくは少なくとも増殖阻害をもたらす限り、いかなるタンパク質またはRNAをコードするものであってもよい。例えば、細胞毒性因子として、アポトーシス誘導遺伝子（Fasなど）、イオンチャネル（ナトリウムチャネルなど）の構成タンパク質をコードする遺伝子、プロドラッグを毒物に変換することによって細胞を傷害しうるタンパク質の遺伝子（自殺遺伝子）（HSV-チミジンキナーゼ、シトシンデアミナーゼなど）、初期化遺伝子に対するアンチセンス核酸（Oct3/4に対するアンチセンス核酸、Nanogに対するアンチセンス核酸など）、アポトーシス促進作用または細胞増殖抑制作用のあるmiRNA若しくはそのmimic、あるいはアポトーシス抑制作用または細胞増殖促進作用のあるmiRNAのアンチセンス核酸、アプタマー、リボザイム等が挙げられる。 In one embodiment, 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. Examples of 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.
　細胞毒性因子をコードする核酸は、それを産生する細胞・組織から自体公知の方法によりcDNAとして単離することができ、癌細胞または未分化細胞特異的遺伝子プロモーターの下流に機能的に連結することができる。癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子をコードする核酸を含む発現カセットは、好ましくは該核酸または遺伝子の下流に適当なポリアデニレーション配列を含む。
　本発明の、細胞毒性因子をコードする核酸と機能的に結合した癌細胞または未分化細胞特異的遺伝子プロモーターを含む発現カセットを有するベクターは、ウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸が癌細胞または未分化細胞特異的遺伝子プロモーター、あるいは癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる外来性プロモーターの制御下に置かれていてもよい。例えば、癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる外来性プロモーターとして、哺乳類において恒常的に発現し得るプロモーターを使用する場合、サイトメガロウイルス (CMV) 由来プロモーター (例: CMV前初期プロモーター)、ヒト免疫不全ウイルス (HIV) 由来プロモーター (例: HIV LTR)、ラウス肉腫ウイルス (RSV) 由来プロモーター (例: RSV LTR)、マウス乳癌ウイルス (MMTV) 由来プロモーター (例: MMTV LTR)、モロニーマウス白血病ウイルス (MoMLV) 由来プロモーター (例: MoMLV LTR)、単純ヘルペスウイルス (HSV) 由来プロモーター (例: HSVチミジンキナーゼ(TK) プロモーター)、SV40由来プロモーター (例: SV40初期プロモーター)、エプスタインバーウイルス(EBV) 由来プロモーター、アデノ随伴ウイルス (AAV) 由来プロモーター (例: AAV p5プロモーター)、アデノウイルス (AdV) 由来プロモーター (Ad2またはAd5主要後期プロモーター) など、ならびにβ-アクチン遺伝子プロモーター、PGK遺伝子プロモーター、トランスフェリン遺伝子プロモーター等の哺乳動物の構成タンパク質の遺伝子プロモーターなどの構成的プロモーターを用いることができる。癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる外来性プロモーターとしては、標的未分化細胞および／または腫瘍化原因細胞が混在する分化細胞で特異的に発現が亢進している因子のプロモーターや誘導性プロモーターを用いることもできる。腫瘍化の原因となる未分化細胞は完全に未分化な状態を維持しているとは限らず、ある程度分化が進んだ状態でなお未分化な特性を保持する場合も多い。そのような細胞では分化マーカーと未分化マーカーとが共に発現しているので、これらのいずれかのマーカー遺伝子のプロモーターを利用することができる。分化細胞特異的に発現が亢進している因子のプロモーターとしては、例えば、肝臓などに特異的なアルブミンおよびα-フェトプロテインのプロモーター、前立腺に特異的な前立腺特異的抗原（PSA）のプロモーター、筋肉や脳など様々な臓器に特異的なミトコンドリア型クレアチンキナーゼ（MCK）のプロモーター、ならびに、脳などの神経系に特異的なミエリン塩基性タンパク質（MB）、グリア繊維酸性タンパク質（GFAP）および神経特異的エノラーゼ（NSE）のプロモーターなどを例示できる。また、誘導性プロモーターとしては、例えば、メタロチオネイン-1遺伝子プロモーターなどを用いることができる。メタロチオネイン-1遺伝子プロモーターを用いた場合、金、亜鉛、カドミウム等の重金属、デキサメサゾン等のステロイド、アルキル化剤、キレート剤またはサイトカインなどの誘導物質を、所望の時期に標的未分化細胞および／または腫瘍化原因細胞の存在する位置（移植部位）に局所投与することにより、任意の時期に標的未分化細胞および／または腫瘍化原因細胞にウイルスタンパク質などの細胞毒性因子の発現を誘導することができる。 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. For example, when using a promoter that can be constitutively expressed in mammals as a foreign promoter that is different from the promoter of cancer cell-specific gene and undifferentiated cell-specific gene, a promoter derived from cytomegalovirus (CMV) (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 (e.g. 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 β-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. In such cells, both the differentiation marker and the undifferentiation marker are expressed, and therefore the promoter of any of these marker genes can be used. Examples of promoters of factors whose expression is specifically increased in differentiated cells include albumin and α-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. Moreover, as an inducible promoter, a metallothionein-1 gene promoter etc. can be used, for example. When using a metallothionein-1 gene promoter, induce 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. 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.
　また、別の態様において、本発明は、少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが癌細胞または未分化細胞特異的遺伝子プロモーターで置換されていることを特徴とする標的未分化細胞および／または腫瘍化原因細胞特異的増殖型ウイルスベクター（conditionally replicating virus：CRV）に関する（以下、「癌細胞または未分化細胞特異的遺伝子プロモーター依存性CRV」ともいう）。即ち、本発明は、標的未分化細胞および／または腫瘍化原因細胞において特異的に（分化細胞や非癌化細胞よりも優位に）増殖することを特徴とするベクターに関する。これらのウイルスベクターは、標的未分化細胞および／または腫瘍化原因細胞で特異的にウイルスの増殖を引き起こすだけでなく、増幅された結果、該標的未分化細胞および／または腫瘍化原因細胞を殺傷（溶解）する。さらに、溶解した細胞から放出されたウイルスは周辺のベクター未導入の未分化細胞および／または腫瘍化原因細胞に感染し、このステップが繰り返されることで、最終的には細胞集団内のすべての未分化細胞および／または腫瘍化原因細胞に本発明のベクターが導入され、未分化細胞および／または腫瘍化原因細胞の殺傷除去効果を得ることができる。 In another embodiment, 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). These 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). In addition, 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.
　本発明の癌細胞または未分化細胞特異的遺伝子プロモーター依存性CRVは、少なくとも１つのウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸を癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におくことにより構築することができる。「ウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸」とは、ウイルスの構造タンパク質などの、ウイルスが自己複製を行うために必須のタンパク質のいずれかをコードする核酸、またはウイルスがアッセンブリを行うために必須のタンパク質のいずれかをコードする核酸を意味する。より具体的には、ウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸は、用いるウイルス種によって異なるが、例えばアデノウイルスの場合、感染初期から転写が開始されて、後のウイルスタンパク質の転写制御に働く初期遺伝子（Early gene）であるE1A、E1B、E2およびE4、または後述のRb結合領域欠損型E1A（E1AΔ24）、p53結合領域欠損型E1B（E1BΔ55K）などが挙げられる。特にE1Aは、アデノウイルスの感染後最初に転写され、E1Aの発現がなければその後のウイルス複製が起こらないことより、癌細胞または未分化細胞特異的遺伝子プロモーターで標的未分化細胞および／または腫瘍化原因細胞特異的にウイルス増殖制御を行なう目的に非常に適している遺伝子であるが、ウイルスの複製に必須のその他の初期遺伝子を制御することでも同様の効果を得ることができる。また、アデノウイルスの構造遺伝子をコードする核酸の後期遺伝子（Late gene）のL1、L2、L3、L4およびL5などは、感染後の細胞***が起こる後期に転写されて、ウイルス構造を構成するタンパク質であるが、これらの後期遺伝子を癌細胞または未分化細胞特異的遺伝子プロモーターで発現制御しても、標的未分化細胞および／または腫瘍化原因細胞特異的にウイルス増殖制御を行なうことができる。このように、本発明の癌細胞または未分化細胞特異的遺伝子プロモーター依存性増殖型ウイルスベクターにおいて癌細胞または未分化細胞特異的遺伝子プロモーターで発現制御されるウイルスタンパク質をコードする遺伝子は、ウイルスの複製またはアッセンブリに必須のウイルス遺伝子であれば、いずれのものでもよい。アデノウイルス以外のウイルスベクターを用いる場合、例えば、アデノ随伴ウイルスの場合であれば、p5プロモーターの制御下にあるRep78およびRep68、p19プロモーターの制御下にあるRep52およびRep40など、単純ヘルペスウイルスの場合であれば、ICP0、ICP4、ICP22、ICP27等の初期遺伝子産物、チミジンキナーゼなど、センダイウイルスの場合であれば、Nタンパク質、Pタンパク質、Lタンパク質などがそれぞれ挙げられる。 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. Can be constructed. “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. For example, in the case of adenovirus, transcription is initiated from the early stage of infection, and transcriptional control of the subsequent viral protein is performed. E1A, E1B, E2 and E4, which are early genes that act on E. coli, or Rb-binding region-deficient E1A (E1AΔ24) and p53-binding region-deficient E1B (E1BΔ55K) described later. In particular, 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. Although it is a gene that is very suitable for the purpose of controlling virus growth specifically in the causative cell, the same effect can be obtained by controlling other early genes essential for virus replication. In addition, 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. However, even if 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. Thus, in the cancer cell or undifferentiated cell-specific gene promoter-dependent propagation viral vector of the present invention, a gene encoding a viral protein whose expression is controlled by a cancer cell or undifferentiated cell-specific gene promoter is a viral replication. Alternatively, any viral gene that is essential for assembly may be used. When using 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. If present, 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.
　かかる癌細胞または未分化細胞特異的遺伝子プロモーター依存性増殖型ウイルスベクターは、ウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸の内因性プロモーターを癌細胞または未分化細胞特異的遺伝子プロモーターで置換することにより得ることができる。好ましくは、本発明の増殖型ウイルスベクターがアデノウイルスベクターの場合、E1Aおよび/またはE1Bをコードする核酸、より好ましくは少なくともE1Aをコードする核酸が癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におかれる。 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. Preferably, when 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.
　細胞内に導入された本発明の癌細胞または未分化細胞特異的遺伝子プロモーター依存性CRVは、癌細胞または未分化細胞特異的遺伝子プロモーターが活性化されない環境下（分化細胞や非癌化細胞）では増殖できないため、当該細胞は傷害を受けない。一方、本発明の癌細胞または未分化細胞特異的遺伝子プロモーター依存性CRVが、癌細胞または未分化細胞特異的遺伝子プロモーターが活性化される環境（標的未分化細胞および／または腫瘍化原因細胞）内に侵入すると、そこでウイルスが増殖し、ウイルスタンパク質の細胞毒性により細胞が傷害される。溶解した細胞から放出されたウイルスは周辺のベクター未導入の未分化細胞および／または腫瘍化原因細胞に次々と感染し、同様のステップが繰り返される。こうして、最終的には細胞集団内のすべての未分化細胞および／または腫瘍化原因細胞に本発明の増殖型ウイルスベクターが導入され得る。 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. On the other hand, 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. Thus, the proliferative virus vector of the present invention can be finally introduced into all undifferentiated cells and / or tumorigenic cells in the cell population.
　ウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸の少なくとも１つが癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におかれていれば、ウイルスの増殖またはアッセンブリは癌細胞または未分化細胞特異的遺伝子プロモーターが活性化される環境下に限定されるので、他のウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸は癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる任意の外来性プロモーターの制御下に置かれてよい。例えば、癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる外来性プロモーターとして、哺乳類において恒常的に発現し得るプロモーターを使用する場合、サイトメガロウイルス (CMV) 由来プロモーター (例: CMV前初期プロモーター)、ヒト免疫不全ウイルス (HIV) 由来プロモーター (例: HIV LTR)、ラウス肉腫ウイルス (RSV) 由来プロモーター (例: RSV LTR)、マウス乳癌ウイルス (MMTV) 由来プロモーター (例: MMTV LTR)、モロニーマウス白血病ウイルス (MoMLV) 由来プロモーター (例: MoMLV LTR）、単純ヘルペスウイルス (HSV) 由来プロモーター (例: HSVチミジンキナーゼ(TK) プロモーター)、SV40由来プロモーター (例: SV40初期プロモーター)、エプスタインバーウイルス(EBV) 由来プロモーター、アデノ随伴ウイルス (AAV) 由来プロモーター (例: AAV p5プロモーター)、アデノウイルス (AdV) 由来プロモーター (Ad2またはAd5主要後期プロモーター) など、ならびにβ-アクチン遺伝子プロモーター、PGK遺伝子プロモーター、トランスフェリン遺伝子プロモーター等の哺乳動物の構成タンパク質の遺伝子プロモーターなどの構成的プロモーターを用いることができる。癌細胞特異的遺伝子および未分化細胞特異的遺伝子のプロモーターとは異なる外来性プロモーターとしては、標的未分化細胞または腫瘍化原因細胞が混在する分化細胞で特異的に発現が亢進している因子のプロモーターや誘導性プロモーターを用いることもできる。分化細胞特異的に発現が亢進している因子のプロモーターとしては、例えば、肝臓などに特異的なアルブミンおよびα－フェトプロテインのプロモーター、前立腺に特異的な前立腺特異的抗原（PSA）のプロモーター、筋肉や脳など様々な臓器に特異的なミトコンドリア型クレアチンキナーゼ（MCK）のプロモーター、ならびに、脳などの神経系に特異的なミエリン塩基性タンパク質（MB）、グリア繊維酸性タンパク質（GFAP）および神経特異的エノラーゼ（NSE）のプロモーターなどを例示できる。また、誘導性プロモーターとしては、例えば、メタロチオネイン－１遺伝子プロモーターなどを用いることができる。メタロチオネイン－１遺伝子プロモーターを用いた場合、金、亜鉛、カドミウム等の重金属、デキサメサゾン等のステロイド、アルキル化剤、キレート剤またはサイトカインなどの誘導物質を、所望の時期に標的未分化細胞および／または腫瘍化原因細胞の存在位置（移植部位）に局所投与することにより、任意の時期に標的未分化細胞および／または腫瘍化原因細胞にウイルスの複製またはアッセンブリに必要なタンパク質の発現を誘導することができる。
　また、2以上のウイルスの複製またはアッセンブリに必要なタンパク質をコードする核酸を癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におく場合、用いるプロモーターは同一のプロモーターであってもよいし、異なるものであってもよい。例えば、TERTプロモーターとサバイビンプロモーターとを、１つのベクター内で併用することもできる。また、癌細胞または未分化細胞特異的遺伝子プロモーター依存性CRVが、更に細胞毒性因子をコードする核酸と機能的に結合した癌細胞または未分化細胞特異的遺伝子プロモーターを含む発現カセットを備えていてもよい。 If at least one 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. For example, when using a promoter that can be constitutively expressed in mammals as a foreign promoter that is different from the promoter of cancer cell-specific gene and undifferentiated cell-specific gene, a promoter derived from cytomegalovirus (CMV) (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 (e.g. AAV p5 promoter), adenovirus (AdV) -derived promoter (Ad2 or Ad5 major late promoter), and constitutive genes such as β-actin gene promoter, PGK gene promoter, transferrin gene promoter, etc. A promoter can be used. As a foreign promoter different from the promoters for cancer cell-specific genes and undifferentiated cell-specific genes, promoters of factors whose expression is specifically enhanced in target undifferentiated cells or differentiated cells in which tumorigenic cells coexist Alternatively, an inducible promoter can be used. Examples of promoters of factors whose expression is specifically enhanced in differentiated cells include, for example, albumin and α-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. As the inducible promoter, for example, a metallothionein-1 gene promoter can be used. When a metallothionein-1 gene promoter is 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. By local administration at the location of transplantation-causing cells (transplantation site), it is possible to induce expression of proteins necessary for virus replication or assembly in target undifferentiated cells and / or tumorigenic cells at any time .
In addition, when a nucleic acid encoding a protein necessary for replication or assembly of two or more viruses is placed under the control of a cancer cell or undifferentiated cell-specific gene promoter, the promoters used may be the same or different. It may be a thing. For example, 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.
　本発明のウイルスベクターは、ウイルスタンパク質の、分化細胞や非癌化細胞におけるウイルスの増殖に必要な細胞環境を誘導するのに必須であるが、標的未分化細胞および／または腫瘍化原因細胞におけるウイルスの増殖には必要でない領域を欠損させてもよい。例えば、分化細胞や非癌化細胞でのアデノウイルス増殖のためには、細胞周期を回すためにRbやp53を不活性化することが必要であるが、未分化細胞および腫瘍化原因細胞ではすでに細胞周期が回っている状態にあるので、未分化細胞および腫瘍化原因細胞でのアデノウイルスの増殖には、E1AのRb結合領域やE1Bのp53結合領域は必要ではない。したがって、例えばアデノウイルスの場合、E1A24KDaの領域を欠損させ（E1AΔ24）、E1B55KDaの領域を欠損させ（E1BΔ55）、またはE1B19KDaの領域を欠損させる（E1BΔ19）ことにより、未分化細胞および／または腫瘍化原因細胞特異的なウイルスの増殖が可能になる。このタイプのウイルスベクターの場合、ウイルスの複製に必要なタンパク質をコードする核酸が癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におかれていなくても未分化細胞および／または腫瘍化原因細胞特異的な増殖を起こすことができる。したがって、本発明は、ウイルスの複製またはアッセンブリに必要なタンパク質をコードするいずれの核酸も癌細胞または未分化細胞特異的遺伝子プロモーターの制御下になく、分化細胞または非癌化細胞におけるウイルスの増殖に必要な細胞環境を誘導するのに必須であるが、標的未分化細胞および／または腫瘍化原因細胞におけるウイルスの増殖には必要でない領域（例えば、E1A24KDaの領域、E1B55KDaの領域、および/またはE1B19KDaの領域）が欠損したウイルスベクターであって、癌細胞特異的遺伝子プロモーターの制御下に細胞毒性因子をコードする核酸が機能的に結合されたベクターを含む。もちろん、分化細胞や非癌化細胞におけるウイルスの増殖に必要な細胞環境を誘導するのに必須であるが、標的未分化細胞および／または腫瘍化原因細胞におけるウイルスの増殖には必要でない領域（例えば、E1A24KDaの領域、E1B55KDaの領域、および/またはE1B19KDaの領域）を欠損させたウイルスタンパク質をコードする核酸が、癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におかれてもよいし、該欠損ウイルスタンパク質以外のウイルスの複製に必要なタンパク質をコードする核酸のいずれかが、癌細胞または未分化細胞特異的遺伝子プロモーターの制御下におかれてもよい。
　本発明のベクターは、宿主細胞で自律増幅するための複製起点や、形質転換細胞選択のための選択マーカー遺伝子 (テトラサイクリン、アンピシリン、カナマイシン、ハイグロマイシン、ホスフィノスリシン等の薬剤に対する抵抗性を付与する遺伝子、栄養要求性変異を相補する遺伝子等) をさらに含有することもできる。 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. Thus, for example, in the case of an adenovirus, the E1A24KDa region is deleted (E1AΔ24), the E1B55KDa region is deleted (E1BΔ55), or the E1B19KDa region is deleted (E1BΔ19). Allows cell-specific virus propagation. In the case of 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. Thus, 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) And a vector in which a nucleic acid encoding a cytotoxic factor is operably linked under the control of a cancer cell-specific gene promoter. Of course, 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 (eg A nucleic acid encoding a viral protein deficient in the E1A24KDa region, the E1B55KDa region, and / or the E1B19KDa region) 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.).
　本発明のベクターは、ウイルスベクターであっても非ウイルスベクターであってもよいが、好ましくはアデノウイルス、レトロウイルス、アデノ随伴ウイルス、ヘルペスウイルス、単純ヘルペスウイルス、レンチウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンドビスウイルス、センダイウイルス等のウイルスベクターである。アデノウイルスは、遺伝子導入効率が極めて高く、非***細胞にも導入可能であり、導入遺伝子の宿主染色体への組込みが極めて稀である等の利点を有する。特に、パッケージングシグナル (ψ) 以外のアデノウイルスゲノムのほぼ全長を導入遺伝子に置換したgutted (gutless) ベクターの開発によって、第一世代ベクターにおける免疫原性の問題が解消され、それに伴い導入遺伝子発現の長期持続性が実現された。同様に、アデノ随伴ウイルスも、比較的遺伝子導入効率が高く、非***細胞にも導入可能で、動物実験で生体内投与により導入遺伝子の発現が長期にわたって持続することが知られているので、本発明におけるウイルスベクターとして好ましい。 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. In particular, the development of a gutted (gutless) vector that replaces almost the entire length of the adenovirus genome other than the packaging signal (ψ) に 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. Similarly, 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. Preferred as a viral vector in the invention.
　本発明の好ましい一実施形態では、本発明者らが開発した多因子がん特異的増殖制御型組換えアデノウイルス系（m-CRA；特開2005-046101号及び国際公開第2005/012536号）の一部として、癌細胞または未分化細胞特異的遺伝子プロモーターを用いる。m-CRAの構築に好適に用いられるプラスミドベクターの例を、図１に提示する。図中で、プラスミドベクターＰ１のうち、プロモーターＡおよび／またはプロモーターＢとして癌細胞特異的遺伝子プロモーターを用い、プラスミドベクターＰ２のプロモーターＣ（細胞毒性因子の発現を制御する）として、癌細胞または未分化細胞特異的遺伝子プロモーターもしくは別の標的未分化細胞および／または腫瘍化原因細胞特異的プロモーターまたは構成的プロモーター等の他の任意のプロモーターを用いることができる。標的未分化細胞および／または腫瘍化原因細胞特異的プロモーターとしては、未分化細胞および／または腫瘍化原因細胞でのみ特異的に発現する上記の各種プロモーターを例示できる。プロモーターＣに制御される細胞毒性因子としては、上記した各種細胞毒性因子を例示できる。
　後述の実施例に示した具体的な実施形態では、TERTまたはサバイビンプロモーターと機能的に連結したE1A遺伝子（24KDa領域を欠損していてもよい）、および構成的プロモーター（CMVプロモーターなど）と機能的に連結したE1B遺伝子（19KDaまたは55KDa領域を欠損していてもよい）を含むプラスミドベクターＰ１、構成的プロモーター（CMVプロモーターなど）と機能的に連結されたレポーター遺伝子（細胞毒性因子のモデル系として）を含むプラスミドベクターＰ２、ならびにE1領域を欠失するアデノウイルスゲノム（ファイバー遺伝子内に標的細胞特異的な変異を有していてもよい）を含むバックボーンプラスミドＰ３が提供される。これら３種のプラスミドを適宜組み合わせ、CreリコンビナーゼloxPシステムを用いてプラスミド融合と、各プラスミドに搭載された薬剤耐性遺伝子とoriを利用した目的プラスミドの選択により、TERTまたはサバイビンプロモーター-E1A発現カセット、構成的プロモーター-E1B発現カセットおよび構成的プロモーター-細胞毒性因子発現カセットを搭載した、未分化細胞特異的増殖型アデノウイルス（CRA）ベクタープラスミドを作製する。続いて該ベクターを用いて、E1Aを相補する細胞株（例：293細胞）にトランスフェクションすることにより、CRAベクターを作製することができる。図２Ｂに示したm-CRAベクターは、E1Aの発現を制御するプロモーター（TERTまたはサバイビン）、E1B遺伝子（E1BΔ55K）の2因子でベクターの増殖が制御されているが、E1A遺伝子、E1Bの発現を制御するプロモーター、細胞毒性因子の発現を制御するプロモーター、細胞毒性因子、さらにはバックボーンのファイバー遺伝子を他の要素に置換することにより、さらに多因子による高度な増殖および発現制御が可能となる。 In a preferred embodiment of the present invention, the multifactor cancer-specific growth-regulated recombinant adenovirus system developed by the present inventors (m-CRA; JP 2005-046101 and WO 2005/012536) As part of this, 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. In the figure, a cancer cell-specific gene promoter is used as promoter A and / or promoter B in plasmid vector P1, and 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. Examples of 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. Examples of the cytotoxic factor controlled by the promoter C include the various cytotoxic factors described above.
In the specific embodiments shown in the examples below, 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) As well as a backbone plasmid P3 containing an adenoviral genome lacking the E1 region (which may have a target cell specific mutation in the fiber gene). By combining these three plasmids as appropriate, plasmid recombination using the Cre recombinase loxP system and selection of the target plasmid using the drug resistance gene and ori loaded on each plasmid, the TERT or survivin promoter-E1A expression cassette, construction An undifferentiated cell-specific proliferating adenovirus (CRA) vector plasmid is constructed that carries a constitutive promoter-E1B expression cassette and a constitutive promoter-cytotoxic factor expression cassette. Subsequently, using this vector, a CRA vector can be prepared by transfection into a cell line (eg, 293 cells) complementary to E1A. In the m-CRA vector shown in FIG. 2B, the growth of the vector is controlled by two factors, the promoter (TERT or survivin) that controls E1A expression and the E1B gene (E1BΔ55K). 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.
　本発明の非ウイルスベクターは、癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子をコードする核酸を含む発現カセットを含む。ベクターとしては、大腸菌由来のプラスミド (例: pBR322、pBR325、pUC12、pUC13)、枯草菌由来のプラスミド (例: pUB110、pTP5、pC194)、酵母由来プラスミド (例: pSH19、pSH15)、動物細胞発現プラスミド (例: pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo) などを用いることができる。ここで「細胞毒性因子」とは上記と同義である。
　本発明の非ウイルスベクターは、上記の他に、所望によりエンハンサー、スプライシングシグナル、ポリA付加シグナル、選択マーカー、SV40複製起点などを含有しているものを用いることができる。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素 (dhfr) 遺伝子 [メソトレキセート (MTX) 耐性]、アンピシリン耐性(Ampr) 遺伝子、ネオマイシン耐性 (Neor) 遺伝子 (G418耐性) 等が挙げられる。
　非ウイルスベクターを使用する場合、該ベクターの導入は、ポリL-リジン-核酸複合体などの高分子キャリアーを用いるか、リポソームに被包して行うことができる。リポソームはリン脂質からなる数10～数100 nmの粒径のカプセルで、その内部に癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子をコードする核酸を含むプラスミド等のベクターを封入できる。あるいは、パーティクルガン法を用いてベクターを標的細胞に直接導入することもできる。 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. As 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 (Examples: pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo) and the like can be used. Here, “cytotoxic factor” has the same meaning as described above.
As the 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. Examples of 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.
When a non-viral vector is used, 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.
　本発明の少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが癌細胞または未分化細胞特異的遺伝子プロモーターで置換されていることを特徴とする標的未分化細胞および／または腫瘍化原因細胞特異的増殖型ウイルスベクター、あるいは癌細胞または未分化細胞特異的遺伝子プロモーターの制御下にある細胞毒性因子を含むベクターは、標的未分化細胞および／または腫瘍化原因細胞で特異的に増殖させあるいは該細胞毒性因子を発現させることができるので、必要に応じて薬理学的に許容し得る担体とともに混合して注射剤などの種々の製剤形態とした後に、未分化細胞／腫瘍化原因細胞殺傷剤、好ましくは、幹細胞、特に多能性幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化原因細胞を選択的に殺傷する薬剤として用いることができる。ここで薬理学的に許容し得る担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤; 液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤などとして配合される。また必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤などの製剤添加物を用いることもできる。 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. Here, as 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.
　賦形剤の好適な例としては、乳糖、白糖、D-マンニトール、D-ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、アラビアゴム、プルラン、軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウムなどが挙げられる。
　滑沢剤の好適な例としては、ステアリン酸マグネシウム、ステアリン酸カルシウム、タルク、コロイドシリカなどが挙げられる。
　結合剤の好適な例としては、α化デンプン、ショ糖、ゼラチン、アラビアゴム、メチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、結晶セルロース、白糖、D-マンニトール、トレハロース、デキストリン、プルラン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドンなどが挙げられる。
　崩壊剤の好適な例としては、乳糖、白糖、デンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、軽質無水ケイ酸、低置換度ヒドロキシプロピルセルロースなどが挙げられる。
　溶剤の好適な例としては、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、ゴマ油、トウモロコシ油、オリーブ油、綿実油などが挙げられる。
　溶解補助剤の好適な例としては、ポリエチレングリコール、プロピレングリコール、D-マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウムなどが挙げられる。
　懸濁化剤の好適な例としては、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、 モノステアリン酸グリセリンなどの界面活性剤、例えばポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロースなどの親水性高分子、ポリソルベート類、ポリオキシエチレン硬化ヒマシ油などが挙げられる。
　等張化剤の好適な例としては、塩化ナトリウム、グリセリン、D-マンニトール、D-ソルビトール、ブドウ糖などが挙げられる。
　緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などが挙げられる。
　無痛化剤の好適な例としては、ベンジルアルコールなどが挙げられる。
　防腐剤の好適な例としては、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸などが挙げられる。
　抗酸化剤の好適な例としては、亜硫酸塩、アスコルビン酸塩などが挙げられる。
　着色剤の好適な例としては、水溶性食用タール色素(例: 食用赤色2号および3号、食用黄色4号および5号、食用青色1号および2号などの食用色素)、水不溶性レーキ色素 (例: 前記水溶性食用タール色素のアルミニウム塩など)、天然色素 (例: β-カロチン、クロロフィル、ベンガラなど) などが挙げられる。
　甘味剤の好適な例としては、サッカリンナトリウム、グリチルリチン酸二カリウム、アスパルテーム、ステビアなどが挙げられる。 Suitable examples of 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.
Preferable examples of 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.
Suitable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
Suitable examples of the suspending agent 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.
Preferable examples of the 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. 1 and 2), water-insoluble lake dyes (Example: Aluminum salt of water-soluble edible tar pigment, etc.), natural pigment (eg, β-carotene, chlorophyll, Bengala, etc.).
Preferable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
　本発明のベクターを含有する未分化細胞／腫瘍化原因細胞殺傷剤の投与は、幹細胞から分化誘導された細胞集団にベクター導入を行い、一定期間培養して残存する未分化細胞および／または腫瘍化原因細胞を殺傷除去した後に、得られた分化細胞を患者に移植するex vivo法と、幹細胞から分化誘導された細胞集団とは別個に、患者に投与するin vivo法のいずれかで行われるが、患者体内に内在する幹細胞をも殺傷するリスクがあるので、in vivo法の場合、該製剤の投与は、例えば、細胞移植部位への局所注入、本発明のベクターを組み込んだインプラントの細胞移植部位への移植などにより行うことが望ましい。ex vivo法の場合、標的細胞へのベクターの導入は、マイクロインジェクション法、リン酸カルシウム共沈殿法、PEG法、エレクトロポレーション法等により行うことができる。in vivo法の場合、本発明の未分化細胞／腫瘍化原因細胞殺傷剤の投与は、細胞移植に先立って行ってもよいし、細胞移植と同時もしくは移植後に行ってもよい。 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. Since there is a risk of killing stem cells existing in the patient body, in the case of in vivo method, 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. In the case of the ex-vivo method, 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. In the case of the in vivo method, 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.
　本発明の未分化細胞／腫瘍化原因細胞殺傷剤を用いることができる、未分化細胞および／または腫瘍化原因細胞が残存するおそれのある分化細胞集団は、例えば、以下のようにして提供される。 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. .
（ａ）幹細胞
　分化細胞を誘導するソースとなる幹細胞は、多能性もしくは多分化能と、未分化状態を保持したまま増殖できる自己複製能とを有する細胞であれば特に限定されず、ES細胞、iPS細胞、始原生殖細胞に由来する胚性生殖（EG）細胞、精巣組織からのGS細胞の樹立培養過程で単離されるmultipotent germline stem（mGS）細胞、骨髄から単離されるmultipotent adult progenitor cell（MAPC）等の多能性幹細胞や、造血幹細胞、間葉系幹細胞、歯髄幹細胞、神経幹細胞、血管内皮前駆細胞等の組織幹細胞が例示されるが、好ましくは多能性幹細胞であり、より好ましくはES細胞およびiPS細胞である。ES細胞は体細胞から核初期化されて生じたES細胞であってもよい。幹細胞の由来する動物種はいずれかの幹細胞が樹立されているか、樹立可能である、任意の哺乳動物であってよく、例えば、ヒト、マウス、サル、ブタ、ラット、イヌ等が挙げられるが、好ましくはヒトまたはマウスである。 (A) Stem cell 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. ES cells and iPS cells. 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.
（ｂ）多能性幹細胞の製造方法
　iPS 細胞は、ある特定の核初期化物質を、核酸又はタンパク質の形態で体細胞に導入することによって作製することができる。
　本明細書中で使用する「体細胞」は、哺乳動物（例えば、ヒト、マウス、サル、ブタ、ラット等）由来の生殖細胞以外のいかなる細胞であってもよく、例えば、角質化する上皮細胞（例、角質化表皮細胞）、粘膜上皮細胞（例、舌表層の上皮細胞）、外分泌腺上皮細胞（例、乳腺細胞）、ホルモン分泌細胞（例、副腎髄質細胞）、代謝・貯蔵用の細胞（例、肝細胞）、境界面を構成する内腔上皮細胞（例、I型肺胞細胞）、内鎖管の内腔上皮細胞（例、血管内皮細胞）、運搬能をもつ繊毛のある細胞（例、気道上皮細胞）、細胞外マトリックス分泌用細胞（例、線維芽細胞）、収縮性細胞（例、平滑筋細胞）、血液と免疫系の細胞（例、Tリンパ球）、感覚に関する細胞（例、桿細胞）、自律神経系ニューロン（例、コリン作動性ニューロン）、感覚器と末梢ニューロンの支持細胞（例、随伴細胞）、中枢神経系の神経細胞とグリア細胞（例、星状グリア細胞）、色素細胞（例、網膜色素上皮細胞）、およびそれらの前駆細胞 (組織前駆細胞) 等が挙げられる。細胞の分化の程度や細胞を採取する動物の齢などに特に制限はなく、未分化な前駆細胞 (体性幹細胞も含む) であっても、最終分化した成熟細胞であっても、同様に本発明における体細胞の起源として使用することができる。ここで未分化な前駆細胞としては、例えば神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）が挙げられる。 (B) Method for Producing Pluripotent Stem Cell iPS cells can be prepared by introducing a specific nuclear reprogramming substance into somatic cells in the form of nucleic acids or proteins.
As used herein, 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 of transporting (Eg, airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T lymphocytes), sensory cells (Eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons) Sensory organs and peripheral neuron support cells (eg, companion cells), central nervous system neurons and glial cells (eg, astrocytes), pigment cells (eg, retinal pigment epithelial cells), and their progenitors ( Tissue precursor cells) and the like. There is no particular limitation on the degree of cell differentiation and the age of the animal from which the cells are collected, and this can be applied to both undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells. It can be used as the source of somatic cells in the invention. Examples of 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.
　核初期化物質は、ES細胞に特異的に発現している遺伝子もしくはES細胞の未分化維持に重要な役割を果たす遺伝子、またはその遺伝子産物であれば良く、特に限定されないが、例えば、Oct3/4, 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またはEsrrgが例示される。これらの核初期化物質は、iPS細胞樹立の際には、組み合わされて使用されてもよい。例えば、上記核初期化物質を、少なくとも１つ、２つもしくは３つ含む組み合わせであり、好ましくは４つを含む組み合わせである。
　上記の各核初期化物質のマウスおよびヒトcDNAのヌクレオチド配列は、WO 2007/069666に記載のNCBI accession numbersを参照すること、またL-Myc、Lin28、Lin28b、EsrrbおよびEsrrgのマウスおよびヒトのcDNA配列情報については、それぞれ下記NCBI accession numbersを参照することにより取得できる。当業者は、当該cDNA配列またはアミノ酸配列情報に基づいて、常法により所望の核初期化物質を調製することができる。
遺伝子名　　マウス　　　　　ヒト　　　
L-Myc　　　NM_008506　　　 NM_001033081
Lin28　　　NM_145833　　　 NM_024674
Lin28b　　 NM_001031772　　NM_001004317
Esrrb　　　NM_011934　　　 NM_004452
Esrrg　　　NM_011935　　　 NM_001438 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. For example, a combination including at least one, two, or three of the nuclear reprogramming substances, preferably a combination including four.
For the 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 The 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.
Gene name mouse human
L-Myc NM_008506 NM_001033081
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
　これらの核初期化物質を、核酸の形態で体細胞へ導入する場合、発現ベクターを用いてもよい。本発明における、発現ベクターは、例えば、プラスミド、人工染色体ベクター、およびウイルスベクターが挙げられる。人工染色体ベクターとしては、例えばヒト人工染色体(HAC)、酵母人工染色体(YAC)、細菌人工染色体(BAC、PAC)などが含まれる。また、ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター（以上、Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007）、アデノウイルスベクター(Science, 322, 945-949, 2008)、アデノ随伴ウイルスベクター、センダイウイルスベクター（Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009）などが例示される。また、プラスミドとしては、哺乳動物細胞用プラスミドを使用しうる(Science, 322:949-953, 2008およびWO 2009/032456)。本発明において発現ベクターは、プラスミド、人工染色体ベクターなどは、リポフェクション、リポソーム、マイクロインジェクション、遺伝子銃法などの手法により体細胞内へ導入することができ、ウイルスベクターの場合は、感染により体細胞内へ導入することができる。発現ベクターには、核初期化物質が発現可能なように、プロモーター、エンハンサー、内部リボソーム進入部位(IRES)、ターミネーター、ポリアデニル化サイトなどの制御配列を含むことができる。 When these nuclear reprogramming substances are introduced into somatic cells in the form of nucleic acids, expression vectors may be used. Examples of 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. Moreover, as 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) . As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008 and WO 2009/032456). In the present invention, an expression vector, such as a plasmid or an artificial chromosome vector, can be introduced into a somatic cell by a method such as lipofection, liposome, microinjection, or gene gun method. Can be introduced. 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.
　使用されるプロモーターとしては、例えばEF1αプロモーター、CAGプロモーター、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが用いられる。なかでも、EF1αプロモーター、CAGプロモーター、MoMuLV LTR、CMVプロモーター、SRαプロモーターなどが挙げられる。
　さらに、必要に応じて、薬剤耐性遺伝子(例えばカナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子など)、チミジンキナーゼ遺伝子、ジフテリアトキシン遺伝子などの選択マーカー配列、緑色蛍光タンパク質(GFP)、赤色蛍光タンパク質、βグルクロニダーゼ(GUS)、FLAGなどのレポーター遺伝子配列などを含むことができる。また、上記発現ベクターには、体細胞への導入後、核初期化物質をコードする遺伝子もしくはプロモーターとそれに結合する核初期化物質をコードする遺伝子を共に切除するために、それらの前後にLoxP配列を有してもよい。別の好ましい一実施態様においては、トランスポゾンを用いて染色体に導入遺伝子を組み込んだ後に、プラスミドベクターもしくはアデノウイルスベクターを用いて細胞に転移酵素を作用させ、導入遺伝子を完全に染色体から除去する方法が用いられ得る。好ましいトランスポゾンとしては、例えば、鱗翅目昆虫由来のトランスポゾンであるpiggyBac等が挙げられる（Kaji, K. et al., Nature, 458: 771-775 (2009)、Woltjen et al., Nature, 458: 766-770 (2009) 、WO 2010/012077）。さらに、上記発現ベクターには、染色体への組み込みがなくとも複製されて、エピソーマルに存在するように、リンパ指向性ヘルペスウイルス（lymphotrophic herpes virus）、BKウイルスおよび牛乳頭腫（Bovine papillomavirus）の起点とその複製に係る配列を含んでいてもよい。例えば、EBNA-1およびoriPもしくはLarge TおよびSV40ori配列を含むことが挙げられる（WO 2009/115295、WO 2009/157201およびWO 2009/149233）。また、複数の核初期化物質を同時に導入するために、ポリシストロニックに発現させる発現ベクターを用いてもよい。ポリシストロニックに発現させるためには、遺伝子をコードする配列の間は、IRESまたはピコルナウイルス（口蹄病ウイルス（FMDV）、馬鼻炎Bウイルス（ERAV）、Thosea asigna ウイルス（TaV）等）由来2A配列により結合されていてもよい（Science, 322:949-953, 2008; BMC Biology, 6:40, 2008; WO 2009/092042およびWO 2009/152529）。 Examples of the promoter used include EF1α promoter, CAG promoter, SRα 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. Among them, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like can be mentioned.
Furthermore, if necessary, 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 , Β glucuronidase (GUS), reporter gene sequences such as FLAG, and the like. In addition, in the above expression vector, after introduction into a somatic cell, in order to excise together a gene encoding a nuclear reprogramming substance or a promoter and a gene encoding a nuclear reprogramming substance that binds to it, a LoxP sequence before and after them. You may have. In another preferred embodiment, there is a method for completely removing a transgene from a chromosome by incorporating a transgene into a chromosome using a transposon and then allowing a transferase to act on the cell using a plasmid vector or an adenovirus vector. Can be used. Preferred 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). In addition, 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. In order to express polycistronically, 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).
　核初期化物質をタンパク質の形態で導入する場合、例えばリポフェクション、細胞膜透過性ペプチドとの結合、マイクロインジェクションなどの手法によって体細胞内に導入してもよい。 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.
　核初期化に際して、iPS細胞の誘導効率を高めるために、上記の因子の他に、例えば、ヒストンデアセチラーゼ（HDAC）阻害剤[例えば、バルプロ酸(VPA)(Nat. Biotechnol., 26(7): 795-797 (2008))、トリコスタチンA、酪酸ナトリウム、MC 1293、M344等の低分子阻害剤、HDACに対するsiRNAおよびshRNA（例、HDAC1 siRNA SmartpoolTM (Millipore)、HuSH 29mer shRNA Constructs against HDAC1 (OriGene)等）等の核酸性発現阻害剤など］、DNAメチルトランスフェラーゼ阻害剤（例えば5’-azacytidine）（Nat. Biotechnol., 26(7): 795-797 (2008))、G9aヒストンメチルトランスフェラーゼ阻害剤［例えば、BIX-01294 (Cell Stem Cell, 2: 525-528 (2008))等の低分子阻害剤、G9aに対するsiRNAおよびshRNA（例、G9a siRNA(human) (Santa Cruz Biotechnology)等）等の核酸性発現阻害剤など］、L-channel calcium agonist (例えばBayk8644) (Cell Stem Cell, 3, 568-574 (2008))、p53阻害剤（例えばp53に対するsiRNAおよびshRNA）(Cell Stem Cell, 3, 475-479 (2008))、Wnt signaling activator（例えばsoluble Wnt3a）（Cell Stem Cell, 3, 132-135 (2008)）、LIFまたはbFGFなどのサイトカイン、ALK5阻害剤（例えば、SB431542）（Nat Methods, 6: 805-8 (2009)）、mitogen-activated protein kinase signalling阻害剤、glycogen synthase kinase-3阻害剤（PloS Biology, 6(10), 2237-2247 (2008))、miR-291-3p、miR-294、miR-295などのmiRNA (R.L. Judson et al., Nat. Biotech., 27:459-461 (2009))、等を使用することができる。
　iPS細胞誘導のための培養培地としては、例えば(1) 10～15% FBS又はノックアウト血清リプレースメント(KSR)を含有するDMEM、DMEM/F12又はDME培地（これらの培地にはさらに、LIF、penicillin/streptomycin、puromycin、L-グルタミン、非必須アミノ酸類、β-メルカプトエタノールなどを適宜含むことができる。）、(2) bFGF又はSCFを含有するES細胞培養用培地、例えばマウスES細胞培養用培地(例えばTX-WES培地、トロンボＸ社)又は霊長類ES細胞培養用培地（例えば霊長類（ヒト＆サル）ES細胞用培地、リプロセル、京都、日本）、などが含まれる。このとき、iPS細胞の誘導効率を高めるために、低タンパク質培地もしくは細胞周期停止剤含有培地を用いても良い（WO 2010/004989）。 In order to increase the induction efficiency of iPS cells upon nuclear reprogramming, in addition to the above factors, for example, histone deacetylase (HDAC) inhibitors [for example, valproic acid (VPA) (Nat. Biotechnol., 26 (7 ): 795-797 (2008)), small molecule inhibitors such as trichostatin A, sodium butyrate, MC 1293, M344, 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) (Nat. Biotechnol., 26 (7): 795-797 (2008)), 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. Biotech., 27: 459-461 (2009)), Etc. can be used.
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, β-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). At this time, in order to increase the induction efficiency of iPS cells, a low protein medium or a cell cycle arrester-containing medium may be used (WO 2010/004989).
　培養法の例としては、例えば、37℃、5% CO2存在下にて、10% FBS含有DMEM又はDMEM/F12培地上で体細胞と核初期化物質 (核酸又はタンパク質) を接触させ約4～7日間培養し、その後、細胞をフィーダー細胞 (例えば、マイトマイシンC処理マウス胎仔線維芽細胞(MEF)、STO細胞、SNL細胞等) 上にまきなおし、体細胞と核初期化物質の接触から約10日後からbFGF含有霊長類ES細胞培養用培地で培養し、該接触から約30～約45日又はそれ以上ののちにiPS様コロニーを生じさせることができる。また、iPS細胞の誘導効率を高めるために、5-10%と低い酸素濃度の条件下で培養してもよい（WO 2010/013845）。 Examples of 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).
　あるいは、その代替培養法として、フィーダー細胞 (例えば、マイトマイシンC処理MEF、STO細胞、SNL細胞等) 上で10% FBS含有DMEM培地（これにはさらに、LIF、ペニシリン/ストレプトマイシン、ピューロマイシン、L-グルタミン、非必須アミノ酸類、β-メルカプトエタノールなどを適宜含むことができる。）で培養し、約25～約30日又はそれ以上の後にES様コロニーを生じさせることができる。
　上記培養の間には、培養開始2日目以降から毎日1回新鮮な培地と培地交換を行う。また、核初期化に使用する体細胞の細胞数は、限定されないが、培養ディッシュ100cm2あたり約5×103～約5×106細胞の範囲である。 Alternatively, as an alternative culture method, DMEM medium containing 10% FBS on feeder cells (eg, mitomycin C-treated MEF, STO cells, SNL cells, etc.) (including LIF, penicillin / streptomycin, puromycin, L- Glutamine, non-essential amino acids, β-mercaptoethanol, etc. can be included as appropriate.) And an ES-like colony can be formed after about 25 to about 30 days or more.
During the culture, 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.
　マーカー遺伝子として薬剤耐性遺伝子を含む遺伝子を用いた場合は、対応する薬剤を含む培地（選択培地）で培養を行うことによりマーカー遺伝子発現細胞を選択することができる。またマーカー遺伝子が蛍光タンパク質遺伝子の場合は蛍光顕微鏡で観察することによって、発光酵素遺伝子の場合は発光基質を加えることによって、また発色酵素遺伝子の場合は発色基質を加えることによって、マーカー遺伝子発現細胞を検出することができる。 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. When 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.
　ES細胞の作製方法としては、例えば、哺乳動物の胚盤胞ステージにおける内部細胞塊を培養する方法（例えば、Manipulating the Mouse Embryo A Laboratory Manual，Second Edition，Cold Spring Harbor Laboratory Press (1994) を参照）、体細胞核移植によって作製された初期胚を培養する方法（Wilmut et al., Nature， 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); 入谷明ら, 蛋白質核酸酵素, 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. Natl. Acad. Sci. USA, 96, 14984 (1999); RideoutIII et al., Nature Genetics, 24, 109 (2000)）などが挙げられるが、これらに限定されない。また、ES細胞は、所定の機関より入手でき、さらには市販品を購入することもできる。例えば、ヒトES細胞であるKhES-1、KhES-2、KhES-3、KhES-4およびKhES-5は、京都大学再生医科学研究所より入手可能である。
　体細胞核移植による場合、体細胞の種類や体細胞を採取するソースは上記iPS細胞の場合に準ずる。
　EG細胞は、常法に従って始原生殖細胞を単離し、これをLIF、bFGFおよびSCFの存在下で培養することにより誘導することができる。また、mGS細胞はWO 2005/100548に記載される方法に従って、精巣細胞から作製することができる。多能性成体前駆細胞（MAPC）はJ. Clin. Invest. 109：337-346 (2002) に記載される方法に従って、骨髄から単離することができる。
　種々の組織幹細胞は、それぞれ自体公知の手法によって単離・培養することができる。 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. Natl. Acad. Sci. USA, 96, 14984 (1999); Rideout III et al., Nature Genetics, 24, 109 (2000)), and the like. In addition, ES cells can be obtained from a predetermined institution, and further commercially available products can be purchased. For example, human ES cells KhES-1, KhES-2, KhES-3, KhES-4 and KhES-5 are available from the Institute of Regenerative Medicine, Kyoto University.
In the case of 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. In addition, mGS cells can be prepared from testis cells according to the method described in WO 2005/100548. Pluripotent adult progenitor cells (MAPC) can be isolated from bone marrow according to the method described in J. Clin. Invest. 109: 337-346 (2002).
Various tissue stem cells can be isolated and cultured by a technique known per se.
（ｃ）幹細胞から各種体細胞への分化誘導
　幹細胞から各種体細胞への分化誘導は、自体公知の任意の方法により実施することができる。例えば、ヒトES細胞を放射線照射したC3H10T1/2細胞株と共培養して嚢状構造体（ES-sac）を誘導することにより造血前駆細胞に分化させることができる（Blood, 111: 5298-306, 2008）。ES細胞からの神経幹細胞・神経細胞の分化誘導法としては、胚様体形成法（Mech Div 59(1) 89-102, 1996）、レチノイン酸法（Dev Biol 168(2) 342-57, 1995）、SDIA法（Neuron 28(1) 31-40, 2000）、NSS法（Neurosci Res 46(2) 241-9, 2003）など様々な方法が知られている。ES細胞から心筋細胞への誘導方法としては、これまでにレチノイン酸、TGFβ1、FGF、dynorphin B、アスコルビン酸、一酸化窒素、FGF2とBMP2、Wnt11、PP2、Wnt3a/Wnt阻害剤などの因子を培地に添加する方法や、Nogginによる心筋分化誘導法（Nat Biotechnol 23(5) 611, 2005）などが報告されている。さらに、SDIA法およびSFEB法によるES/iPS細胞からの網膜細胞の分化誘導法（Nat Neurosci 8 288-96, 2005）なども知られているが、これらに限定されない。また、造血幹細胞から各種造血細胞への分化誘導法、血管内皮前駆細胞から血管細胞への分化誘導法、神経幹細胞からの各種神経細胞への分化誘導法、間葉系幹細胞からの脂肪細胞や筋管細胞などの分化誘導法など、組織幹細胞から体細胞への分化誘導法も当該技術分野において周知である。 (C) Differentiation induction from stem cells to various somatic cells Differentiation induction from stem cells to various somatic cells can be performed by any method known per se. For example, human 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). 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), and the NSS method (Neurosci Res 46 (2) 241-9, 2003) are known. As the method of induction from ES cells to cardiomyocytes, factors such as retinoic acid, TGFβ1, FGF, dynorphin B, ascorbic acid, nitric oxide, FGF2 and BMP2, Wnt11, PP2, Wnt3a / Wnt inhibitors have been used in the medium And a method for inducing myocardial differentiation by Noggin (Nat Biotechnol 23 (5) 611, 2005) have been reported. Furthermore, methods for inducing differentiation of retinal cells from ES / iPS cells by the SDIA method and SFEB method (Nat Neurosci 8 288-96, 2005) are known, but are not limited thereto. In addition, differentiation induction methods from hematopoietic stem cells to various hematopoietic cells, differentiation induction methods from vascular endothelial progenitor cells to vascular cells, differentiation induction methods from neural stem cells to various neural cells, adipocytes and muscles from mesenchymal stem cells Methods for inducing differentiation from tissue stem cells to somatic cells, such as methods for inducing differentiation of duct cells and the like, are also well known in the art.
（ｄ）分化細胞集団と本発明の未分化細胞／腫瘍化原因細胞殺傷剤との接触
　上記のようにして得られる幹細胞から分化誘導された細胞集団と、本発明の未分化細胞／腫瘍化原因細胞殺傷剤との接触（ex vivo法）は、通常の細胞へのベクター導入法に準じて行うことができる。
　一方、in vivo法における本発明の未分化細胞／腫瘍化原因細胞殺傷剤の投与量は、ベクターの種類、標的細胞におけるプロモーター活性、細胞毒性因子の種類、投与経路、病気の重篤度、投与対象となる動物種、投与対象の薬物受容性、体重、年齢等によって異なるが、例えば、ウイルスベクターとして未分化細胞または腫瘍化原因細胞特異的増殖型アデノウイルスを用いる場合、従来のがん遺伝子治療の臨床試験において、ウイルス粒子 (particle)で1x1010～1012 particle/腫瘍を用いて安全性が確認されているため、同量が投与の目安となる（Molecular Therapy, 18: 429-434, 2010）。一方、非ウイルスベクターをリポソームに被包して用いる場合には、体重約4 kgのカニクイザルを用いた臨床研究では666 μgのDNAを静脈投与して安全性が確認されているため同量が目安となる。例えば成人1回投与量は約2～約10 mgで、好ましくは約5～約8 mgである。 (D) Contact of differentiated cell population with undifferentiated cell / tumor causing cell killing agent of the present invention Cell population derived from stem cells obtained as described above and undifferentiated cell / tumorized cause of the present invention Contact with a cell killing agent (ex vivo method) can be carried out according to a normal method for introducing a vector into a cell.
On the other hand, 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 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. In clinical trials of this drug, 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 ). On the other hand, when using non-viral vectors encapsulated in liposomes, in clinical studies using cynomolgus monkeys weighing approximately 4 kg, 666 μg of DNA has been intravenously administered and safety has been confirmed. It becomes. For example, a single adult dose is about 2 to about 10 mg, preferably about 5 to about 8 mg.
　上記のようにして得られる、未分化細胞および／または腫瘍化原因細胞が殺傷除去された均一な分化細胞は、常套手段にしたがって医薬上許容される担体と混合するなどして、注射剤、懸濁剤、点滴剤等の非経口製剤として製造される。当該非経口製剤に含まれ得る医薬上許容される担体としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、D-ソルビトール、D-マンニトール、塩化ナトリウムなど）などの注射用の水性液を挙げることができる。本発明の移植療法剤は、例えば、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤、酸化防止剤などと配合しても良い。本発明の移植療法剤を水性懸濁液剤として製剤化する場合、上記水性液に約1×106～約1×108細胞/mLとなるように、分化細胞を懸濁させればよい。
　本発明の移植療法剤は、細胞の凍結保存に通常使用される条件で凍結保存された状態で提供され、用時融解して用いることもできる。その場合、血清もしくはその代替物、有機溶剤（例、DMSO）等をさらに含んでいてもよい。この場合、血清もしくはその代替物の濃度は、特に限定されるものではないが約1～約30% (v/v)、好ましくは約5～約20% (v/v) であり得る。有機溶剤の濃度は、特に限定されるものではないが0～約50% (v/v)、好ましくは約5～約20% (v/v) であり得る。 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. Manufactured as a parenteral preparation such as a suspension or infusion. Examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation 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. When the transplantation therapeutic agent of the present invention is formulated as an aqueous suspension, the differentiated cells may be suspended in the aqueous solution so as to be about 1 × 10 6 to about 1 × 10 8 cells / mL.
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. In that case, serum or an alternative thereof, an organic solvent (eg, DMSO) and the like may further be included. In this case, 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).
　本発明はまた、幹細胞から分化誘導された細胞集団に本発明の未分化細胞／腫瘍化原因細胞殺傷剤を接触させ、該細胞集団内に残存する未分化細胞および／または腫瘍化原因細胞の殺傷の程度を検定することによる、幹細胞からの分化誘導における腫瘍化リスクの評価方法を提供する。後述の実施例に示されるとおり、形態学的には分化した形態を呈する細胞であっても、RT-PCR解析によると未分化マーカーの発現を依然として認めるものが少なくない。本発明の未分化細胞／腫瘍化原因細胞殺傷剤は、そのような形態学的には分化細胞と判断されるが未分化状態をある程度保持している細胞をも殺傷し得るので、当該殺傷の程度を検定することにより、ある方法によって分化誘導された細胞集団が移植後に腫瘍化するリスクが高いか否かを、分化/未分化マーカーの発現解析を行うことなく評価することができる。 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.
　本発明はまた、幹細胞から分化誘導された細胞集団に本発明の未分化細胞／腫瘍化原因細胞殺傷剤を接触させることにより得られる、残存する未分化細胞および／または腫瘍化原因細胞が低減された分化細胞（目的細胞）を、当該目的細胞において病態を発現する疾患のメカニズムの解明や治療薬の開発などといった、基礎研究並びに創薬研究・開発のための各種細胞実験への利用に関する。
　かかる目的において、好ましくは目的細胞を誘導するための幹細胞もしくはその由来となる体細胞は、目的の疾患に罹患した患者由来の細胞であることが望ましい。例えば、患者由来の体細胞や細胞バンクの疾患細胞から疾患特異的ヒトiPS細胞を樹立する方法は、Nature, 461: 402-406, 2009 (家族性自律神経失調症); Science, 321: 1218-1221, 2008 (ALS); Cell, 134: 877-886, 2008 (ADA-SCID、Shwachman-Bodian-Diamond syndrome、ゴーシェ病III型、デュシェンヌ型筋ジストロフィー、ベッカー型筋ジストロフィー、パーキンソン病、ハンチントン病、I型糖尿病、ダウン症候群); Cell, 136: 964-977, 2009 (パーキンソン病); Nature, 457(7227): 277-280, 2009 (脊髄性筋萎縮症); Proc. Natl. Acad. Sci. USA, 106: 9826-9830, 2009 (サラセミア); Proc. Natl. Acad. Sci. USA, 106: 15768-15773, 2009 (I型糖尿病); Cell, 143(4): 527-539, 2010 (レット症候群); Nature. 465(7299): 808-812, 2010 (LEOPARD症候群) 等に記載されている。
　得られた疾患特異的ヒトiPS細胞から疾患標的組織あるいは細胞へ分化誘導することにより、その病態を初期から経時的に解析することができる。また、健常者から樹立した正常iPS細胞との間で、分化過程における遺伝子発現や細胞機能を比較検討することにより、疾患に関する臨床的知見の検証や、疾患に関与する分子の探索が可能となる。
　上記のような疾患のメカニズム解明等の研究に関し、本発明はまた、幹細胞から分化誘導された細胞集団に本発明の未分化細胞／腫瘍化原因細胞殺傷剤を接触させることにより得られる、残存する未分化細胞および／または腫瘍化原因細胞が低減された分化細胞を含有してなる生物学的研究用試薬を提供する。当該試薬は、上記移植療法剤と同様に、当該分化細胞に必要に応じて薬理学的上許容される各種添加剤を配合して製造することができる。
　さらに、ヒトiPS細胞から分化誘導し、本発明の未分化細胞／腫瘍化原因細胞殺傷剤を接触させて残存する未分化細胞および／または腫瘍化原因細胞を低減させた分化細胞に化合物ライブラリーを接触させ、目的の疾患に対する治療効果の指標となる細胞機能の変化を測定することにより、当該疾患に対して治療活性を有する化合物を医薬品候補化合物として選択することができる。例えば、ヒトiPS細胞から神経細胞を分化誘導し（例えばProc. Natl. Acad. Sci. USA, 105: 5856, 2008参照）、本発明の未分化細胞／腫瘍化原因細胞殺傷剤を用いて未分化細胞および／または腫瘍化原因細胞を殺傷除去した後、当該神経細胞に被検物質を接触させ、神経細胞死、神経突起伸長、電気生理などに関した機能アッセイを行うことにより、神経機能の改善効果を示した被検物質を神経変性疾患等の治療薬候補として選択することができる。さらに、選択的な分化培養法により得られた各種成熟神経（ドーパミン神経、アセチルコリン神経等）を用いて神経伝達物質に関したアッセイ系を構築することもできる。
　また、前記疾患特異的ヒトiPS細胞を利用した薬効評価系として、例えば、遺伝性の変異を有するALS患者やAD患者等から疾患特異的iPS細胞を樹立し、病態（例えば、Aβ産生異常、タウ蓄積異常、神経変性等）を反映する神経細胞へと分化させ、本発明の未分化細胞／腫瘍化原因細胞殺傷剤を用いて未分化細胞および／または腫瘍化原因細胞を殺傷除去した後、当該神経細胞に被検物質を接触させ、当該病態の改善の有無やその程度を指標として、孤発性のALSやADの治療薬の候補化合物を選択することもできる。 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.
For this purpose, 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. For example, methods for establishing disease-specific human iPS cells from patient-derived somatic cells or disease cells in a cell bank are described in Nature, 461: 402-406, 2009 (familial autonomic ataxia); Science, 321: 1218- 1221, 2008 (ALS); Cell, 134: 877-886, 2008 (ADA-SCID, Shwachman-Bodian-Diamond syndrome, Gaucher type III, Duchenne muscular dystrophy, Becker muscular dystrophy, Parkinson's disease, Huntington's disease, type I diabetes Down syndrome); Cell, 136: 964-977, 2009 (Parkinson's disease); Nature, 457 (7227): 277-280, 2009 (spinal muscular atrophy); Proc. Natl. Acad. Sci. USA, 106 : 9826-9830, 2009 (thalassemia); Proc. Natl. Acad. Sci. USA, 106: 15768-15773, 2009 (type I diabetes); Cell, 143 (4): 527-539, 2010 (Rett syndrome); Nature. 465 (7299): 808-812, 2010 (LEOPARD syndrome) and the like.
By inducing differentiation from the obtained disease-specific human iPS cells into disease target tissues or cells, the pathological condition can be analyzed from the initial stage over time. In addition, by comparing gene expression and cell function in the differentiation process with normal iPS cells established from healthy individuals, it becomes possible to verify clinical knowledge about the disease and search for molecules involved in the disease. .
Regarding the research for elucidating the mechanism of the disease as described above, 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.
Further, 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. For example, 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. Furthermore, an assay system relating to a neurotransmitter can be constructed using various mature nerves (dopamine nerve, acetylcholine nerve, etc.) obtained by selective differentiation culture.
In addition, as a drug efficacy evaluation system using the disease-specific human iPS cells, for example, disease-specific iPS cells are established from ALS patients and AD patients having inherited mutations, and pathological conditions (for example, abnormal Aβ 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.
　一方、薬物の毒性評価試験の中で創薬の比較的初期の段階から細胞評価系を用いた心毒性や肝毒性の予測が行われている。薬剤による重篤な副作用の１つにQT延長に伴う心室性不整脈があるが、QT延長の主なメカニズムとしてHERGチャネルの阻害が知られており、HERGチャネルを発現させた動物培養細胞を用いて電気生理学的に薬物の心毒性を評価する方法が広く活用されている（Biophys. J., 74: 230, 1998）。しかしながら、より生理的な条件を反映したiPS細胞由来の心筋細胞を用いれば、効率的でより精度の高い心毒性評価が可能となる。例えば、ヒトiPS細胞から心筋細胞を分化誘導し（例えばCirculation, 118: 498, 2008参照）、本発明の未分化細胞／腫瘍化原因細胞殺傷剤を用いて未分化細胞および／または腫瘍化原因細胞を殺傷除去した後、当該心筋細胞コロニーから拍動領域をピックアップして多電極測定機器の電極上に配置し、被検物質の存在下で細胞外電位測定を行い、QT間隔の延長の有無やその程度を検定することにより、QT延長作用を示した被検物質を心毒性を有する物質であるとして、医薬品候補から除外することができる。 On the other hand, 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. Using cultured animal cells expressing HERG channel 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. For example, 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.
　本発明を以下の実施例を用いてさらに詳細に説明するが、本発明の範囲はこれらの実施例によってなんら限定されるものではない。 The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited by these examples.
参考例１　ES/iPS細胞における癌細胞特異的遺伝子の発現
（１）FACSによる未分化細胞の分取
　Rock阻害剤（10 μM Y27632）で一晩処理したヒトES細胞（KhES1: 京都大学より譲渡）、ヒトiPS細胞（201B7、253G1: 以上、理研セルバンク）をトリプシン処理し単細胞へ分散した。細胞数のカウント後、必要量の細胞を遠心し(1,000rpm、5 min) 、10 μM Y27632を含むPBSで再懸濁した。Alexa Fluor（登録商標）488 anti-human SSEA4 (BioLegend) とAlexa Fluor（登録商標）647 anti-human TRA-1-81 (BioLegend) の二種類の未分化細胞標識抗体を用い細胞懸濁液100 μl (1×106cell) あたり5 μlの標識抗体を加え氷上で30分間静置して、細胞のラべリングを行った。10 μM Y27632を含むPBSで2回洗浄したのち、10 μM Y27632を含むES/iPS用培地に懸濁し、両抗体に標識されたiPS/ES細胞を未分化細胞として分取した。分取した細胞は直ちに遠心し(1,000rpm、 7 min)、新しい培地に再懸濁し、MEF細胞と共培養を行った。細胞分取には、BD FACSAriaTM IIを用いた。
（２）RT-PCR解析
　（１）で調製した細胞から以下の方法でtotal RNAを抽出した。細胞をPBSで洗浄、上清を除去した後Sepazol RNA Ｉ (nacalai tesque) を2 ml加え、ボルテックスミキサーで激しく撹拌し、5分間室温で静置した。クロロホルム200 μlを加えて転倒混和し、室温で3分間静置した。そして、4℃、12,000 gで15分間遠心し、水相(上相)を別のサンプルに移し、イソプロパノールを加えて混和し、室温で10分間静置した。再度、4℃、12,000 gで15分間遠心し、残った沈殿に75%エタノールを1-2 ml 加えて撹拌し、沈殿を十分懸濁させ、再度、4℃、12,000 gで15分間遠心し、上清を除き、得られた沈殿を5分間ほど自然乾燥した。DEPC水を加え、60℃で15分間加熱した後、4℃で静置した。次に、PrimerScriptTM II 1st strand cDNA synthesis kit (Takara)を用いて逆転写反応を行った。5 μgのtotal RNAをOligo dT primer(1 μl), dNTP mixture(1 μl), RNase free dH2O(up to 10 μl)に加え、これらの混合液を65℃で5分間静置した後、すぐに氷で冷却後、遠心し、5×PrimerScriptII Buffer(4 μl)、RNase Inhibitor(0.5 μl)、PrimerScriptIIRTase(1 μl)、RNase free dH2O(up to 20 μl)を加えて穏やかに撹拌した。42℃で30分静置し、95℃で5分間加熱した後氷上で冷却した。得られたcDNAを鋳型とし、EXTaq (Takara) を用いて、サバイビン、TERTそれぞれの遺伝子について発現の有無を確認した。GAPDHをコントロールとして用いた。その結果、サバイビン、TERTとも未分化細胞で発現していることが確認できた（図３）。尚、今回用いたプライマーのセットとそれぞれのPCR条件（アニーリング温度、時間、cycle数）については表１の通りである。 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 μM 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 μM Y27632. 100 μl of cell suspension using two types of undifferentiated cell labeled antibodies: Alexa Fluor (registered trademark) 488 anti-human SSEA4 (BioLegend) and Alexa Fluor (registered trademark) 647 anti-human TRA-1-81 (BioLegend) Labeling of cells was performed by adding 5 μl of labeled antibody per 1 × 10 6 cells and allowing to stand on ice for 30 minutes. After washing twice with PBS containing 10 μM Y27632, the suspension was suspended in an ES / iPS medium containing 10 μM Y27632, and iPS / ES cells labeled with both antibodies were collected as undifferentiated cells. The sorted cells were immediately centrifuged (1,000 rpm, 7 min), resuspended in fresh medium, and co-cultured with MEF cells. For cell sorting, BD FACSAria ™ II was used.
(2) RT-PCR analysis Total RNA was extracted from the cells prepared in (1) by the following method. The cells were washed with PBS, the supernatant was removed, 2 ml of Sepazol RNA I (nacalai tesque) was added, and the mixture was vigorously stirred with a vortex mixer and allowed to stand at room temperature for 5 minutes. 200 μl of chloroform was added and mixed by inversion, and left at room temperature for 3 minutes. Then, the mixture was centrifuged at 4 ° C. and 12,000 g for 15 minutes, 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. Next, reverse transcription reaction was performed using PrimerScript ™ II 1 st strand cDNA synthesis kit (Takara). Add 5 μg of total RNA to Oligo dT primer (1 μl), dNTP mixture (1 μl), RNase free dH 2 O (up to 10 μl), and leave these mixtures at 65 ° C for 5 minutes. Immediately after cooling with ice, centrifuge, add 5 × PrimerScriptII Buffer (4 μl), RNase Inhibitor (0.5 μl), PrimerScriptIIRTase (1 μl), RNase free dH 2 O (up to 20 μl) and mix gently. . The mixture was allowed to stand at 42 ° C for 30 minutes, heated at 95 ° C for 5 minutes, and then cooled on ice. Using the obtained cDNA as a template, the presence or absence of expression of each survivin gene and TERT gene was confirmed using EXTaq (Takara). GAPDH was used as a control. As a result, it was confirmed that both survivin and TERT were expressed in undifferentiated cells (FIG. 3). The primer sets used this time and the respective PCR conditions (annealing temperature, time, number of cycles) are as shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例１　プロモーターアッセイと未分化細胞特異的な外来遺伝子発現
　Rock阻害剤で一晩処理したヒトES細胞（KhES1）およびヒトiPS細胞（201B7、253G1）を6 well plateに3×105で播種した。翌日、1 wellあたりの細胞数を計測し、サバイビン、TERT、RSVおよびCMVプロモーターの制御下にあるlacZ遺伝子を搭載した非増殖型アデノウイルス（Ad.Survivn-LacZ、Ad.TERT-LacZ、Ad.RSV-LacZ、Ad.CMV-LacZ; 図２ａ）をMOI 10で感染させ、プロモーターアッセイを行った。
　感染2日後に細胞をPBSで洗浄後、各細胞に5倍希釈したReporter Lysis Buffer 5X (Promega) 250 μl/well加え、室温で15分間静置した。スクレーパーで細胞を1.5 mlチューブに回収し、10-15秒の間ボルテックスで撹拌した後、4℃、15,000 rpm、2分間遠心した。得られた上清を用いてβ-Galactosidase測定を行った。タンパク定量は、0.1% BSAをStandardとして用いた。得られた各細胞の上清5 μlを96 well plateに入れ、4 倍希釈したBio Rad Protein Assay 試薬 (Bio Rad Laboratories,Inc.)を100 μl加え、吸光度595 nmで測定した。段階希釈したBSAにより得られた検量線からタンパク量を算出した。β-Galactosidase測定では、Standardとして、10,000倍希釈したβ-Galactosidase (Promega)を段階希釈したものを用いた。各細胞の上清50 μlおよびStandard を96 well plateに入れ、Assay 2×Buffer(Promega)を50 μl加え、37℃、30分間インキュベートした。30分経過後直ちに、1 M sodium Carbonate(Promega) 150 μlを加えて反応を止め、420 nmでの吸光度を測定し、プロモーター活性を比較した。その結果、サバイビン、TERTプロモーターとも、未分化細胞において、ポジティブコントロールとして用いた恒常的プロモーター（RSVおよびCMVプロモーター）に匹敵するプロモーター活性を示した（図４）。
　これらの結果より、癌細胞特異的遺伝子のプロモーターを用いて未分化細胞特異的な外来遺伝子の発現が可能であることが明らかとなった。また、本発明者等の過去の詳細な研究により、例えばHSV-tk（Herpes simplex virus thymidine kinase）遺伝子などの細胞傷害性遺伝子は、RSVプロモーターの活性強度があれば必要十分以上の（RSVプロモーターの活性強度よりかなり低いプロモーター活性でも十分な）、細胞傷害効果を誘導できることが明らかとなっている（Hepatology. 2003 37(1):155-63.）。よって、上記の結果から、SurvivinやTERTなどの癌細胞特異的プロモーターにHSV-tk遺伝子などの細胞傷害性遺伝子を繋げれば、ES細胞やiPS細胞から分化誘導した細胞集団内に残存する未分化細胞を十分に殺傷できることは明らかである。 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.
Two days after infection, the cells were washed with PBS, Reporter Lysis Buffer 5X (Promega) 250 μl / well diluted 5-fold was added to each cell, and allowed to stand at room temperature for 15 minutes. Cells were collected in a 1.5 ml tube with a scraper, vortexed for 10-15 seconds, and then centrifuged at 15,000 rpm for 2 minutes at 4 ° C. Β-Galactosidase measurement was performed using the obtained supernatant. For protein quantification, 0.1% BSA was used as a standard. 5 μl of the obtained supernatant of each cell was placed in a 96-well plate, 100 μl of 4-fold diluted Bio Rad Protein Assay reagent (Bio Rad Laboratories, Inc.) was added, and the absorbance was measured at 595 nm. The amount of protein was calculated from a calibration curve obtained with serially diluted BSA. In β-Galactosidase measurement, a standard diluted β-Galactosidase (Promega) diluted 10,000 times was used as Standard. 50 μl of each cell supernatant and Standard were placed in a 96-well plate, 50 μl of Assay 2 × Buffer (Promega) was added, and the mixture was incubated at 37 ° C. for 30 minutes. Immediately after 30 minutes, 150 μl of 1 M sodium Carbonate (Promega) was added to stop the reaction, the absorbance at 420 nm was measured, and the promoter activity was compared. As a result, both survivin and TERT promoters showed promoter activity comparable to constitutive promoters (RSV and CMV promoters) used as positive controls in undifferentiated cells (FIG. 4).
From these results, it became clear that the expression of foreign genes specific to undifferentiated cells is possible using a promoter for cancer cell-specific genes. In addition, according to detailed studies by the present inventors, for example, 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.
実施例２　in vitroにおけるm-CRAの未分化細胞殺傷効果
　Rock阻害剤で一晩処理したヒトES細胞（KhES1）およびヒトiPS細胞（201B7、253G1）を96 well plateに1 wellあたり4～6x104で播種した。翌日、1 wellあたりの細胞数を計測後、二種類のm-CRA（TERT.m-CRA、Survivin.m-CRA; 図２ｂ）または非増殖型アデノウイルスベクター(Ad.CA-EGFP; 図２ａ)を、それぞれMOI 3、またはMOI 10で感染させ、ウイルス感染1日後、2日後、3日後、4日後、7日後にWST-8により非感染細胞群に対するm-CRA感染細胞の生細胞の割合を計測した。
　感染1日後では細胞へのウイルス導入はみられるものの、非増殖型ウイルス、m-CRAともに細胞傷害効果はみられなかった。感染2日後以降になると、非増殖型ウイルスと比較してm-CRAでは細胞傷害効果が顕著に見られ、m-CRA感染細胞では、生細胞数が減少していることが確認でき、有意な細胞傷害効果が確認できた（図５－１～５－３）。また、Survivin.m-CRAとTERT.m-CRAを比較すると、Survivin.m-CRAの方がより強い細胞傷害効果を示した。この結果は、サバイビンプロモーターの方がTERTプロモーターより活性が高いという参考例２のプロモーターアッセイ結果を反映していた。hES細胞（図５－３）とhiPS細胞（図５－１、５－２）との細胞傷害効果を比較すると、hiPS細胞の方がm-CRAによる細胞傷害効果が強く現れた。 Example 2 In vitro m-CRA killing effect of undifferentiated cells In vitro, human ES cells (KhES1) and 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. 2a) ) With MOI 3 or MOI 10 respectively, and the ratio of viable cells of m-CRA-infected cells to non-infected cell groups by WST-8 after 1, 2, 3, 4, and 7 days after virus infection Was measured.
One day after the infection, although the virus was introduced into the cells, neither the non-replicating virus nor m-CRA showed any cytotoxic effect. After 2 days after infection, m-CRA showed a significant cytotoxic effect compared to non-proliferative viruses, and m-CRA-infected cells confirmed that the number of viable cells had decreased. The cytotoxic effect was confirmed (FIGS. 5-1 to 5-3). Moreover, when comparing Survivin.m-CRA and TERT.m-CRA, 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.
実施例３　サバイビンまたはTERTで増殖制御されるm-CRAを用いた未分化細胞の除去
　ヒト多能性幹細胞の維持培養プロトコル（理化学研究所）に従い、matrigel上に、Rock阻害剤で一晩処理したKhES1、201B7株、253G1株を分散培養し、DMEM (10% FCS) で培養することにより自発的な分化を促した。1週間後にサバイビンまたはTERTで増殖制御されるm-CRA（TERT.m-CRA、Survivin.m-CRA; 図２ｂ）をMOI 10で1時間感染させ、feeder細胞であるマイトマイシンC処理したMEF上に播きなおし、ES/iPS細胞用培地で培養することにより残存未分化細胞が増殖できる環境下に置いた。さらに約2週間後に培地を除去後、アルカリフォスファターゼ染色を行い、未分化細胞を選択的に染色した後、10%ホルマリンで固定し、肉眼下で未分化細胞のコロニー数を計測した。その結果を図６に示す。
　全ての細胞種において、ウイルス非感染群（No virus）には残存未分化細胞由来のコロニーが確認できたが、KhES1では平均35.5個（図６ａ）と少ないのに対しiPS細胞群では375(201B7; 図６ｂ)、195(253G1; 図６ｃ)とその数に大きな差が見られた。Ad.dE1.3感染群においても残存未分化細胞のコロニーの出現が認められたが、その数はネガティブコントロール（No virus）の群と比較して有意に少ないものであった。一方、TERT.m-CRAおよびSurvivin.m-CRA感染群ではどの細胞種においてもAd.dE1.3感染群と比較して、有意に分化抵抗性細胞のコロニー出現数が少なく、ES細胞においてはTERT.m-CRA感染群には分化抵抗性細胞のコロニーの出現は確認できなかった。このことから、TERT.m-CRAおよびSurvivin.m-CRAはいずれも未分化細胞で増殖し、当該細胞を殺傷し得ることが明らかとなった。 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). One week later, m-CRA (TERT.m-CRA, Survivin.m-CRA; Fig. 2b), whose growth is controlled by survivin or TERT, was infected with MOI 10 for 1 hour, and on mitomycin C-treated MEF feeder cells. After re-seeding and culturing in a medium for ES / iPS cells, the cells were placed in an environment where the remaining undifferentiated cells could grow. Further, after about 2 weeks, the medium was removed, and alkaline phosphatase staining was performed. Undifferentiated cells were selectively stained, then fixed with 10% formalin, and the number of colonies of undifferentiated cells was counted under the naked eye. The result is shown in FIG.
In all cell types, colonies derived from residual undifferentiated cells were confirmed in the virus-uninfected group (No virus), but the average of KhES1 was as small as 35.5 cells (Fig. 6a), whereas the iPS cell group was 375 (201B7). FIG. 6b), 195 (253G1; FIG. 6c) and a large difference were observed. In the Ad.dE1.3 infected group, colonies of remaining undifferentiated cells were observed, but the number was significantly smaller than that in the negative control (No virus) group. On the other hand, in the TERT.m-CRA and Survivin.m-CRA infected groups, the number of colonies of differentiation-resistant cells was significantly smaller in all cell types compared to the Ad.dE1.3 infected group, and in ES cells The appearance of colonies of differentiation-resistant cells in the TERT.m-CRA-infected group was not confirmed. From this, it became clear that both TERT.m-CRA and Survivin.m-CRA can proliferate in undifferentiated cells and kill the cells.
実施例４　自発的分化誘導後のES/iPS細胞におけるm-CRAの影響
　実施例２では出現コロニー数のみで判断しているため、m-CRAが本当に分化抵抗性細胞のみを除去したかが不明である。そこで、m-CRAが分化抵抗性細胞のみに有効かどうかを確認するために分化後の細胞の遺伝子発現を確認し、さらにm-CRA感染実験を行った。7日間分化誘導を行ったiPS細胞（201B7株）およびES細胞（KhES-1株）、15日間分化誘導を行った201B7株およびKhES-1株、並びに20日間分化誘導を行ったKhES-1株の5種類の細胞よりtotal RNAを抽出し、cDNAへ逆転写し、Oct3/4およびNanog（未分化マーカー）、Nestin（外胚葉マーカー）、Brachyury（中胚葉マーカー）、並びにGATA4（内胚葉マーカー）についてRT-PCRを行った。また、内因性のコントロールとしてGAPDHを用いた。
　これらの細胞は、顕微鏡下の確認では未分化の状態の細胞とは異なり、dishに張り付いて広がっており、分化した形態を示していた。また、RT-PCRにおいて、ほぼ全ての細胞種で分化マーカーの発現が確認できた。しかし、全ての細胞種において未分化マーカーであるOct3/4、Nanogの発現も認められた(図７)。さらに、TERT、サバイビンの発現を確認した結果、TERTは発現の弱いものもあったが、サバイビンは全ての細胞種で発現が残っていた(図７)。
　未分化マーカーやTERT、サバイビンは、未分化細胞では発現が認められるが、分化するとその発現は消失する。従って、これらの遺伝子の発現が検出できたこと、各分化マーカーの遺伝子の発現も検出できたことから、7～21日間自発的に分化誘導を行ったES/iPS分化細胞群の中には、未分化細胞が残存することが考えられる。25日間分化誘導を行ったKhES1にTERT.m-CRA、Survivin.m-CRA、及びAd.CApr-EGFPを感染させたところ、細胞は分化形態を示していたにもかかわらず、感染2日後にはm-CRAを感染させた細胞群でプラークが出現し始め、3日後にはほぼすべての細胞が死滅していた（図８）。これは、bFGF非存在下での自発的な分化では、形態上は分化しているように見えるものの、内因性のTERT、サバイビンの遺伝子が残っており、m-CRAによる細胞傷害効果が見られたものと考えられる。 Example 4 Effect of m-CRA on ES / iPS cells after induction of spontaneous differentiation In Example 2, it was determined only by the number of appearance colonies, so it was unknown whether m-CRA really removed only differentiation-resistant cells. It is. Therefore, in order to confirm whether m-CRA is effective only against differentiation-resistant cells, the gene expression of differentiated cells was confirmed, and further m-CRA infection experiments were performed. IPS cells (201B7 strain) and ES cells (KhES-1 strain) induced to differentiate for 7 days, 201B7 and KhES-1 strains induced to differentiate for 15 days, and 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. However, expression of Oct3 / 4 and Nanog, which are undifferentiated markers, was also observed in all cell types (FIG. 7). Furthermore, as a result of confirming the expression of TERT and survivin, TERT was weakly expressed, but survivin remained in all cell types (FIG. 7).
Undifferentiated markers, TERT, and survivin are expressed in undifferentiated cells, but their expression disappears upon differentiation. Therefore, since the expression of these genes could be detected and the expression of genes for each differentiation marker could also be detected, among the ES / iPS differentiated cell groups that spontaneously induced differentiation for 7 to 21 days, It is considered that undifferentiated cells remain. KhES1, which was induced to differentiate for 25 days, was infected with TERT.m-CRA, Survivin.m-CRA, and Ad.CApr-EGFP. In the group of cells infected with m-CRA, plaques started to appear, and almost all cells died after 3 days (FIG. 8). This is because spontaneous differentiation in the absence of bFGF appears to be differentiated in morphology, but endogenous TERT and survivin genes remain, and the cytotoxic effect of m-CRA is observed. It is thought that.
実施例５　胚様体（EB）形成後の分化状態
　実施例４の結果から、多能性幹細胞をbFGF非存在下で培養するという自発的な分化誘導では分化が十分ではなく、m-CRAの効果を検討するには不十分であった。そこで、より自然な分化方法に近いEB形成による分化誘導を行い、m-CRAの効果について検討した。
　ROCK阻害剤で処理した各多能性幹細胞を、低接着の96 well plate上に1x104/wellずつ培養し、胚様体（EB）を形成させた。分化後7日目（Day7）にゼラチンコートした24 well plate上に1 wellあたり1つのEBを播種し、さらに分化を進めた。分化後15日目（Day15）にTERT.m-CRA、Survivin.m-CRA、Ad.CA-EGFPをMOI 10で1時間感染させ、感染後の細胞の様子を2週間観察した。Day0、7、15、22にtotal RNAを回収し、RT-PCRにより遺伝子の発現状況を確認した。
　DNAの増幅にはHuman Pluripotent Stem Cell Assessment Primer Pair Panelにある以下のプライマーを用いた。外胚葉系のマーカーとしてTP63、内胚葉系のマーカーとしてGATA4、内因性コントロールとしてGAPDH。また、キットに含まれないTERT、サバイビン、未分化細胞のマーカーであるNanog、Oct4については以下のプライマーを使用した。
TERT；S-TERT 5’-GCCTTCAAGAGCCACGTC-3’（配列番号１２）
　　　AS-TERT 5’-AGGTGAGCCACGAACTGTC-3’（配列番号１３）
Survivin；S-Surv 5’-CCAGTGTTTCTTCTGCTTCAA-3’（配列番号１４） 
　　　　　AS-Surv 5’-GAATGCTTTTTATGTTCCTCTATG-3’（配列番号１５）
Nanog;S-Nanog 5’-AGATGCCTCACACGGAGACT-3’（配列番号１６）
　　　AS-Nanog 5’-TTTGCGACACTCTTCTCTGC-3’（配列番号１７）
Oct4;S-Oct4 5’-GCTAGAGCAAAACCCGGAGA-3’（配列番号１８）
　　 AS-Oct4 5’-CCACATCGGCCTGTGTATATC-3’（配列番号１９）
　また、PCRはアニーリング55℃、10秒、サイクル数は35回で行った。 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. 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.
For amplification of DNA, the following primers in the Human Pluripotent Stem Cell Assessment Primer Pair Panel were used. 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.
TERT; S-TERT 5'-GCCTTCAAGAGCCACGTC-3 '(SEQ ID NO: 12)
AS-TERT 5'-AGGTGAGCCACGAACTGTC-3 '(SEQ ID NO: 13)
Survivin; S-Surv 5'-CCAGTGTTTCTTCTGCTTCAA-3 '(SEQ ID NO: 14)
AS-Surv 5'-GAATGCTTTTTATGTTCCTCTATG-3 '(SEQ ID NO: 15)
Nanog; S-Nanog 5'-AGATGCCTCACACGGAGACT-3 '(SEQ ID NO: 16)
AS-Nanog 5'-TTTGCGACACTCTTCTCTGC-3 '(SEQ ID NO: 17)
Oct4; S-Oct4 5'-GCTAGAGCAAAACCCGGAGA-3 '(SEQ ID NO: 18)
AS-Oct4 5'-CCACATCGGCCTGTGTATATC-3 '(SEQ ID NO: 19)
PCR was performed at an annealing temperature of 55 ° C. for 10 seconds and a cycle number of 35 times.
　ウイルス感染の翌日（day16）よりKhES1由来の分化細胞では次々と拍動する細胞を確認した。また、253G1株由来の分化細胞ではday22より拍動する細胞が確認できるようになった。これらの拍動する細胞はウイルス感染の有無にかかわらず観察されたが、観察を続けたday31までに201B7株では拍動する細胞は見受けられなかった。分化細胞にはEGFPの発現が認められたことからm-CRAの感染は認められたが、EGFP陽性細胞数がほとんど増えていないことから、m-CRAは増殖していないことが分かった（図９－１～９－３）。さらにウイルス感染2-5日後（day17-20）には一部のEGFP陽性細胞、特にEB由来コロニーの周囲の細胞の死滅が観察されたが、コロニー中央付近の分化細胞は死滅していなかった。これらのことから、EB由来コロニーの周囲の細胞は分化が不十分であるが、m-CRAは分化が進んだ細胞には影響を与えないと考えられた。
　RT-PCRを行った結果、KhES1においてはTERT.m-CRA感染群でポジティブコントロールに比べNanogやOct4の発現が低下していることが確認され、TERT.m-CRAにより残存未分化細胞が除去されていることが示唆された(図１０)。 From the day after virus infection (day 16), KhES1-derived differentiated cells were confirmed to be beating one after another. In addition, 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). Furthermore, 2-5 days after virus infection (day 17-20), some EGFP positive cells, particularly cells around the EB-derived colonies, were observed to die, but differentiated cells near the center of the colonies were not killed. From these results, it was considered that cells around the EB-derived colonies were insufficiently differentiated, but m-CRA had no effect on the cells that had undergone differentiation.
As a result of RT-PCR, it was confirmed that the expression of Nanog and Oct4 was decreased in KhES1 in the TERT.m-CRA-infected group compared to the positive control, and residual undifferentiated cells were removed by TERT.m-CRA. (Fig. 10).
　本発明の未分化細胞／腫瘍化原因細胞殺傷剤は、ES細胞やiPS細胞などの多能性幹細胞から分化誘導された細胞集団の中に残存する未分化細胞および／または腫瘍化原因細胞を選択的に殺傷除去することができるので、当該薬剤を用いて腫瘍化リスクの低減された安全な移植細胞・薬効・毒性評価系としての信頼性の高い均一な分化細胞の提供が可能となり、ひいては多能性幹細胞を用いた細胞移植治療、並びに効率的かつ高精度な医薬品候補化合物のスクリーニングの実現のために極めて有用である。 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.
　本出願は、日本で出願された特願2011-073690（出願日：2011年3月29日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2011-073690 (filing date: March 29, 2011) filed in Japan, the contents of which are incorporated in full herein.

Claims (30)

  1. 　少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする遺伝子のプロモーターが癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターで置換されているウイルスベクターを含有してなる、未分化細胞および／または腫瘍化の原因となる細胞の殺傷剤。 An undifferentiated cell comprising a viral vector in which a promoter of a gene encoding a factor essential for at least one virus replication or assembly is replaced with a promoter of a gene specifically expressed in cancer cells or undifferentiated cells And / or cell killers that cause tumorigenesis.
  2. 　幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を選択的に殺傷することを特徴とする、請求項１記載の剤。 2. The agent according to claim 1, which selectively kills undifferentiated cells remaining in the cell population induced to differentiate from stem cells and / or cells causing tumorigenesis.
  3. 　幹細胞が多能性幹細胞である、請求項２記載の剤。 The agent according to claim 2, wherein the stem cells are pluripotent stem cells.
  4. 　多能性幹細胞がES細胞またはiPS細胞である、請求項３記載の剤。 The agent according to claim 3, wherein the pluripotent stem cells are ES cells or iPS cells.
  5. 　癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターがテロメラーゼ逆転写酵素（TERT）、サバイビン、Auroraキナーゼ、がん胎児性抗原（CEA）、低酸素応答性領域（HRE）、Grp78、L-プラスチン、ヘキソキナーゼII、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3B、miR-290クラスターまたはmiR-302クラスターのプロモーターである、請求項１～４のいずれか１項に記載の剤。 The promoters of genes specifically expressed in cancer cells or undifferentiated cells are 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 The agent according to any one of claims 1 to 4, which is a promoter of a cluster.
  6. 　更に、少なくとも１つの他のウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターで置換されていることを特徴とする、請求項１～５のいずれか１項に記載の剤。 Furthermore, the promoter of a nucleic acid encoding a factor essential for replication or assembly of at least one other virus is replaced with a promoter that can be constitutively expressed in mammals or a promoter of a factor that is specifically expressed in differentiated cells. The agent according to any one of claims 1 to 5, which is characterized in that
  7. 　少なくとも１つのウイルスの複製またはアッセンブリに必須の因子が、Ｅ１Ａ、Ｅ１ＡΔ２４、Ｅ１Ｂ、およびＥ１ＢΔ５５Ｋから選択される因子である、請求項１～６のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 6, wherein the factor essential for at least one virus replication or assembly is a factor selected from E1A, E1AΔ24, E1B, and E1BΔ55K.
  8. 　更に、癌細胞もしくは未分化細胞で特異的に発現する遺伝子のプロモーター、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターの制御下にある、細胞毒性因子をコードする核酸を含む発現カセットを含有する、請求項１～７のいずれか１項に記載の剤。 Furthermore, cytotoxicity under the control of promoters of genes that are specifically expressed in cancer cells or undifferentiated cells, promoters that can be constitutively expressed in mammals, or promoters of factors that are specifically expressed in differentiated cells The agent according to any one of claims 1 to 7, comprising an expression cassette comprising a nucleic acid encoding the factor.
  9. 　ウイルスベクターが細胞溶解性ウイルスベクターである、請求項１～８のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 8, wherein the viral vector is a cytolytic viral vector.
  10. 　細胞溶解性ウイルスベクターがアデノウイルスベクターである、請求項９に記載の剤。 The agent according to claim 9, wherein the cytolytic virus vector is an adenovirus vector.
  11. 　癌細胞または未分化細胞で特異的に発現する遺伝子のプロモーターの制御下にある細胞毒性因子をコードする核酸を含む発現ベクターを含有してなる、未分化細胞および／または腫瘍化の原因となる細胞の殺傷剤。 An undifferentiated cell and / or a cell that causes tumorigenesis, comprising an expression vector containing a nucleic acid encoding a cytotoxic factor under the control of a promoter of a gene specifically expressed in cancer cells or undifferentiated cells Killing agent.
  12. 　幹細胞から分化誘導された細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を選択的に殺傷することを特徴とする、請求項１１記載の剤。 The agent according to claim 11, which selectively kills undifferentiated cells remaining in a cell population induced to differentiate from stem cells and / or cells causing tumorigenesis.
  13. 　幹細胞が多能性幹細胞である、請求項１２記載の剤。 The agent according to claim 12, wherein the stem cell is a pluripotent stem cell.
  14. 　多能性幹細胞がES細胞またはiPS細胞である、請求項１３記載の剤。 The agent according to claim 13, wherein the pluripotent stem cells are ES cells or iPS cells.
  15. 　ベクターがウイルスベクターである、請求項１１～１４のいずれか１項に記載の剤。 The agent according to any one of claims 11 to 14, wherein the vector is a viral vector.
  16. 　ウイルスベクターがアデノウイルスベクターである、請求項１５記載の剤。 The agent according to claim 15, wherein the viral vector is an adenoviral vector.
  17. 　プロモーターがテロメラーゼ逆転写酵素（TERT）、サバイビン、Auroraキナーゼ、がん胎児性抗原（CEA）、低酸素応答性領域（HRE）、Grp78、L-プラスチン、ヘキソキナーゼII、Oct3/4、Nanog、Sox2、Cripto、Dax1、ERas、Fgf4、Esg1、Rex1、Zfp296、UTF1、GDF3、Sall4、Tbx3、Tcf3、DNMT3L、DNMT3B、miR-290クラスターまたはmiR-302クラスターのプロモーターである、請求項１１～１６のいずれか１項に記載の剤。 Promoters are telomerase reverse transcriptase (TERT), survivin, Aurora kinase, carcinoembryonic antigen (CEA), hypoxia responsive region (HRE), Grp78, L-plastin, hexokinase II, Oct3 / 4, Nanog, Sox2, The promoter of Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, miR-290 cluster or miR-302 cluster Item 1. The agent according to item 1.
  18. 　更に、少なくとも１つのウイルスの複製またはアッセンブリに必須の因子をコードする核酸のプロモーターが癌細胞もしくは未分化細胞で特異的に発現する遺伝子のプロモーター、哺乳類において恒常的に発現し得るプロモーターまたは分化細胞特異的に発現が亢進している因子のプロモーターで置換されていることを特徴とする、請求項１５または１６記載の剤。 Furthermore, a promoter of a gene in which a nucleic acid encoding a factor essential for at least one virus replication or assembly is specifically expressed in cancer cells or undifferentiated cells, a promoter capable of being constitutively expressed in mammals, or differentiated cell-specific The agent according to claim 15 or 16, wherein the agent is replaced with a promoter of a factor whose expression is enhanced.
  19. 　少なくとも１つのウイルスの複製またはアッセンブリに必須の因子が、Ｅ１Ａ、Ｅ１ＡΔ２４、Ｅ１Ｂ、またはＥ１ＢΔ５５Ｋである、請求項１８記載の剤。 The agent according to claim 18, wherein the factor essential for at least one virus replication or assembly is E1A, E1AΔ24, E1B, or E1BΔ55K.
  20. 　幹細胞から分化誘導された細胞集団に請求項１～１９のいずれか１項に記載の剤を接触させることにより、該細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞を殺傷することを特徴とする、腫瘍化リスクの低減された分化細胞の製造方法。 The undifferentiated cells remaining in the cell population and / or cells causing tumorigenesis are brought into contact with the cell population induced to differentiate from stem cells by the agent according to any one of claims 1 to 19. A method for producing differentiated cells with reduced risk of tumorigenesis, characterized by killing.
  21. 　幹細胞が多能性幹細胞である、請求項２０記載の方法。 21. The method according to claim 20, wherein the stem cell is a pluripotent stem cell.
  22. 　多能性幹細胞がES細胞またはiPS細胞である、請求項２１記載の方法。 The method according to claim 21, wherein the pluripotent stem cells are ES cells or iPS cells.
  23. 　請求項２０～２２のいずれか１項に記載の方法により得られる有効量の分化細胞を、該分化細胞の移植を必要とする哺乳動物に移植すること、あるいは、幹細胞から誘導された分化細胞集団を移植されたか、もしくは移植される哺乳動物に、有効量の請求項１～１９のいずれか１項に記載の剤を投与することを特徴とする、腫瘍化リスクが低減された細胞移植療法。 23. Transplanting an effective amount of differentiated cells obtained by the method according to any one of claims 20 to 22 into a mammal in need of transplantation of the differentiated cells, or a differentiated cell population derived from stem cells A cell transplantation therapy with reduced risk of tumor formation, comprising administering an effective amount of the agent according to any one of claims 1 to 19 to a mammal to be transplanted or a mammal to be transplanted.
  24. 　請求項２０～２２のいずれか１項に記載の方法により得られる分化細胞を含有してなる、細胞移植療法剤。 A cell transplantation therapeutic agent comprising a differentiated cell obtained by the method according to any one of claims 20 to 22.
  25. 　幹細胞から誘導された分化細胞集団と組み合わせて投与される、細胞移植療法のための請求項１～１９のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 19, for cell transplantation therapy, administered in combination with a differentiated cell population derived from stem cells.
  26. 　幹細胞から分化誘導された細胞集団に請求項１～１９のいずれか１項に記載の剤を接触させ、該細胞集団内に残存する未分化細胞および／または腫瘍化の原因となる細胞の殺傷の程度を検定することを特徴とする、幹細胞からの分化誘導における腫瘍化リスクの評価方法。 The agent according to any one of claims 1 to 19 is brought into contact with a cell population induced to differentiate from a stem cell, and killing of undifferentiated cells remaining in the cell population and / or cells causing tumorigenesis. A method for evaluating a risk of tumorigenesis in inducing differentiation from a stem cell, characterized by testing the degree.
  27. 　請求項２０～２２のいずれか１項に記載の方法により得られる分化細胞と、被検物質とを接触させる工程、および該細胞における目的の薬効または毒性の発現を検定する工程を含む、被検物質の薬効または毒性試験方法。 A test comprising the steps of bringing a differentiated cell obtained by the method according to any one of claims 20 to 22 into contact with a test substance, and assaying the expression of a target drug effect or toxicity in the cell. Method for testing the efficacy or toxicity of a substance.
  28. 　分化細胞が患者由来の多能性幹細胞から誘導されたものである、請求項２７記載の方法。 28. The method according to claim 27, wherein the differentiated cells are derived from patient-derived pluripotent stem cells.
  29. 　請求項２０～２２のいずれか１項に記載の方法により得られる分化細胞を含有してなる、生物学的研究試薬。 A biological research reagent comprising a differentiated cell obtained by the method according to any one of claims 20 to 22.
  30. 　分化細胞が患者由来の多能性幹細胞から誘導されたものである、請求項２９記載の試薬。 30. The reagent according to claim 29, wherein the differentiated cell is derived from a pluripotent stem cell derived from a patient.
PCT/JP2012/058414 2011-03-29 2012-03-29 Method for removing tumorigenesis-causing cells among stem cells WO2012133674A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2013507734A JP6037393B2 (en) 2011-03-29 2012-03-29 Methods for removing tumorigenic cells in stem cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-073690 2011-03-29
JP2011073690 2011-03-29

Publications (1)

Publication Number Publication Date
WO2012133674A1 true WO2012133674A1 (en) 2012-10-04

Family

ID=46931376

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/058414 WO2012133674A1 (en) 2011-03-29 2012-03-29 Method for removing tumorigenesis-causing cells among stem cells

Country Status (2)

Country Link
JP (2) JP6037393B2 (en)
WO (1) WO2012133674A1 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014157257A1 (en) * 2013-03-25 2014-10-02 公益財団法人先端医療振興財団 Cell sorting method
WO2014200117A1 (en) * 2013-06-13 2014-12-18 Sucampo Ag Method for suppressing tumorigenicity of stem cells
WO2015107888A1 (en) 2014-01-14 2015-07-23 国立大学法人鹿児島大学 Novel method for labelling cancerization-causing cell in stem cells, and therapy method
CN108342366A (en) * 2017-01-25 2018-07-31 上海元宋生物技术有限公司 Recombination oncolytic gene-the adenovirus and its construction method of target on cancer and application
KR101909905B1 (en) * 2016-10-21 2018-10-19 한양대학교 산학협력단 Gene Expression System for Targeting Cancer Stem Cell and Cancer Cell
JP2018177648A (en) * 2017-04-04 2018-11-15 利章 大原 Agents and methods for inhibiting tumorigenesis of pluripotent stem cell
WO2018225705A1 (en) 2017-06-05 2018-12-13 テルモ株式会社 Method for producing cell culture
WO2020067438A1 (en) 2018-09-27 2020-04-02 国立大学法人大阪大学 Sheeting method for pluripotent stem cell-derived cells
WO2023106306A1 (en) * 2021-12-08 2023-06-15 株式会社八神製作所 Method for isolating stem cells using in-vitro boron-neutron reaction

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004523217A (en) * 2000-11-27 2004-08-05 ジェロン・コーポレーション Differentiated cells suitable for human therapy
WO2005115476A1 (en) * 2004-05-25 2005-12-08 Univ Kurume Drug comprising as the active ingredient proliferative vector containing survivin promoter

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004523217A (en) * 2000-11-27 2004-08-05 ジェロン・コーポレーション Differentiated cells suitable for human therapy
WO2005115476A1 (en) * 2004-05-25 2005-12-08 Univ Kurume Drug comprising as the active ingredient proliferative vector containing survivin promoter

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLUM B ET AL.: "The tumorigenicity of diploid and aneuploid human pluripotent stem cells", CELL CYCLE, vol. 8, no. 23, December 2009 (2009-12-01), pages 3822 - 3830, XP009142200 *
NAUJOK 0 ET AL.: "Selective Removal of Undifferentiated Embryonic Stem Cells from Differentiation Cultures Through HSV1 Thymidine Kinase and Ganciclovir Treatment", STEM CELL REVIEWS AND REPORTS, vol. 6, no. 3, September 2010 (2010-09-01), pages 450 - 461 *
SHORT JJ ET AL.: "Oncolytic adenoviruses targeted to cancer stem cells", MOLECULAR CANCER THERAPEUTICS, vol. 8, no. 8, August 2009 (2009-08-01), pages 2096 - 2102 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10072242B2 (en) 2013-03-25 2018-09-11 Foundation For Biomedical Research And Innovation At Kobe Cell sorting method
WO2014157257A1 (en) * 2013-03-25 2014-10-02 公益財団法人先端医療振興財団 Cell sorting method
JPWO2014157257A1 (en) * 2013-03-25 2017-02-16 公益財団法人先端医療振興財団 Cell sorting method
WO2014200117A1 (en) * 2013-06-13 2014-12-18 Sucampo Ag Method for suppressing tumorigenicity of stem cells
JPWO2015107888A1 (en) * 2014-01-14 2017-03-23 国立大学法人 鹿児島大学 New labeling and treatment of tumorigenic cells in stem cells
WO2015107888A1 (en) 2014-01-14 2015-07-23 国立大学法人鹿児島大学 Novel method for labelling cancerization-causing cell in stem cells, and therapy method
EP3690055A1 (en) 2014-01-14 2020-08-05 Kagoshima University Method for labelling cancerization-causing cell in stem cells
KR101909905B1 (en) * 2016-10-21 2018-10-19 한양대학교 산학협력단 Gene Expression System for Targeting Cancer Stem Cell and Cancer Cell
CN108342366A (en) * 2017-01-25 2018-07-31 上海元宋生物技术有限公司 Recombination oncolytic gene-the adenovirus and its construction method of target on cancer and application
CN108342366B (en) * 2017-01-25 2022-01-21 上海元宋生物技术有限公司 Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof
JP2018177648A (en) * 2017-04-04 2018-11-15 利章 大原 Agents and methods for inhibiting tumorigenesis of pluripotent stem cell
WO2018225705A1 (en) 2017-06-05 2018-12-13 テルモ株式会社 Method for producing cell culture
WO2020067438A1 (en) 2018-09-27 2020-04-02 国立大学法人大阪大学 Sheeting method for pluripotent stem cell-derived cells
WO2023106306A1 (en) * 2021-12-08 2023-06-15 株式会社八神製作所 Method for isolating stem cells using in-vitro boron-neutron reaction

Also Published As

Publication number Publication date
JPWO2012133674A1 (en) 2014-07-28
JP6264670B2 (en) 2018-01-24
JP2017036278A (en) 2017-02-16
JP6037393B2 (en) 2016-12-07

Similar Documents

Publication Publication Date Title
JP6264670B2 (en) Methods for removing tumorigenic cells in stem cells
US20200172875A1 (en) Method of nuclear reprogramming
Stadtfeld et al. Induced pluripotency: history, mechanisms, and applications
KR101657318B1 (en) Multipotent/pluripotent cells and methods
JP6473686B2 (en) Cell sorting method
JP6381442B2 (en) Efficient method for establishing induced pluripotent stem cells
US20150184131A1 (en) Enhanced Efficiency of Induced Pluripotent Stem Cell Generation
Chen et al. Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway
WO2009114949A1 (en) Methods for deprogramming somatic cells and uses thereof
WO2009032456A2 (en) Non-viral delivery of transcription factors that reprogram human somatic cells into a stem cell-like state
Jin et al. Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology
JP2018076385A (en) Transplantation adjuvant in cell therapy using neural progenitor cells
US20160376559A1 (en) Nuclear receptor and mutant thereof and the use of the same in the reprogramming of cells
Ahmad et al. Generation of two induced pluripotent stem cell (iPSC) lines from patients with Duchenne muscular dystrophy (IGIBi006-A and IGIBi008-A) carrying exonic deletions in the dystrophin gene
KR20210027290A (en) Neural stem cell composition and method of treating neurodegenerative disorder
WO2013022022A1 (en) Method for efficient establishment of artificial pluripotent stem cells
JP2019162054A (en) Efficient method for establishing induced pluripotent stem cell
JP6083874B2 (en) Mitochondrial disease-specific induced pluripotent stem cells, production method thereof and use
US20170183633A1 (en) Methods using reprogrammed cells for regenerative, restorative, and rejuvenative therapies
CA3216719A1 (en) Methods of generating mature corneal endothelial cells
De Miguel et al. Toward Induced Pluripotent Stem Cells for Clinical Use: Sources, Methods and Selection
CN116457469A (en) Methods and assays for analyzing compositions containing secretory groups
Goes Barbosa Buskin Improving our understanding of autosomal dominant Retinitis Pigmentosa using PRPF31 patient-specific induced pluripotent stem cells (iPSCs)
Chichagova Investigating retinal pathology in patients carrying m. 3243A> G mutation using human induced pluripotent stem cells
WO2011121636A1 (en) Method for producing induced pluripotent stem cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12763649

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2013507734

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12763649

Country of ref document: EP

Kind code of ref document: A1