Mutant KLF protein and method for producing induced pluripotent stem cells

Abstract

Provided is a mutant KLF protein capable of inducing reprogramming of somatic cells at higher efficiency than a KLF protein composed of the natural amino acid sequence. Also provided is a method for producing iPS cells efficiently using the mutant KLF protein.　Provided is a mutant KLF protein including an amino acid substitution, or a peptide fragment thereof that includes the amino acid substitution.

Description

[Technical field]

[0001] The present invention relates to a mutant KLF protein, an inducer of induced pluripotent stem cells, and a method for producing induced pluripotent stem cells. [Background technology]

[0002] Cell reprogramming technology, typified by the generation of induced pluripotent stem cells (iPS cells), has rapidly developed as an innovative platform technology in the fields of life science, drug discovery, and regenerative medicine.

[0003] iPS cells are induced by introducing reprogramming factors such as OCT3 / 4, SOX2, KLF4, and C-MYC into somatic cells (Patent Documents 1 and 2; Non-Patent Documents 1 and 2). All of these reprogramming factors act as transcription factors and are thought to control the expression of genes involved in self-renewal and pluripotency, thereby inducing the reprogramming of somatic cells.

[0004] However, the efficiency of iPS cell production is extremely low. According to conventional techniques, among mammalian somatic cells into which reprogramming factors such as OCT3 / 4, SOX2, KLF4, and C-MYC are introduced, the percentage of cells in which reprogramming is actually induced is less than 1% (Non-Patent Document 2).

[0005] Such low efficiency of iPS cell production is a major obstacle to clinical application. For example, when preparing tissue for transplantation, it is desirable to use iPS cells prepared from the patient's own somatic cells from the viewpoint of immune responsiveness. However, if the efficiency of iPS cell production remains low, it will take time to prepare a sufficient number of iPS cells for the preparation of tissue for transplantation, during which time the disease will progress. Conversely, if the efficiency of iPS cell production can be improved, it will be possible to rapidly prepare tissue for transplantation. Furthermore, if fewer somatic cells are required to prepare iPS cells, it will be possible to reduce the number of somatic cells to be collected from the patient, thereby reducing the burden on the patient's body.

[0006] In research aimed at the clinical application of iPS cells, technologies to improve safety are also being explored. For example, a method has been devised to replace the cancer gene C-MYC with another factor to reduce the risk of tumor formation in iPS cells. However, this method results in extremely low efficiency in iPS cell production (Non-Patent Document 3). In order to compensate for the shortcomings of such technologies to improve safety, improving the efficiency of iPS cell production has become an important issue.

[0007] The general theory behind the low efficiency of iPS cell production is that the epigenetic state of somatic cells is a barrier to reprogramming. Therefore, approaches that change the epigenetic state of somatic cells are being explored as a method for improving the efficiency of iPS cell production. Methods that additionally use cytokines and chemicals have also been developed (Non-Patent Document 4).

[0008] On the other hand, no attempt has been made to modify the natural sequences of the reprogramming factors used in reprogramming induction, such as OCT3 / 4, SOX2, KLF4, and C-MYC, especially to improve their DNA-binding function. Since all of these reprogramming factors are transcription factors, it is generally believed that their DNA-binding functions have already been optimized during the evolution of life. Therefore, it is unlikely that there is any room to increase the activity of transcription factors and improve the efficiency of iPS cell production by artificially modifying their sequences, and no attempt has been made to optimize reprogramming factors based on their molecular structure. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2007 / 069666 [Patent Document 2] JP 2008-283972 A [Non-patent literature]

[0010] [Non-Patent Document 1] Takahashi K. and Yamanaka, S., Cell, 2006, Aug 25;126(4):663-76. [Non-Patent Document 2] Takahashi K. et al., Cell, 2007, Nov 30;131(5):861-72. [Non-Patent Document 3] Nakagawa M., et al., Nat Biotechnol. 2008 Jan;26(1):101-6. [Non-Patent Document 4] Takahashi K. and Yamanaka S., Nat Rev Mol Cell Biol. 2016 Mar;17(3):183-93. Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a mutant KLF protein that can induce the reprogramming of somatic cells more efficiently than a KLF protein consisting of a natural amino acid sequence, and to provide a method for efficiently producing iPS cells using the mutant KLF protein. [Means for solving the problem]

[0012] Until now, reprogramming factors used for reprogramming induction have been exclusively composed of natural amino acid sequences. It was thought that changing the amino acid sequence in the DNA-binding domain of a reprogramming factor would likely affect its ability to recognize the target DNA sequence, reducing or eliminating its activity as a transcription factor.

[0013] In the presence of such technical common knowledge, the present inventors artificially modified the amino acid sequence of KLF4, one of the reprogramming factors, based on the molecular structure of the reprogramming factor, and attempted to design a mutant reprogramming factor that can improve reprogramming efficiency. The present inventors first identified 19 amino acid residues that are commonly conserved in the reprogramming factors KLF1, KLF2, KLF4, and KLF5 proteins (hereinafter often collectively referred to as "KLF proteins") and can directly interact with DNA. Then, a group of mutant KLF4 proteins in which the 19 amino acid residues were replaced with alanine were prepared, and a mutant having the activity of reprogramming somatic cells more efficiently than the wild-type KLF4 protein was searched for. As a result, a specific substitution mutation that increases the reprogramming efficiency of somatic cells was found, and the present invention was completed. The present invention is based on the above findings and provides the following.

[0014] (1) A mutant KLF protein having an amino acid substitution or a peptide fragment thereof having the amino acid substitution, The amino acid substitutions are (a) serine at position 349 and / or leucine at position 356 in the amino acid sequence shown in SEQ ID NO:1; (b) serine at position 342 and / or leucine at position 349 in the amino acid sequence shown in SEQ ID NO:3; (c) a serine at position 500 and / or a leucine at position 507 in the amino acid sequence shown in SEQ ID NO:5, or (d) serine at position 443 and / or leucine at position 450 in the amino acid sequence shown in SEQ ID NO: 7 The mutant KLF protein or a peptide fragment thereof, (2) The substitution in (a) is S349A, and / or L356A, L356N, L356D, L356C, L356E, L356G, L356K, L356M, L356S, or L356T, the substitution in (b) is S342A, and / or L349A, L349N, L349D, L349C, L349E, L349G, L349K, L349M, L349S, or L349T, and the substitution in (c) is S500A. and / or L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, or L507T, or the substitution in (d) is S443A, and / or L450A, L450N, L450D, L450C, L450E, L450G, L450K, L450M, L450S, or L450T. (3) A nucleic acid encoding the mutant KLF protein or a peptide fragment thereof according to (1) or (2). (4) A gene expression vector comprising the nucleic acid according to (3) in an expressible state. (5) An agent for inducing induced pluripotent stem cells (iPS cells), comprising the mutant KLF protein or peptide fragment thereof according to (1) or (2), the nucleic acid according to (3), or the gene expression vector according to (4). (6) The iPS cell inducer according to (5), further comprising the following (i) and / or (ii): (i) Any of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state. (ii) any one of a nucleic acid encoding a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein, or a SOX17 protein, or a gene expression vector containing the nucleic acid in an expressible state; (7) The iPS cell inducer according to (6), further comprising the following (iii): (iii) A C-MYC protein, a T58A mutant C-MYC protein, an N-MYC protein, or an L-MYC protein, or a nucleic acid encoding any of them, or a gene expression vector comprising the nucleic acid in an expressible state. (8) A direct reprogramming agent comprising the mutant KLF protein or peptide fragment thereof according to (1) or (2), the nucleic acid according to (3), or the gene expression vector according to (4). (9) Use of the iPS cell inducer according to any one of (5) to (7) for producing iPS cells from somatic cells. (10) A method for producing iPS cells, comprising the steps of: An introduction step of introducing an iPS cell-inducing agent into a somatic cell, the agent comprising the following [1] to [3]: [1] Any of the mutant KLF4 protein or peptide fragment thereof according to (1) or (2), the nucleic acid according to (3), or the gene expression vector according to (4), [2] An OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state, and [3] A nucleic acid encoding any one of SOX1 protein, SOX2 protein, SOX3 protein, SOX15 protein, and SOX17 protein, or a gene expression vector containing the nucleic acid in an expressible state; a culturing step of culturing the somatic cells after the introduction step in the presence of any one or more of basic fibroblast growth factor, TGF-β1 protein, BMP protein, Wnt3 protein, GSK3β inhibitor, Wnt inhibitor, retinoic acid, ascorbic acid, and ROCK inhibitor. The manufacturing method comprising the steps of: (11) A method for producing iPS cells, comprising the steps of: An introduction step of introducing an iPS cell-inducing agent into a somatic cell, the agent comprising the following [1] to [4]: [1] Any of the mutant KLF4 protein or peptide fragment thereof according to (1) or (2), the nucleic acid according to (3), or the gene expression vector according to (4), [2] Any of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state; [3] A nucleic acid encoding any one of SOX1 protein, SOX2 protein, SOX3 protein, SOX15 protein, and SOX17 protein, or a gene expression vector containing the nucleic acid in an expressible state; and [4] A nucleic acid encoding a C-MYC protein, an N-MYC protein, an L-MYC protein, or a T58A mutant protein of a C-MYC protein, or any of these, or a gene expression vector containing the nucleic acid in an expressible state; and A culturing step of culturing the somatic cells after the introduction step. The manufacturing method comprising the steps of: (12) The method according to (10) or (11), further comprising a selection step of selecting iPS cells induced in the culture step. (13) The method according to any one of (10) to (12), wherein the somatic cells are of human origin. (14) A cancer therapeutic agent comprising, as an active ingredient, the mutant KLF protein or peptide fragment thereof according to (1) or (2), the nucleic acid according to (3), or the gene expression vector according to (4). This specification includes the disclosures of Japanese Patent Application No. 2020-005399, which is the priority basis of this application. Effect of the Invention

[0015] The mutant KLF proteins of the present invention are capable of inducing somatic cell reprogramming more efficiently than KLF proteins consisting of a natural amino acid sequence. [Brief description of the drawings]

[0016] [Figure 1]FIG. 1 shows the structure of KLF protein. (A) shows the domain structure of human wild-type KLF4 protein and the positions of 19 amino acid residues that can directly interact with DNA in zinc finger domains 1 to 3 (ZF1, ZF2, and ZF3). In the figure, "PEST" means a sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T), and "NLS" means a nuclear localization signal. (B) shows an alignment of the amino acid sequences of the C-terminal regions of wild-type KLF1 protein, KLF2 protein, KLF4 protein, and KLF5 protein. The positions of amino acid residues conserved among KLF proteins (S349 and L356 in KLF1, S342 and L349 in KLF2, and S443 and L450 in KLF5) corresponding to S500 and L507 in the wild-type KLF4 protein are indicated by black boxes. The amino acid substitution positions in the mutant KLF protein of the present invention correspond to the amino acid residues boxed in black. [Diagram 2] This is a diagram showing the results of performing reprogramming induction using a mutant KLF4 protein in which each of 19 amino acid residues that can directly interact with DNA is replaced with alanine. Reprogramming induction was performed by introducing the mutant KLF4 protein together with other reprogramming factors (OCT3 / 4, SOX2, and L-MYC) into Nanog-GFP mouse embryonic fibroblasts using a retroviral vector. (A) is a diagram showing the number of Nanog-GFP positive colonies on the 25th day after virus infection formed from 10,000 Nanog-GFP mouse embryonic fibroblasts into which reprogramming factors were introduced. (B) is a diagram showing the ratio of the number of Nanog-GFP positive colonies to the total number of colonies on the 25th day after virus infection. [Diagram 3]This figure shows the results of flow cytometry analysis of the percentage of cells expressing the pluripotent stem cell marker TRA1-60 in human fibroblasts that had undergone reprogramming induction. For cell populations obtained by performing reprogramming induction using wild-type KLF4 protein or KLF4 (L507A) mutant, the percentage of cells expressing the pluripotent stem cell marker TRA1-60 was measured 7 days after retroviral infection. "Negative control" shows human fibroblasts that had not been transfected with a reprogramming factor. The figure shows the average value of measurement data of n = 6, and error bars indicate standard error. [Figure 4] This figure shows the results of inducing reprogramming of Nanog-GFP mouse embryonic fibroblasts using a Sendai virus vector. In this experiment, the KLF4 protein was fused to a destabilization domain (DD), and was degraded in the absence of Shield1 (0 nM), but not in the presence of Shield1 (100 nM). The bright areas indicated by the arrowheads indicate Nanog-GFP-positive colonies. [Diagram 5] This figure shows the predicted results of protein stability when the leucine at position 507 of the KLF4 protein is replaced with various amino acid residues. The vertical axis shows the predicted free energy change (ΔΔG) when each amino acid residue is replaced. The higher the ΔΔG, the more stable the structure of the KLF4 protein is. [Figure 6]This figure shows the results of inducing reprogramming of normal human fibroblasts using a Sendai virus vector. iPS cell clones obtained using wild-type KLF4 protein (WT iPS clones, n=13) and iPS cell clones obtained using KLF4(L507A) mutant (L507A iPS clones, n=16) were compared. (A) The results of quantification of NANOG mRNA expression level and Sendai virus NP mRNA expression level in each iPS cell clone by RT-qPCR are shown. The expression levels are normalized to the expression level in the standard human iPS cell line (HiPS-WTc11) as 1.0. (B) The results of quantification of RNA expression levels of HERV-H and lincRNA-RoR, which are differentiation resistance markers, in each iPS cell clone by RT-qPCR are shown. The expression levels are normalized to the expression level in the standard human iPS cell line (HiPS-WTc11) as 1.0. [Figure 7] This figure shows the results of reprogramming induction using KLF4 mutants in which the 507th leucine in the KLF4 protein was replaced with various amino acid residues. (A) The number of Nanog-GFP positive iPS cell colonies on day 15 after retrovirus infection. (B) The number of Nanog-GFP positive iPS cell colonies on day 25 after retrovirus infection. (C) The ratio of Nanog-GFP positive iPS cell colonies to the total colonies on day 25 after retrovirus infection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. Mutant KLF protein or peptide fragment thereof 1-1. Overview A first aspect of the present invention is a mutant KLF protein or a peptide fragment thereof. The mutant KLF protein or a peptide fragment thereof of the present invention contains a specific amino acid substitution, and when introduced into a somatic cell together with other reprogramming factors, it can induce reprogramming of the somatic cell with high efficiency.

[0018] 1-2.Definition The following definitions are provided for terms frequently used in this specification. "KLF protein" is a zinc finger transcription factor belonging to the Krueppel-like factor (KLF) family, and 17 types, KLF1 to KLF17, are known in humans. In this specification, "KLF protein" means any of KLF1, KLF2, KLF4, and KLF5 proteins. KLF1, KLF2, KLF4, and KLF5 proteins have an activity of inducing somatic cell reprogramming when introduced into somatic cells together with other reprogramming factors (e.g., OCT3 / 4, SOX2, and C-MYC proteins). In the present invention, the KLF protein is preferably derived from a mammal. For example, it is derived from mouse, rat, rabbit, cow, cynomolgus monkey, marmoset, or human, and is preferably derived from human. Examples of human-derived KLF proteins include human wild-type KLF1 protein consisting of the amino acid sequence shown in SEQ ID NO: 1, human wild-type KLF2 protein consisting of the amino acid sequence shown in SEQ ID NO: 3, human wild-type KLF4 protein consisting of the amino acid sequence shown in SEQ ID NO: 5, and human wild-type KLF5 protein consisting of the amino acid sequence shown in SEQ ID NO: 7. In this specification, the term "KLF" refers to either a KLF protein, a gene or nucleic acid encoding a KLF protein, or a gene expression vector containing the nucleic acid. Similarly, the term "mutant KLF" refers to either a mutant KLF protein, a gene or nucleic acid encoding a mutant KLF protein, or a gene expression vector containing the nucleic acid. The same applies to "KLF4" and "mutant KLF4", etc.

[0019] "Induced pluripotent stem cells (iPSCs)" refer to cells that are obtained from somatic cells by induction treatment and have differentiation potential similar to that of embryonic stem cells (ESCs). Normally, iPS cells have pluripotency, which allows them to differentiate into any type of cell in the body except for extraembryonic tissues, and proliferation potential, which allows them to grow almost indefinitely in culture. iPS cells can be obtained from various types of cells by various methods. For example, they are usually produced by introducing four reprogramming factors, OCT3 / 4, SOX2, KLF4, and C-MYC proteins, into somatic cells.

[0020] As used herein, the term "somatic cells" refers to cells other than germ cells that constitute an individual animal. As used herein, somatic cells are not limited as long as they are capable of acquiring pluripotency through reprogramming induction. In addition, the animal species from which the somatic cells are derived is not important. The animal species from which the somatic cells are derived is, for example, a mammalian species. For example, any mammalian species such as mouse, rat, rabbit, cow, cynomolgus monkey, marmoset, and human may be used, but human is preferred. The tissue or organ from which the somatic cells are derived is not particularly limited, but those that are easy to collect and from which reprogramming can be efficiently induced are preferred. For example, the tissue or organ may be an organ such as skin or liver, blood, urine, cancer tissue, dental pulp cells, etc. In addition, the somatic cells may be differentiated cells or undifferentiated cells, and may be established cells or primary culture cells isolated from tissues. Differentiated cells are preferred. Examples of somatic cells in this specification include human fibroblasts, human epithelial cells, human hepatocytes, human blood cells, mesenchymal cells, nerve cells, muscle cells, and the like. In this specification, somatic cells used for reprogramming induction are particularly referred to as "somatic cells to be targeted for reprogramming."

[0021] As used herein, "reprogramming" refers to an operation or process that changes a somatic cell into another cell type. In general, it refers to dedifferentiating a differentiated cell into an undifferentiated cell state. As used herein, unless otherwise specified, it refers to an operation or process that changes a somatic cell into an iPS cell.

[0022] As used herein, "reprogramming induction" or "inducing reprogramming" means that a cell is given an operation that can cause reprogramming, and is actually reprogrammed. In contrast, "performing reprogramming induction" means giving a cell an operation that can cause reprogramming, regardless of whether or not the cell is actually reprogrammed. For example, "performing reprogramming induction" refers to introducing a reprogramming factor required for reprogramming into a somatic cell, and culturing the somatic cell after the introduction under a specified condition.

[0023] As used herein, the term "pluripotency" is synonymous with multipotency and refers to the property of a cell that can differentiate into cells of multiple lineages through differentiation. In particular, it refers to the property of being able to differentiate into all of the endoderm, mesoderm, and ectoderm, but does not include the ability to differentiate into extraembryonic tissues such as the placenta.

[0024] As used herein, the term "reprogramming factor (reprogramming factor)" refers to a factor that can cause somatic cell reprogramming by being introduced into somatic cells alone or together with other factors. In this specification, when the term "reprogramming factor" is used without specifying whether it is a protein or a gene, it means either a protein corresponding to the reprogramming factor, a nucleic acid encoding the protein, or a gene expression vector containing the nucleic acid. Examples of reprogramming factors include any of the four factors OCT3 / 4, SOX2, KLF4, and C-MYC (often referred to as the "reprogramming four factors" in this specification), and factors related to any of the reprogramming four factors.

[0025] As used herein, the term "related factor" of the four reprogramming factors refers to a factor that can induce the reprogramming of somatic cells by introducing it into somatic cells in place of any one of the four reprogramming factors.

[0026] In the present specification, the animal species from which the reprogramming factor is derived is not important. The animal species from which the reprogramming factor is derived is, for example, a mammalian species. For example, any mammalian species such as mouse, rat, rabbit, cow, cynomolgus monkey, marmoset, human, etc. is acceptable. Preferably, the reprogramming factor is a human.

[0027] Examples of reprogramming factors and their associated factors are given below, but the reprogramming factors and their associated factors in this specification are not limited to the following examples.

[0028] A specific example of OCT3 / 4 is the human OCT3 / 4 protein consisting of the amino acid sequence shown in SEQ ID NO: 13. Factors related to OCT3 / 4 include NR5A2 (LRH1), TBX3, and the like.

[0029] A specific example of KLF4 is human KLF4 protein consisting of the amino acid sequence shown in SEQ ID NO: 5. Factors related to KLF4 include KLF1, KLF2, KLF5, the mutant KLF of the present invention, etc. Examples include human KLF1 protein consisting of the amino acid sequence shown in SEQ ID NO: 1, human KLF2 protein consisting of the amino acid sequence shown in SEQ ID NO: 3, and human KLF5 protein consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0030] A specific example of SOX2 is human SOX2 protein consisting of the amino acid sequence shown in SEQ ID NO: 15. In addition, SOX2-related factors include SOX1, SOX3, SOX15, and SOX18. For example, human SOX1 protein consisting of the amino acid sequence shown in SEQ ID NO: 14, human SOX3 protein consisting of the amino acid sequence shown in SEQ ID NO: 16, human SOX15 protein consisting of the amino acid sequence shown in SEQ ID NO: 17, and human SOX18 protein consisting of the amino acid sequence shown in SEQ ID NO: 18 are included.

[0031] A specific example of C-MYC is the human C-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 19. Factors related to C-MYC include the T58A mutant of C-MYC, N-MYC, L-MYC, etc. Examples include the human N-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 20, and the human L-MYC protein consisting of the amino acid sequence shown in SEQ ID NO: 21.

[0032] Other examples of reprogramming factors and related factors include LIN28A, LIN28B, LIN41, GLIS1, FOXH1, HMGA2, etc.

[0033] As a method for reprogramming somatic cells, a method for replacing some of the reprogramming factors with alternative reprogramming factors is also known. In this specification, the term "alternative reprogramming factor" refers to a factor other than the above-mentioned reprogramming factors that can cause reprogramming when used in place of any of the above-mentioned reprogramming factors. For example, a method for inducing somatic cell reprogramming using an alternative reprogramming factor instead of C-MYC among the four reprogramming factors of OCT3 / 4, SOX2, KLF4, and C-MYC is known. Specific examples of alternative reprogramming factors that can induce somatic cell reprogramming when used in place of C-MYC include basic fibroblast growth factor (bFGF), TGF-β1 protein, BMP protein, Wnt3 protein, GSK3β inhibitor, Wnt inhibitor, retinoic acid, ascorbic acid, and ROCK inhibitor. A specific example of a basic fibroblast growth factor (bFGF) is a human bFGF protein consisting of the amino acid sequence shown in SEQ ID NO: 22; a specific example of a TGF-β1 protein is a human TGF-β1 protein consisting of the amino acid sequence shown in SEQ ID NO: 23; a specific example of a BMP protein is a human BMP protein consisting of the amino acid sequence shown in SEQ ID NO: 24; a specific example of a Wnt3 protein is a human Wnt3 protein consisting of the amino acid sequence shown in SEQ ID NO: 25; a specific example of a GSK3β inhibitor is CHIR99021, etc.; an example of a Wnt inhibitor is IWR-1-endo, etc.; and an example of a ROCK inhibitor is Y-27632, etc.

[0034] As used herein, "multiple" refers to, for example, 2 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. "Amino acid identity" refers to the percentage (%) of the number of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides to be compared are aligned by inserting appropriate gaps into one or both of them as necessary so that the number of identical amino acid residues is maximized. The alignment of two amino acid sequences to calculate the amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW.

[0035] As used herein, unless otherwise specified, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties such as charge, side chain, polarity, and aromaticity among the 20 types of amino acids that constitute natural proteins. For example, substitutions within the uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr) with a low polarity side chain, the branched chain amino acid group (Leu, Val, Ile), the neutral amino acid group (Gly, Ile, Val, Leu, Ala, Met, Pro), the neutral amino acid group with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), the acidic amino acid group (Asp, Glu), the basic amino acid group (Arg, Lys, His), and the aromatic amino acid group (Phe, Tyr, Trp) are included. Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of the polypeptide. However, in the KLF protein of the present invention, the amino acid substitution of serine at position 500 or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein (i.e., the amino acid sequence shown in SEQ ID NO:5), or the amino acid substitution at a corresponding position in another KLF protein, is not limited to substitutions within a conservative amino acid group having similar properties such as charge, side chain, polarity, aromaticity, etc.

[0036] 1-3.Configuration The configuration of the mutant KLF protein or peptide fragment thereof of the present invention is specifically explained below.

[0037] The mutant KLF protein of the present invention is a mutant KLF protein that has the activity of inducing somatic cell reprogramming more efficiently than the wild-type KLF protein.

[0038] As used herein, the phrase "mutant KLF protein has an activity of inducing somatic cell reprogramming more efficiently than wild-type KLF protein" means that when the mutant KLF protein is introduced into somatic cells, the efficiency of inducing somatic cell reprogramming is significantly higher than that of the wild-type KLF protein introduced under the same conditions. For example, when the mutant KLF protein is introduced into somatic cells together with OCT3 / 4, SOX2, and C-MYC under the same conditions, the efficiency of inducing somatic cell reprogramming is significantly higher.

[0039] In this specification, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measurement results of multiple measurement targets when a statistical analysis is performed on the measurement results from the mutant KLF protein and the wild-type KLF protein. In the present invention, the significant difference between the two when a statistical analysis is performed on the measurement results from the mutant KLF protein and the wild-type KLF protein corresponds to this. For example, the risk rate (significance level) of the obtained value is small, specifically, less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). The "p (value)" shown here indicates the probability that the assumption will be correct by chance in the distribution in which the statistic is assumed in a statistical test. Therefore, the smaller "p" means that the assumption is closer to the truth. The test method for statistical processing is not particularly limited, and any known test method capable of determining the presence or absence of significance may be used as appropriate. For example, the Student t-test method, covariate variance analysis, etc. can be used.

[0040] The mutant KLF proteins of the present invention have amino acid substitutions at specific positions in the amino acid sequence of the wild-type KLF protein.

[0041] In a specific configuration, the mutant KLF protein of the present invention may include an amino acid substitution of either serine at position 349 and / or leucine at position 356 in the amino acid sequence of human wild-type KLF1 protein (i.e., the amino acid sequence shown in SEQ ID NO: 1). The mutant KLF protein of the present invention may also include an amino acid substitution of either serine at position 342 and / or leucine at position 349 in the amino acid sequence of human wild-type KLF2 protein (i.e., the amino acid sequence shown in SEQ ID NO: 3). The mutant KLF protein of the present invention may also include an amino acid substitution of either serine at position 500 and / or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein (i.e., the amino acid sequence shown in SEQ ID NO: 5). The mutant KLF protein of the present invention may also include an amino acid substitution of serine at position 443 and / or leucine at position 450 in the amino acid sequence of human wild-type KLF5 protein (i.e., the amino acid sequence shown in SEQ ID NO: 7). The mutant KLF protein may be derived from a human wild-type KLF protein and include the above amino acid substitutions in its sequence, or may be derived from a wild-type KLF protein of an animal species other than human and include amino acid substitutions in its sequence that correspond to the above amino acid substitutions. Hereinafter, the positions of the above amino acid substitutions are collectively referred to as "amino acid substitution positions in the mutant KLF protein of the present invention."

[0042] The amino acid residue after the above amino acid substitution may be any of the 20 types of amino acid residues, namely, an alanine (Ala / A) residue, a cysteine ​​(Cis / C) residue, an aspartic acid (Asp / D) residue, a glutamic acid (Glu / E) residue, a phenylalanine (Phe / F) residue, a glycine (Gly / G) residue, a histidine (His / H) residue, an isoleucine (Ile / I) residue, a lysine (Lys / K) residue, a leucine (Leu / L) residue, a methionine (Met / M) residue, an asparagine (Asn / N) residue, a proline (Pro / P) residue, a glutamine (Gln / Q) residue, an arginine (Arg / R) residue, a serine residue (Ser / S), a threonine residue (Thr / T), a valine (Val / V) residue, a tryptophan (Trp / W) residue, or a tyrosine (Tyr / Y) residue.

[0043] In a preferred embodiment, in the mutant KLF protein of the present invention, the amino acid substitution of either serine at position 349 and / or leucine at position 356 in the amino acid sequence of human wild-type KLF1 protein (i.e., the amino acid sequence shown in SEQ ID NO: 1) is S349A and / or L356A, L356N, L356D, L356C, L356E, L356G, L356K, L356M, L356S, or L356T, and the amino acid substitution of either serine at position 342 and / or leucine at position 349 in the amino acid sequence of human wild-type KLF2 protein (i.e., the amino acid sequence shown in SEQ ID NO: 3) is S342A and / or L349A, L349N, L349D, L349C, L349E, L349G, L349K, L349M, L356S, or L356T. the amino acid substitution of either serine at position 500 and / or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein (i.e., the amino acid sequence shown in SEQ ID NO:5) is S500A, and / or L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, or L507T; or the amino acid substitution of serine at position 443 and / or leucine at position 450 in the amino acid sequence of human wild-type KLF5 protein (i.e., the amino acid sequence shown in SEQ ID NO:7) is S443A, and / or L450A, L450N, L450D, L450C, L450E, L450G, L450K, L450M, L450S, or L450T.

[0044] In a more preferred embodiment, in the mutant KLF protein of the present invention, the amino acid substitution of either serine at position 349 and / or leucine at position 356 in the amino acid sequence of human wild-type KLF1 protein (i.e., the amino acid sequence shown in SEQ ID NO: 1) is S349A and / or L356A, L356C, L356G, L356K, or L356S, and the amino acid substitution of either serine at position 342 and / or leucine at position 349 in the amino acid sequence of human wild-type KLF2 protein (i.e., the amino acid sequence shown in SEQ ID NO: 3) is S342A and / or L349A, L349C, L349G, L356K, or L356S. and the amino acid substitution of either serine at position 500 and / or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein (i.e., the amino acid sequence shown in SEQ ID NO: 5) is S500A and / or L507A, L507C, L507G, L507K, or L507S, or the amino acid substitution of serine at position 443 and / or leucine at position 450 in the amino acid sequence of human wild-type KLF5 protein (i.e., the amino acid sequence shown in SEQ ID NO: 7) is S443A and / or L450A, L450C, L450G, L450K, or L450S. For example, a human mutant KLF4 (S500A) protein consisting of the amino acid sequence shown in SEQ ID NO: 28, or a human mutant KLF4 (L507A) protein consisting of the amino acid sequence shown in SEQ ID NO: 30 is exemplified.

[0045] In a further preferred embodiment, in the mutant KLF protein of the present invention, the amino acid substitution at either the serine at position 349 and / or the leucine at position 356 in the amino acid sequence of the human wild-type KLF1 protein (i.e., the amino acid sequence shown in SEQ ID NO: 1) is L356A, L356G, or L356S, and the amino acid substitution at either the serine at position 342 and / or the leucine at position 349 in the amino acid sequence of the human wild-type KLF2 protein (i.e., the amino acid sequence shown in SEQ ID NO: 3) is L349A, and the amino acid substitution of either serine at position 500 and / or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein (i.e., the amino acid sequence shown in SEQ ID NO: 5) is L507A, L507G, or L507S, or the amino acid substitution of serine at position 443 and / or leucine at position 450 in the amino acid sequence of human wild-type KLF5 protein (i.e., the amino acid sequence shown in SEQ ID NO: 7) is L450A, L450G, or L450S. For example, a human mutant KLF4 (L507A) protein consisting of the amino acid sequence shown in SEQ ID NO: 30 is exemplified.

[0046] The mutant KLF protein of the present invention may contain an addition, deletion, or substitution at a position other than the position of the amino acid substitution of the mutant KLF protein of the present invention. Such a mutant KLF protein is preferably a polypeptide having an activity of inducing somatic cell reprogramming more efficiently than the wild-type KLF protein. For example, the mutant KLF protein of the present invention is a polypeptide consisting of an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 with respect to the full length sequence excluding the position of the amino acid substitution of the mutant KLF protein of the present invention, and having an activity of inducing somatic cell reprogramming more efficiently than the wild-type KLF protein. Alternatively, the mutant KLF protein of the present invention may be a polypeptide in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, with respect to the full-length sequence of the mutant KLF protein of the present invention excluding the positions of amino acid substitutions, and which has the activity of inducing somatic cell reprogramming more efficiently than the wild-type KLF protein.

[0047] As used herein, the term "peptide fragment" of a mutant KLF protein refers to a polypeptide fragment that contains an amino acid residue substituted at the amino acid substitution position of the mutant KLF protein of the present invention in the above mutant KLF protein, and that retains the activity of inducing somatic cell reprogramming more efficiently than the wild-type KLF protein. For example, a polypeptide fragment containing a DNA binding site of a mutant KLF protein is included. The length of amino acids of the polypeptide constituting the present active fragment is not particularly limited. For example, it may be a region of at least 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or 450 consecutive amino acids in the KLF protein.

[0048] 1-4.Effects The mutant KLF protein of the present invention or a peptide fragment thereof has enhanced transcription factor activity compared to the wild-type KLF protein. For example, when the mutant KLF protein of the present invention or a peptide fragment thereof is introduced into somatic cells, the expression level of the target gene is increased compared to the case where the wild-type KLF protein is introduced. This results in increased efficiency of iPS cell production and cancer treatment compared to the wild-type KLF protein.

[0049] The mutant KLF protein of the present invention or a peptide fragment thereof can induce somatic cell reprogramming more efficiently than the wild-type KLF protein. For example, when the mutant KLF protein of the present invention or a peptide fragment thereof, which has been expressed and / or purified by a known method using E. coli or the like, is directly introduced into somatic cells together with other reprogramming factors (e.g., OCT3 / 4, SOX2, and C-MYC), somatic cell reprogramming can be induced more efficiently than when the wild-type KLF protein is introduced together with the other reprogramming factors.

[0050] Moreover, the mutant KLF protein or peptide fragment thereof of the present invention can produce iPS cells with high homogeneity.Furthermore, the mutant KLF protein or peptide fragment thereof of the present invention can produce iPS cells with low differentiation resistance (e.g., iPS cells with low expression levels of differentiation resistance markers such as HERV-H and / or lincRNA-RoR).

[0051] 2. Nucleic acid encoding a mutant KLF protein or a peptide fragment thereof 2-1. Overview A second aspect of the present invention is a nucleic acid. The nucleic acid of the present invention is a nucleic acid, such as DNA or mRNA, that encodes the mutant KLF protein of the first aspect or a peptide fragment thereof.

[0052] 2-2.Configuration The nucleic acid of the present invention encodes the mutant KLF protein of the first aspect or a peptide fragment thereof. Examples of the nucleic acid include a nucleic acid that contains or consists of a nucleotide sequence that is identical to the nucleotide sequence of the wild-type KLF gene except for codons that code for the amino acid substitution of serine at position 500 or leucine at position 507 in the amino acid sequence of human wild-type KLF4 protein, or an amino acid substitution at a corresponding position in another KLF protein, and a nucleic acid that contains or consists of a nucleotide sequence in which the nucleotide sequence has been codon-optimized to match the codon usage frequency in a somatic cell into which the nucleic acid is introduced.

[0053] The nucleic acid of the present invention may be DNA or RNA such as mRNA.

[0054] Specific examples of DNA corresponding to the nucleic acid of the present invention include DNA consisting of the base sequence shown in SEQ ID NO: 29, which encodes a mutant KLF4 protein in which the serine at position 500 in the amino acid sequence shown in SEQ ID NO: 5 is replaced with alanine, and DNA consisting of the base sequence shown in SEQ ID NO: 31, which encodes a mutant KLF4 protein in which the leucine at position 507 in the amino acid sequence shown in SEQ ID NO: 5 is replaced with alanine.

[0055] The mRNA corresponding to the nucleic acid of the present invention is an mRNA containing an RNA base sequence corresponding to the above DNA as a coding region. The term "RNA base sequence corresponding to the above DNA" as used herein refers to a base sequence in which thymine (T) in the base sequence of the above DNA is replaced with uracil (U). The mRNA corresponding to the nucleic acid of the present invention may contain, in addition to the coding region, a cap structure at the 5' end, a polyA tail at the 3' end, a 5' untranslated region (5' UTR) upstream of the start codon, and / or a 3' untranslated region (3' UTR) downstream of the stop codon, etc. The 5' UTR and / or 3' UTR, etc. may contain a sequence for regulating the amount of translation from the mRNA. For example, the 3' UTR may contain a sequence that increases the amount of translation from the mRNA, such as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), etc.

[0056] 2-3.Effects A DNA corresponding to the nucleic acid of the present invention can be used as the coding region (protein translation region) in the gene expression vector of the third embodiment.

[0057] The mRNA corresponding to the nucleic acid of the present invention can be introduced directly into somatic cells together with mRNAs encoding other reprogramming factors, for example, to induce somatic cell reprogramming with high efficiency. In such cases, the reprogramming factors are translated and expressed transiently, and then the mRNA is rapidly degraded, so that the genetic factors encoding the reprogramming factors are not maintained in the cells after reprogramming. Therefore, a reprogramming technique with low risk of tumor formation and high safety can be provided.

[0058] 3. Gene Expression Vectors 3-1. Overview A third aspect of the present invention is a gene expression vector. The gene expression vector of the present invention contains a nucleic acid encoding the mutant KLF protein of the present invention or a peptide fragment thereof in an expressible state. The gene expression vector of the present invention allows the mutant KLF protein of the present invention or a peptide fragment thereof to be expressed in somatic cells.

[0059] 3-2.Configuration 3-2-1. Overview of configuration The gene expression vector of the present invention comprises, as essential components, a promoter and the nucleic acid according to the second aspect.

[0060] As used herein, the term "gene expression vector" refers to a vector that contains a gene or a gene fragment in an expressible state and includes an expression unit that can control the expression of the gene, etc. As used herein, the term "expressible state" refers to a state in which the gene to be expressed is located downstream of a promoter under the control of the promoter. The gene expression vector of the present invention is a vector that contains the nucleic acid of the second aspect in an expressible state and is capable of expressing a mutant KLF protein or a peptide fragment thereof in somatic cells.

[0061] Vectors that can be used as the gene expression vector of the present invention, as well as promoters and other optional components contained in the gene expression vector of the present invention, are described below.

[0062] 3-2-2.Vector Vectors that can be used as gene expression vectors of the present invention are not particularly limited as long as they can express the mutant KLF protein of the present invention or a peptide fragment thereof in somatic cells, and examples of such vectors include virus vectors, plasmid vectors, and artificial chromosome vectors.

[0063] The viral vector usable as the gene expression vector of the present invention is not particularly limited as long as it can infect somatic cells to be reprogrammed and can express the mutant KLF protein of the present invention or a peptide fragment thereof in the somatic cells. Examples include adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, etc. Depending on the type of viral vector, there are differences in the size of DNA that can be carried, the type of cells that can be infected, cytotoxicity, whether or not it is integrated into the host genome, and the expression period, and the like, and can be appropriately selected depending on the type of somatic cells to be reprogrammed, etc. For example, a replication-defective and persistent Sendai virus vector (SeVdp vector) is highly safe and particularly preferred because it does not integrate into the host genome and remains persistently in the cytoplasm (Nishimura K., et al., J Biol Chem. 2011 Feb 11;286(6):4760-71.; Fusaki N., et al., Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62.).

[0064] The plasmid vector usable as the gene expression vector of the present invention is not particularly limited as long as it can express the mutant KLF protein of the present invention or a peptide fragment thereof in a somatic cell to be reprogrammed when introduced into the somatic cell. The plasmid vector may be a shuttle vector capable of replicating between a mammalian cell and a bacterium such as Escherichia coli. Specific examples of the plasmid vector include plasmids derived from Escherichia coli (pBR322, pUC18, pUC19, pUC118, pUC119, pBluescript, etc.), plasmids derived from actinomycetes (pIJ486, etc.), plasmids derived from Bacillus subtilis (pUB110, pSH19, etc.), and plasmids derived from yeast (YEp13, YEp24, Ycp50, etc.), as well as commercially available vectors. Specific examples of commercially available vectors include CMV6-XL3 (OriGene), EGFP-C1, pGBT-9 (Clontech), pcDNA, pcDM8, and pREP4 (Thermo Fisher Scientific).

[0065] Examples of artificial chromosome vectors that can be used as gene expression vectors of the present invention include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).

[0066] Promoter The promoter contained in the gene expression vector of the present invention is a promoter that has the activity of inducing gene expression in somatic cells to be initialized. Since the somatic cells into which the gene expression vector of the present invention is introduced are, in principle, mammalian cells, particularly cells derived from humans, any promoter that can express downstream genes in those cells may be used. Examples of such promoters include CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, HSV-TK promoter, EF1α promoter, Ub promoter, metallothionein promoter, SRα promoter, and CAG promoter. Other examples include inducible promoters such as a heat shock promoter that can be controlled by temperature and a tetracycline responsive promoter that can be controlled by the presence or absence of tetracycline.

[0067] 3-2-4. Other optional components The gene expression vector of the present invention may contain, as optional components, a regulatory sequence other than the above-mentioned promoter, a selection marker gene, and / or a reporter gene.

[0068] The control sequences other than the promoter that may be included in the gene expression vector of the present invention include expression control sequences, intron sequences, nuclease recognition sequences, replication origin sequences, etc. Examples of the expression control sequences include expression control sequences such as enhancers, ribosome binding sequences, terminators, and polyA addition signals. Examples of the nuclease recognition sequences include restriction enzyme recognition sequences, loxP sequences recognized by Cre recombinase, sequences that are targets of artificial nucleases such as ZFN and TALEN, or sequences that are targets of the CRISPR / Cas9 system. Examples of the replication origin sequence include the SV40 replication origin sequence.

[0069] For example, in the gene expression vector of the present invention, a nuclease recognition sequence can be introduced before and after the coding region of the reprogramming factor, in which case, after somatic cell reprogramming is completed, the coding region of the reprogramming factor can be removed by introducing a nuclease.

[0070] The selectable marker gene that may be contained in the gene expression vector of the present invention is a selectable marker gene that allows the selection of somatic cells into which the gene expression vector of the present invention has been introduced. Specific examples of the selectable marker gene include drug resistance genes such as ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, neomycin resistance gene, puromycin resistance gene, and hygromycin resistance gene.

[0071] The reporter gene that can be contained in the gene expression vector of the present invention is a gene encoding a reporter capable of identifying a somatic cell into which the gene expression vector of the present invention has been introduced. Examples of reporter genes include genes encoding fluorescent proteins such as GFP and RFP, and luciferase genes.

[0072] 3-3.Effects The gene expression vector of the present invention can express the mutant KLF protein of the present invention or a peptide fragment thereof in somatic cells to be reprogrammed. Furthermore, by using the gene expression vector together with other reprogramming factors (e.g., OCT3 / 4, SOX2, and C-MYC), reprogramming of somatic cells can be induced with high efficiency.

[0073] 4. iPS cell inducers 4-1. Overview A fourth aspect of the present invention is an agent for inducing induced pluripotent stem cells (iPS cells). The iPS cell inducer of the present invention contains, as an essential component, the mutant KLF protein of the present invention or a peptide fragment thereof, a nucleic acid encoding the mutant KLF protein or a peptide fragment thereof, or a gene expression vector containing the nucleic acid, and contains other reprogramming factors as optional components.

[0074] 4-2.Configuration 4-2-1. Overview of configuration The iPS cell inducer of the present invention comprises, as an essential component, any one of the mutant KLF protein of the first aspect or a peptide fragment thereof, the nucleic acid of the second aspect, or the gene expression vector of the third aspect (hereinafter collectively referred to as the "mutant KLF of the present invention").

[0075] The iPS cell induction agent of the present invention may contain one or more other reprogramming factors as optional components.

[0076] The essential components are the same as those described in the first to third embodiments, so only optional components will be described below.

[0077] 4-2-2.Selective components The iPS cell inducer of the present invention may contain, as optional components, (1) one or more other reprogramming factors and / or (2) reprogramming cofactors. (1) and (2) are specifically described below.

[0078] (1) Other reprogramming factors The iPS cell inducer of the present invention may contain one or more other reprogramming factors as selective components. The "other reprogramming factors" referred to here are reprogramming factors other than the mutant KLF of the present invention. There is no limitation on the reprogramming factors other than the mutant KLF of the present invention as long as they can induce the reprogramming of somatic cells. Examples of the reprogramming factors include OCT3 / 4, SOX2, C-MYC, and KLFs other than the mutant KLF of the present invention (e.g., wild-type KLF), and any of their related factors. Examples of the related factors include SOX1, SOX3, SOX15, SOX18, the T58A mutant of C-MYC, N-MYC, and L-MYC, as well as wild-type KLF1, wild-type KLF2, wild-type KLF4, and wild-type KLF5. These other reprogramming factors may be any of proteins or peptide fragments thereof corresponding to the other reprogramming factors, nucleic acids encoding the proteins or peptide fragments thereof, or gene expression vectors containing the nucleic acids in an expressible state. The configuration of the nucleic acid encoding the protein or peptide fragment thereof corresponding to the other reprogramming factor, and the gene expression vector containing the nucleic acid in an expressible state, shall be similar to the second and third aspects described above for mutant KLF.

[0079] The number of other reprogramming factors contained in the iPS cell inducer of the present invention is not limited. For example, the iPS cell inducer of the present invention may contain one, two, three, four, five, six, or more types of reprogramming factors as other reprogramming factors.

[0080] Furthermore, when the iPS cell-inducing agent of the present invention contains two or more gene expression vectors, the two or more gene expression vectors may be contained in the same vector or may be separate vectors.

[0081] In a preferred embodiment, the iPS cell inducer of the present invention may further comprise, in addition to the essential components, any of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector comprising the nucleic acid in an expressible state, and / or any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein, or a SOX17 protein, a nucleic acid encoding any of them, or a gene expression vector comprising the nucleic acid in an expressible state. In addition to these, the iPS cell inducer may further comprise any of a C-MYC protein, a T58A mutant of a C-MYC protein, an N-MYC protein, or an L-MYC protein, a nucleic acid encoding any of them, or a gene expression vector comprising the nucleic acid in an expressible state.

[0082] (2) Reprogramming cofactors The "reprogramming cofactor" that the iPS cell inducer of the present invention may include as a selective component is a factor other than those falling under (1) above that is not essential for the reprogramming induction of somatic cells but that can increase the efficiency of reprogramming induction when introduced into somatic cells. Examples include NANOG, NR5A2, LIN28A, LIN28B, LIN41, GLIS1, TBX3, HMGA2, FOXH1, mir-302, mir-367, mir-106a, mir-363, shRNA or siRNA against TP53, dominant negative TP53, or shRNA or siRNA against P21.

[0083] Effects According to the iPS cell inducer of the present invention, it is possible to induce iPS cells from somatic cells. Furthermore, the iPS cell inducer of the present invention can be used to produce iPS cells from somatic cells.

[0084] 5. Direct Reprogramming Agents 5-1. Overview The fifth aspect of the present invention is a direct reprogramming agent. The direct reprogramming agent of the present invention contains, as an essential component, either the mutant KLF protein of the present invention or a peptide fragment thereof, a nucleic acid encoding the mutant KLF protein or a peptide fragment thereof, or a gene expression vector containing said nucleic acid (hereinafter collectively referred to as "mutant KLF of the present invention"), and contains other direct reprogramming factors as optional components. The direct reprogramming agent of the present invention can directly induce various other cell types from differentiated cells.

[0085] 5-2.Definition As used herein, "direct reprogramming" refers to directly inducing another cell type (e.g., a cell type other than iPS cells) from a specific cell type. More specifically, it refers to inducing another cell type from a specific cell type without going through the iPS cell stage, for example, inducing various other cell types such as nerve cells, hepatocytes, pancreatic β cells, cardiomyocytes, or endothelial cells from differentiated cells (Ieda M., Keio J Med. 2013;62(3):74-82.). Note that, as used herein, direct reprogramming includes transdifferentiation.

[0086] As used herein, the term "direct reprogramming factor" refers to a factor that can cause direct reprogramming by being introduced into a specific cell type, alone or together with other factors. The direct reprogramming factor varies depending on the type of cell to be subjected to direct reprogramming and / or the type of cell obtained by induction by direct reprogramming. For example, MyoD, which induces direct reprogramming from fibroblasts to muscle cells; a combination of Ascl1, Brn2, and Myt1l, which induces direct reprogramming from fibroblasts to nerve cells; a combination of Gata4, Mef2c, and Tbx5, a combination of Gata4, Mef2c, Tbx5, and Hand2, a combination of Gata4, Mef2c, Tbx5, and VEGF, or a combination of Mef2c, Myocardin, and Tbx5, which induces direct reprogramming from fibroblasts to cardiomyocytes, are known. In particular, specific examples of direct reprogramming involving KLF4 as a direct reprogramming factor include a combination of PAX6, OVOL2, and KLF4 that induces direct reprogramming from fibroblasts to corneal epithelial cells; a combination of SOX2, KLF4, C-MYC, and POU3F4 (BRN4) that induces direct reprogramming from fibroblasts to neural stem cells; a combination of KLF4, C-MYC, and SOX9 that induces direct reprogramming from skin fibroblasts to chondrocytes; and a combination of OCT3 / 4, SOX2, KLF4, and C-MYC that induces direct reprogramming from fibroblasts to endothelial cells by partial reprogramming for 4 days (Kitazawa K., et al., Cornea, 2019 Nov;38 Suppl 1:S34-S41.; Kim SM, et al., Nat Protoc., 2014 Apr;9(4):871-81.; Outani H., et al., PLoS One, 2013 Oct 16;8(10):e77365.;Margariti A, et al., Proc Natl Acad Sci US A., 2012;109(34):13793-13798.).

[0087] 5-3.Configuration The direct reprogramming agent of the present invention comprises, as an essential component, the mutant KLF of the present invention. More specifically, as an essential component, the mutant KLF protein of the first aspect or a peptide fragment thereof, the nucleic acid of the second aspect, or the gene expression vector of the third aspect.

[0088] The direct reprogramming agent of the present invention may further include one or more other direct reprogramming factors as selective components. The other direct reprogramming factors included in the direct reprogramming agent of the present invention vary depending on the cell type of the cells to be subjected to direct reprogramming and / or the cell type induced by direct reprogramming, and can be appropriately selected by those skilled in the art. Examples include, but are not limited to, OCT3 / 4, SOX2, C-MYC, PAX6, VOL2, POU3F4 (BRN4), and SOX9. The other direct reprogramming factors included in the direct reprogramming agent of the present invention may be any of a protein or a peptide fragment thereof corresponding to the other direct reprogramming factor, a nucleic acid encoding the protein or the peptide fragment thereof, or a gene expression vector containing the nucleic acid in an expressible state. The configurations of the nucleic acid encoding the protein or the peptide fragment thereof of the other direct reprogramming factor and the gene expression vector containing the nucleic acid in an expressible state shall conform to the descriptions of the second and third aspects described for the mutant KLF protein, respectively.

[0089] The number of other direct reprogramming factors that the direct reprogramming agent of the present invention includes is not limited. For example, the direct reprogramming agent of the present invention may include one, two, three, four, five, six, or more other direct reprogramming factors.

[0090] Furthermore, when the direct reprogramming agent of the present invention contains two or more gene expression vectors, the two or more gene expression vectors may be contained in the same vector or may be separate vectors.

[0091] In one embodiment, the direct reprogramming agent of the present invention includes, in addition to the mutant KLF of the present invention, OCT3 / 4, SOX2, and C-MYC.

[0092] In another embodiment, the direct reprogramming agent of the present invention comprises, in addition to the mutant KLF of the present invention, PAX6 and OVOL2.

[0093] In another embodiment, the direct reprogramming agent of the present invention includes, in addition to the mutant KLF of the present invention, SOX2, C-MYC, and POU3F4 (BRN4).

[0094] In another embodiment, the direct reprogramming agent of the present invention includes, in addition to the mutant KLF of the present invention, C-MYC and SOX9.

[0095] Effects When the direct reprogramming agent of the present invention includes mutant KLF, OCT3 / 4, SOX2, and C-MYC, for example, partial reprogramming using the direct reprogramming agent of the present invention for 4 days induces direct reprogramming from fibroblasts to endothelial cells with high efficiency.

[0096] When the direct reprogramming agent of the present invention comprises mutant KLF, PAX6, and OVOL2, the direct reprogramming agent of the present invention induces direct reprogramming from fibroblasts to corneal epithelial cells with high efficiency.

[0097] When the direct reprogramming agent of the present invention comprises mutant KLF, SOX2, C-MYC, and POU3F4 (BRN4), the direct reprogramming agent of the present invention induces direct reprogramming from fibroblasts to neural stem cells with high efficiency.

[0098] When the direct reprogramming agent of the present invention comprises mutant KLF, C-MYC, and SOX9, the direct reprogramming agent of the present invention induces direct reprogramming from skin fibroblasts to chondrocytes with high efficiency.

[0099] According to the direct reprogramming agent of the present invention, direct reprogramming from somatic cells can be carried out in a culture system or in vivo.

[0100] The direct reprogramming agent of the present invention can induce other cell types without going through the pluripotent stem cell stage, providing a highly safe cell reprogramming technique with reduced risk of tumorigenesis.

[0101] 6.iPS cell production method 6-1. Overview A sixth aspect of the present invention is a method for producing iPS cells. The method for producing iPS cells of the present invention comprises, as essential steps, (1) an introduction step of introducing an iPS cell inducer into somatic cells, and (2) a culture step of culturing the somatic cells after the introduction step. According to the method for producing iPS cells of the present invention, iPS cells can be produced with high efficiency.

[0102] 6-2. Method The method for producing iPS cells of the present invention includes, as essential steps, (1) an introduction step of introducing an iPS cell inducer into somatic cells, and (2) a culture step of culturing the somatic cells after the introduction step, and also includes, as an optional step, (3) an iPS cell selection step.

[0103] The steps of the introduction step and the culture step are different between "6-2-1. An embodiment using C-MYC or a C-MYC-related factor" and "6-2-2. An embodiment not using C-MYC or a C-MYC-related factor" described below. The "C-MYC-related factor" here refers to a factor that has a similar structure to C-MYC and can reprogram somatic cells by introducing it into somatic cells together with other reprogramming factors instead of C-MYC (hereinafter, the same applies to "OCT3 / 4-related factor" and "SOX2-related factor"). Specific examples of each related factor are as described in "1-2. Definitions".

[0104] Each step in the above two cases will be specifically explained below.

[0105] 6-2-1.Embodiments using C-MYC or C-MYC-related factors In the iPS cell production method of this embodiment, C-MYC or a C-MYC-related factor is introduced into a somatic cell in the introduction step.

[0106] (1) Introduction process The iPS cell inducer introduced into somatic cells in the introduction step in this embodiment corresponds to a combination capable of inducing reprogramming of somatic cells, among the combinations of reprogramming factors constituting the iPS cell inducer of the fourth aspect. In this embodiment, the iPS cell inducer introduced into somatic cells may be, for example, a combination of factors (a) to (d) below: (a) any one of the mutant KLF protein or peptide fragment thereof of the first aspect, the nucleic acid of the second aspect, or the gene expression vector of the third aspect (hereinafter collectively referred to as "mutant KLF of the present invention"), (b) OCT3 / 4 or an OCT3 / 4-related factor, (c) SOX2 or a SOX2-related factor, and (d) C-MYC or a C-MYC-related factor.

[0107] The iPS cell inducer introduced into the somatic cells in this introduction step may contain, in addition to the combination of reprogramming factors described above, reprogramming cofactors that can increase the efficiency of iPS cell production. The reprogramming cofactors are as described in "(2) Reprogramming cofactors" in "4-2-2. Optional components".

[0108] In this introduction step, the method of introducing the iPS cell inducer into somatic cells is not limited. The introduction method may be appropriately selected depending on the type of iPS cell inducer to be introduced (plasmid DNA, mRNA, protein, viral vector, etc.). For example, the iPS cell inducer can be introduced into somatic cells by viral infection, lipofection, liposome, electroporation, calcium phosphate, DEAE-Dextran, microinjection, or electroporation. In addition, gene introduction methods (transformation methods) known in the art, such as those described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., can be used.

[0109] (2)Culture process The culture step in this embodiment is a step of culturing the somatic cells after the introduction step. In this culture step, the reprogramming factor introduced in the introduction step induces reprogramming of the somatic cells, and iPS cells are induced. Furthermore, by observing colonies formed from the induced iPS cells in this step, it can be determined whether iPS cells have been produced.

[0110] The culture method in the main culture step is not limited. For example, the somatic cells after the introduction step may be cultured using feeder cells. Specifically, there are a method of culturing the somatic cells after the introduction step together with feeder cells in a cell culture medium, a method of maintaining the somatic cells after the introduction step in a cell culture medium for 30 to 40 days and then culturing them together with feeder cells, and the like.

[0111] The feeder cells may be, but are not limited to, cells whose growth has been stopped by irradiation or antibiotic treatment (e.g., mouse embryonic fibroblasts (MEF), cells derived from a human embryo, or fibroblasts).

[0112] When feeder cells are not used, a method using a culture dish coated with a basement membrane matrix, laminin, vitronectin, etc., or a method using a medium containing a basement membrane matrix, laminin, vitronectin, etc. can be used.

[0113] The cell culture medium may be appropriately selected from known media. For example, a commercially available basal medium for mammalian cells, such as DMEM, to which serum or a serum replacement solution has been added, may be used. An example of a serum replacement solution is KnockOut. TM Serum Replacement: KSR (ThermoFisher, SCIENTIFIC) may be used. In addition, commercially available primate ES cell medium or primate ES / iPS cell medium may be used. These media may contain known additives suitable for culturing pluripotent stem cells such as ES cells or iPS cells, such as N2 supplement, B27(R) supplement, insulin, bFGF, activin A, heparin, ROCK (Rho-associated coiled-coil forming kinase / Rho binding kinase) inhibitor, and / or GSK-3 inhibitor.

[0114] In order to increase the efficiency of iPS cell production, TGF-β, histone deacetylase (HDAC) inhibitors, G9a histone methyltransferase inhibitors, and / or p53 inhibitors may be added to the medium in the introduction step (1) and / or the main culture step. Examples of HDAC inhibitors that can be used include small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, and M344, and siRNAs against HDAC. Examples of G9a histone methyltransferase inhibitors that can be used include small molecule inhibitors such as BIX-01294, and siRNAs against G9a. Examples of p53 inhibitors that can be used include small molecule inhibitors such as pifithrin-α, and siRNAs against p53.

[0115] In the main culturing step, the culturing conditions such as temperature, CO2 concentration, culturing period, and frequency of medium exchange are not limited. For example, the cells may be statically cultured at 37°C and 5% CO2, with half of the medium exchanged every two days, for 10 to 40 days depending on the state of colony formation.

[0116] (3) iPS cell selection process In the method for producing iPS cells of this embodiment, the iPS cells induced after the introduction step and the culture step may be selected.

[0117] The method of selecting iPS cells in this step is not limited. For example, the method of selecting using the expression of an iPS cell marker gene as an index, the method of selecting by a selection marker gene, or the method of selecting by a reporter gene can be mentioned. The "iPS cell marker gene" is a gene that is not expressed in somatic cells to be reprogrammed, but is expressed in iPS cells. Preferably, it is a gene that is specifically expressed in iPS cells. Examples of the gene include Oct3 / 4, Sox2, Nanog, ERas, Esg1, TRA1-60, or TRA-1-85 genes, and endogenous alkaline phosphatase genes. Examples of the method of detecting the expression of the marker gene of iPS cells include a method of detecting mRNA, an immunological detection method (e.g., immunostaining, Western blotting, ELISA), and the like. The selection marker gene and the reporter gene are as described in "3-2-4. Other optional components" in "3. Gene expression vector". That is, iPS cells can be selected by selecting using a drug corresponding to a selection marker gene (e.g., a drug resistance gene such as an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a neomycin resistance gene, a puromycin resistance gene, or a hygromycin resistance gene) contained in the gene expression vector introduced in the introduction step, or by detecting a reporter corresponding to a reporter gene (e.g., a gene encoding a fluorescent protein such as GFP or RFP, or a luciferase gene) contained in the gene expression vector introduced in the introduction step.

[0118] 6-2-2.Embodiments not using C-MYC or C-MYC-related factors In the iPS cell production method of this embodiment, C-MYC or a C-MYC-related factor is not introduced into somatic cells in the introduction step, and a reprogramming alternative factor is used in the culture step. Here, the "reprogramming alternative factor" means a factor that can cause reprogramming when used in place of a reprogramming factor, as described in "1-2. Definition". In this embodiment, the reprogramming alternative factor is a reprogramming alternative factor that can induce reprogramming of somatic cells by adding it to a medium and culturing it instead of introducing C-MYC or a C-MYC-related factor into somatic cells. Examples of the reprogramming alternative factor include, but are not limited to, basic fibroblast growth factor (bFGF), TGF-β1 protein, BMP protein, Wnt3 protein, GSK3β inhibitor, Wnt inhibitor, retinoic acid, ascorbic acid, and ROCK inhibitor.

[0119] (1) Introduction process The iPS cell inducer introduced into somatic cells in the introduction step in this embodiment corresponds to the combination of reprogramming factors in the "(1) Introduction step" in the above "6-2-1. Embodiments using C-MYC or a C-MYC-related factor", excluding C-MYC or a C-MYC-related factor. Although not limited thereto, the iPS cell inducer introduced into somatic cells in this embodiment is, for example, a combination of the following factors (a) to (c): (a) mutant KLF, (b) OCT3 / 4 or an OCT3 / 4-related factor, and (c) SOX2 or a SOX2-related factor.

[0120] Other conditions for this introduction step are as described in "(1) Introduction step" in "6-2-1. Embodiments using C-MYC or a C-MYC-related factor" above.

[0121] (2)Culture process The culture step in this embodiment is a step of culturing the somatic cells after the introduction step. In the culture step of this embodiment, a reprogramming replacement factor is added to the medium. Other culture conditions in the main culture step are as described in "(2) Culturing step" in "6-2-1. Embodiments using C-MYC or a C-MYC-related factor."

[0122] (3) iPS cell selection process The iPS cell selection step in this embodiment is similar to the description in "(3) iPS cell selection step" in "6-2-1. Embodiments using C-MYC or a C-MYC-related factor."

[0123] Effects According to the method for producing iPS cells of the present invention, iPS cells can be produced with high efficiency. According to "6-2-2. An embodiment in which C-MYC or a C-MYC-related factor is not used" in this aspect, the oncogene C-MYC or a C-MYC-related factor is not introduced into somatic cells, thereby reducing the risk of tumorigenesis and providing a safer method for producing iPS cells. This method is known to have extremely low efficiency of iPS cell production in conventional methods using wild-type KLF. In contrast, this embodiment uses mutant KLF of the present invention, making it possible to improve the efficiency of iPS cell production, providing a method for producing iPS cells that is both safe and efficient. The present invention also provides a method for producing iPS cells with high homogeneity and a method for producing iPS cells with low differentiation resistance.

[0124] 7. Cancer treatments 7-1. Overview The seventh aspect of the present invention is a cancer therapeutic agent. The cancer therapeutic agent of the present invention contains, as an active ingredient, any one of the mutant KLF protein of the first aspect or a peptide fragment thereof, the nucleic acid of the second aspect, or the gene expression vector of the third aspect (hereinafter collectively referred to as "mutant KLF of the present invention"). The cancer therapeutic agent of the present invention has a cancer therapeutic effect.

[0125] Regarding the cancer therapeutic effects of KLF4, it has been shown that the survival, invasion, and migration of papillary thyroid cancer are suppressed by KLF4 overexpression, that KLF4 suppresses the progression of prostate tumors, and that KLF4 overexpression can suppress chemotherapy resistance of colorectal cancer (Wang Q., et al., Exp Ther Med., 2019 Nov;18(5):3493-3501.; Oncogene. 2019 Jul;38(29):5766-5777.; Yadav SS, et al., Life Sci., 2019 Mar 1;220:169-176.).

[0126] 7-2.Configuration The cancer therapeutic agent of the present invention includes an active ingredient as an essential component, and a pharma- ceutically acceptable carrier or other drug as an optional component. The cancer therapeutic agent of the present invention may be composed of only the active ingredient. However, in order to facilitate the formation of a dosage form and to maintain the pharmacological effect and / or dosage form of the active ingredient, it is preferable that the agent is composed as a pharmaceutical composition containing a pharma- ceutically acceptable carrier, which will be described later.

[0127] 7-2-1. Components Each component constituting the cancer therapeutic agent of the present invention will now be specifically described.

[0128] (1) Active ingredient The active ingredient in the cancer therapeutic agent of the present invention is the mutant KLF of the present invention. The details of these components have already been described in the first to third aspects, so a detailed description thereof will be omitted here. In this aspect, it is preferable that the active ingredient is mutant KLF4, among the mutant KLFs of the present invention.

[0129] (2) A pharma- ceutically acceptable carrier The term "pharmaceutical acceptable carrier" refers to a solvent and / or additive that can be commonly used in the technical field of pharmaceutical formulations and that is little or not harmful to living organisms.

[0130] Examples of pharma- ceutically acceptable solvents include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. These are desirably sterilized, and preferably adjusted to be isotonic with blood as necessary.

[0131] Furthermore, examples of pharma- ceutically acceptable additives include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, and the like.

[0132] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, including, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrins, maltodextrins, starch, and cellulose), metal salts (e.g., sodium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, or high molecular weight polyethylene glycols (PEGs), Pluronic®, kaolin, silicic acid, or combinations thereof.

[0133] Examples of binders include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone.

[0134] Disintegrants include, for example, the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearate monoglyceride, or salts thereof.

[0135] Fillers include, for example, the sugars and / or calcium phosphate (eg, tricalcium phosphate or calcium hydrogen phosphate).

[0136] Examples of the emulsifier include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0137] Flow regulators and lubricants include, for example, silicates, talc, stearates or polyethylene glycol.

[0138] In addition to the above additives, the composition may contain, as necessary, flavoring agents, dissolution aids (solubilizers), suspending agents, diluents, surfactants, stabilizers, absorption promoters (e.g., quaternary ammonium salts, sodium lauryl sulfate), bulking agents, moisturizing agents (e.g., glycerin, starch), adsorbents (e.g., starch, lactose, kaolin, bentonite, colloidal silicic acid), disintegration inhibitors (e.g., sucrose, stearin, cocoa butter, hydrogenated oils), coating agents, coloring agents, preservatives, antioxidants, fragrances, flavorings, sweeteners, buffers, and the like.

[0139] (3) Other drugs The cancer therapeutic agent of the present invention may contain other drugs to the extent that the pharmacological effects of the above-mentioned active ingredient are not lost. Here, "other drugs" include drugs that have a cancer therapeutic effect through the same mechanism of action as the mutant KLF of the present invention, and drugs that have a cancer therapeutic effect through a mechanism of action different from that of the mutant KLF of the present invention. In addition, the other drugs may have a pharmacological effect unrelated to the cancer therapeutic effect. For example, gastric mucosa protective agents and antibiotics are included.

[0140] When the cancer therapeutic agent of the present invention is a combination preparation containing other drugs, it is convenient because a synergistic effect such as multifaceted cancer suppression can be expected.

[0141] 7-2-2. Dosage form The dosage form of the cancer therapeutic agent of the present invention is not particularly limited as long as it does not or is unlikely to inactivate the mutant KLF of the present invention, which is the active ingredient, and is capable of fully exerting its pharmacological effect in the body after administration.

[0142] Dosage forms can be classified into liquid dosage forms or solid dosage forms (including semi-solid dosage forms such as gels) depending on their form, and the cancer therapeutic agent of the present invention may be either of them. Furthermore, dosage forms can be roughly classified into oral dosage forms and parenteral dosage forms depending on the administration method, and either of them may be used.

[0143] Specific dosage forms include, for oral dosage forms, liquid dosage forms such as suspensions, emulsions, and syrups, and solid dosage forms such as powders (including powders, powders, and candy powders), granules, tablets, capsules, sublingual tablets, and troches. For parenteral dosage forms, liquid dosage forms such as injections, suspensions, emulsions, eye drops, and nasal drops, and solid dosage forms such as creams, ointments, plasters, patches, and suppositories. Preferred dosage forms are any of the oral dosage forms, or, for parenteral dosage forms, liquid dosage forms such as injections.

[0144] 7-2-3. Method of administration The cancer therapeutic agent of the present invention can be prepared by any method known in the art, so long as it is capable of administering an effective amount of the mutant KLF of the present invention, which is the active ingredient, to a living body for cancer treatment.

[0145] In this specification, the term "effective amount" refers to the amount of the active ingredient required to exert its function, i.e., the amount of the cancer therapeutic agent in the present invention required to treat cancer and that does not cause any or no harmful side effects to the living body to which it is applied. This effective amount may vary depending on conditions such as subject information, administration route, and number of administrations. "Subject" refers to an individual animal to which the cancer therapeutic agent of the present invention is applied. The preferred subject is a human. "Subject information" refers to various individual information of the subject, including, for example, the subject's age, weight, sex, general health condition, drug sensitivity, and the presence or absence of medications being taken. The effective amount and the dosage calculated based on it are determined by the judgment of a doctor or veterinarian depending on the information of each individual subject. When it is necessary to administer a large amount of the cancer therapeutic agent of the present invention to obtain a sufficient effect of cancer treatment, it can be administered in several divided doses to reduce the burden on the subject.

[0146] The method of administration of the cancer therapeutic agent of the present invention may be either systemic administration or local administration. Examples of systemic administration include intravascular injection such as intravenous injection and oral administration. Examples of local administration include local injection. The active ingredient of the cancer therapeutic agent of the present invention is composed of the mutant KLF of the present invention. Therefore, in the case of oral administration, it is preferable to take appropriate measures, such as using an appropriate DDS (drug delivery system) to protect the active ingredient from degradation by digestive enzymes.

[0147] As an example of a specific dosage, when administered to a human adult male (body weight 60 kg) suffering from cancer, the effective amount of the cancer therapeutic agent per day is usually in the range of 1 to 2000 mg, preferably 1 to 1000 mg, and more preferably 1 to 500 mg. When the cancer therapeutic agent of the present invention is administered to a subject, the effective dosage of the mutant KLF of the present invention is selected from the range of 0.001 mg to 1000 mg per kg of body weight per administration. Alternatively, a dosage of 0.01 to 100,000 mg / body per subject can be selected. However, the dosage is not limited to these dosages.

[0148] Effects The cancer therapeutic agent of the present invention has a cancer therapeutic effect. The cancer therapeutic agent of the present invention has a therapeutic effect against, for example, papillary thyroid cancer, prostate tumor, colon cancer, etc. More specifically, it is possible to highly efficiently inhibit the survival, invasion, and migration of papillary thyroid cancer, highly efficiently inhibit the progression of prostate tumor, and highly efficiently inhibit the resistance of colon cancer to chemotherapy. EXAMPLES

[0149] <Example 1: Search and identification of KLF4 mutants that induce somatic cell reprogramming with high efficiency> (the purpose) We will introduce mutations into amino acid residues in the wild-type KLF4 protein that can directly interact with DNA, and identify KLF4 mutants that induce somatic cell reprogramming with high efficiency.

[0150] (method) The protein structure database Protein Data Bank (PDB) has nine structures of the KLF4 protein C2H2 zinc finger domain (KLF4ZFD) complexed with DNA (accession numbers: 2WBS, 2WBU, 4M9E, 5KE6, 5KE7, 5KE8, 5KE9, 5KEA, and 5KEB). We identified 19 amino acid residues that directly interact with DNA in these crystal structures (Figure 1).

[0151] Next, 19 types of retroviral vectors were constructed for expressing KLF4 mutants in which each of the 19 amino acid residues in the human wild-type KLF4 protein consisting of the amino acid sequence shown in SEQ ID NO: 5 was replaced with alanine. Furthermore, retroviral vectors were prepared for expressing human wild-type OCT3 / 4 consisting of the amino acid sequence shown in SEQ ID NO: 13, human wild-type SOX2 consisting of the amino acid sequence shown in SEQ ID NO: 15, and human wild-type L-MYC consisting of the amino acid sequence shown in SEQ ID NO: 21.

[0152] Specifically, in pMXs-hKLF4 (Addgene plasmid #17219), a 3xFLAG tag for detecting protein expression was fused to the N-terminus of KLF4, which was used as a template for site-directed mutagenesis. The sequences of the primers used for adding the N-terminal 3xFLAG tag are shown in SEQ ID NOs: 26 to 27. For site-directed mutagenesis, PrimeSTAR Mutagenesis Basal Kit (TAKARA BIO INC) was used together with PrimeSTAR Max DNA Polymerase (TAKARA BIO INC) according to the manufacturer's protocol for a PCR thermal cycler (Applied Biosystems SimpliAmp). The specific primer pair used for site-directed mutagenesis was designed so that the 15 bp regions encompassing the substitution site overlapped each other. The PCR conditions were 98°C for 10 seconds (denaturation), 55°C for 15 seconds (annealing), and 72°C for 30 seconds (extension). The sequence after the introduction of the mutations was confirmed by sequencing using the pMX-s1811 (FW) and pMX-as3205 (RV) primers.

[0153] Retroviral vectors were prepared as follows: Plat-E cells (Cell Biolab Inc., RV-101) were cultured at 3.6 × 10 per 100 mm dish. 6 cells or 5.4 x 10 per well on a 6-well plate 5The next day, in the case of 100 mm dishes, 27 μl of FuGENE 6 transfection reagent (Promega INC, E2691) was used to separately transfect Plat-E cells with pMX-based retroviral vectors (9 μg each) for expressing human OCT4 (Addgene plasmid #17217), human SOX2 (Addgene plasmid #17218), human L-MYC (Addgene plasmid #26022), human wild-type KLF4 (pMXs-hKLF4, Addgene plasmid #17219), or KLF4 mutants. In the case of 6-well plates, 4.5 μl of FuGENE 6 transfection reagent and 1.5 μg of each retroviral vector were used. After 24 hours, the medium was replaced with 10 ml of DMEM containing 10% FBS (Biosera, FB-1365 / 500), P / S (Nacalai Tesque, 26253-84), and the cells were incubated for another 24 hours. The supernatants containing the retroviruses were collected in two batches at 48 and 72 hours after lipofection, and filtered through a 0.45 μm pore size cellulose acetate filter (As One, RJN1345NH). 0.5 ml of each supernatant was mixed in equal volumes (ratio 1:1:1:1), and polybrene (Nacalai Tesque, 12996-81) was added at a final concentration of 4 μg / ml for cell infection.

[0154] Next, mouse fetal fibroblasts were infected with the retroviral vector to induce reprogramming. Mouse fetal fibroblasts isolated by a known method from Nanog-GFP mice (RIKEN BioResource Research Center Experimental Animal Development Division, STOCK Tg(Nanog-GFP, Puro)1Yam, accession number RBRC02290) were used. Infection of Nanog-GFP mouse fetal fibroblasts with the retroviral vector was performed on 100,000 to 200,000 Nanog-GFP mouse fetal fibroblasts with a low passage number in a 6-well dish. Nanog-GFP mouse fetal fibroblasts were incubated with the prepared retroviral vector for 24 hours, and then returned to normal fibroblast culture medium (high glucose DMEM (Nacalai Tesque, 08458-16) supplemented with 10% FBS (Biosera, FB-1365 / 500), P / S (Nacalai Tesque, 26253-84)). The medium was changed every 2 days. Furthermore, on the 6th day, 10,000 cells were reseeded on SL10 feeder cells (REPROCELL USA Inc) and the next day, the medium was changed to mES complete medium (high glucose DMEM (Nacalai Tesque, 08458-16) supplemented with 15% FBS (Biosera, FB-1365 / 500), MEM non-essential amino acid solution (Nacalai Tesque, 06344-56), LIF (Wako Pure Chemical Industries, 129-05601), and P / S (Nacalai Tesque, 26253-84)). The cells were cultured for 25 days with the medium changed every 2 days.

[0155] (result) On day 25 after infection with the retroviral vector, the number of colonies exhibiting green fluorescence derived from Nanog-GFP (iPS cell marker) was counted.

[0156] As a result, many of the alanine substitution mutants formed a reduced number of Nanog-GFP-positive colonies compared to wild-type KLF4 (Fig. 2A). On the other hand, the KLF4(S500A) mutant and the KLF4(L507A) mutant showed a significant increase in the number of Nanog-GFP-positive colonies formed compared to wild-type KLF4 (Fig. 2A). Furthermore, the ratio of Nanog-GFP-positive colonies to the total number of colonies was increased in the KLF4(S500A) mutant and the KLF4(L507A) mutant compared to wild-type KLF4 (Fig. 2B).

[0157] The increase in the percentage of Nanog-GFP positive colonies means that the percentage of fully reprogrammed iPS cells has increased relative to partially reprogrammed iPS cells. Partially reprogrammed iPS cells have low differentiation potential and pluripotency. In contrast, fully reprogrammed iPS cells have high differentiation potential and pluripotency. Therefore, it was shown that by using the KLF4(S500A) mutant or KLF4(L507A) mutant for reprogramming induction, it is possible to produce high-quality iPS cells with higher efficiency and provide a population of iPS cells with high differentiation potential and high homogeneity.

[0158] In addition, the amino acid identity between the zinc finger domains of KLF1, KLF2, and KLF5 with respect to KLF4 is extremely high, at 85%, 93%, and 81%, respectively. Furthermore, when KLF1, KLF2, KLF4, and KLF5 are introduced into somatic cells together with other reprogramming factors (e.g., OCT3 / 4, SOX2, and C-MYC), they all have the activity of inducing somatic cell reprogramming. Therefore, mutant KLF1, mutant KLF2, and mutant KLF5 containing amino acid substitutions at positions corresponding to positions 500 and 507 of KLF4 (e.g., mutant KLF1 containing an L356A mutation, mutant KLF2 containing an L349A mutation, and mutant KLF5 containing an L450A mutation) are also likely to induce somatic cell reprogramming more efficiently than wild-type KLF1, KLF2, and KLF5, respectively.

[0159] <Example 2: Creation of iPS cells from normal human fibroblasts using KLF4 mutants> (the purpose) We will perform reprogramming induction in normal human fibroblasts using the KLF4(L507A) mutant, and analyze the expression of the pluripotent stem cell marker tumor-related antigen-1-60 (TRA-1-60) by flow cytometry at an early stage after reprogramming induction.

[0160] The pluripotent stem cell marker TRA-1-60 is a glycoprotein that is specifically expressed in human iPS cells and ES cells, but not in somatic cells. TRA-1-60-positive human cells obtained by inducing reprogramming with four reprogramming factors (OCT3 / 4, SOX2, KLF4, and c-MYC) are cells at an intermediate stage of reprogramming into high-quality iPS cells. TRA-1-60-positive cells have a gene expression profile similar to that of primitive streak-like mesendodermal (PSMN), and the PSMN-like state is known to be important for maturation in the later stages of reprogramming (Chan EM, et al., Nat Biotechnol. 2009 Nov;27(11):1033-7.). Therefore, by using TRA-1-60 as an expression marker, high-quality reprogrammed cells can be detected at the earliest stage.

[0161] (method) Normal human fibroblasts (NB1RGB cells) were cultured in Dulbecco's modified Eagle's medium (4.5 g / L glucose, Nacalai Tesque, 08458-16) containing 10% FBS (Biosera, FB-1365 / 500), P / S, penicillin (100 units / ml), and streptomycin (100 μg / ml) (Nacalai Tesque, 26253-84).

[0162] Reprogramming induction from normal human fibroblasts was performed as follows. Human OCT4 (Addgene plasmid #17217), human SOX2 (Addgene plasmid #17218), human L-MYC (Addgene plasmid #26022), human wild-type KLF4 (pMXs-hKLF4, Addgene plasmid #17219) or KLF4 mutant-containing pMX vectors were combined with pCMV-VSV-G viral envelope vector (RDB04392, RIKEN BRC lentiviral vector plasmid) at a ratio of 3:1 and transfected into Plat-GP (Cell Biolab INC, RV-103) cells using FuGENE 6 transfection reagent. The resulting viral supernatant was used to infect 10,000 low-passage NB1RGB cells overnight in a 6-well dish.

[0163] Flow cytometry analysis was performed 7 days after virus infection. After infection, the cells were washed with 1 ml PBS and incubated with Accutase (Nacalai Tesque, 12679-54) for approximately 10 minutes until the cells detached. The cells were resuspended in flow cytometry buffer (PBS containing 0.5% EDTA supplemented with 1% fetal bovine serum) and incubated with Alexa Fluor 488 mouse anti-human TRA-1-60 antibody (BD Pharmingen) and human TRA-1-85 / CD147 APC conjugate antibody (R&D systems) for 1 hour. After washing the cells, they were used for flow cytometry (Guava easyCyte Flow Cytometer).

[0164] The presence or absence of TRA-1-60 expression was determined based on the fluorescence intensity measured by flow cytometry, specifically, cells were considered TRA-1-60 positive when the fluorescence intensity was higher than that measured in the original fibroblast population.

[0165] (result) Flow cytometry showed that the KLF4(L507A) mutant produced more TRA-1-60 positive cells than the wild-type KLF4 on day 7 after infection (Fig. 3). That is, the positive rate of TRA1-60 expression on day 7 after infection was about 1.5% with the KLF4(L507A) mutant, which was about 5-fold higher than that with the wild-type KLF4 (Fig. 3).

[0166] These results demonstrated that the KLF4 mutant of the present invention increases the efficiency of reprogramming induction in normal human fibroblasts, and also that the KLF4 mutant of the present invention accelerates the reprogramming process at the early stage of reprogramming induction.

[0167] <Example 3: Generation of iPS cells from Nanog-GFP mouse embryonic fibroblasts using Sendai virus vector and KLF4 mutant> (the purpose) We will examine whether the KLF4 mutant of the present invention can increase the efficiency of reprogramming induction even when a highly safe, replication-deficient, persistently expressing Sendai virus vector (SeVdp vector) is used.

[0168] (method) In this Example 3, the expression level of KLF4 protein is controlled using a destabilizing domain (DD tag). That is, the stability of KLF4 protein with a DD tag added to the N-terminus side is controlled by adding a small molecule ligand, Shield1, to the medium (Nishimura K., et al., Stem Cell Reports. 2014 Nov 11;3(5):915-929.). When Shield1 is not added to the medium, the DD-tagged KLF4 protein is degraded. Conversely, when Shield1 is added to the medium, the degradation of the DD-tagged KLF4 protein is blocked.

[0169] A KLF4(L507A) mutant with a FLAG tag added to the C-terminus and a DD tag added to the N-terminus, and a SeVdp(fK[L507A]OSM) vector, which is a SeVdp vector for introducing other reprogramming factors, were prepared, and reprogramming induction was performed using the vector. The same experiment was also performed on wild-type KLF4 with a FLAG tag added to the C-terminus and a DD tag added to the N-terminus, and the KLF4(K483A) mutant, and the iPS cell production efficiency was compared. The KLF4(K483A) mutant is a mutant that did not increase the reprogramming efficiency of somatic cells in Example 1, and was used as a control group in this Example 3.

[0170] Nanog-GFP mouse embryonic fibroblasts (MEFs) at 125,000 cells / well were infected with SeVdp vector at 32°C for 24 hours to introduce four reprogramming factors (OCT3 / 4, SOX2, KLF4, and c-MYC). The day after infection, the SeV-containing medium was replaced with fibroblast culture medium (Dulbecco's modified Eagle medium (Sigma, D5796) containing 10% FBS (Gibco, 10437028), 100 U / ml penicillin-streptomycin (Nacalai, 26253-94)) supplemented with 100 nM Shield1 (Takara Bio, 632189) (Shield1 was added at each medium change for sustained expression of KLF4). On the third day, blasticidin S (Wako Pure Chemical Industries, 029-18701) (final concentration 5 μg / ml) was added to the medium. Then on the 7th day, 1.0 × 10 4 pieces, 5.0×10 3 pieces, 2.5×10 3Infected cells were replated on SL10 feeder cells in 6-well plates. Cells were cultured in mES medium (Sigma, D5796) supplemented with 15% fetal bovine serum (Gibco, 10437028), 0.1 mM non-essential amino acids (Nacalai, 06344-56), 55 μM 2-mercaptoethanol (Gibco, 21985023100), 100 U / ml penicillin-streptomycin (Nacalai, 26252-94), and 1,000 U / ml LIF (Wako, 125-05603) in the presence or absence of Shield1 for 25 days with medium changes every 2 days. iPS cell colonies expressing GFP were observed by fluorescence microscopy.

[0171] (result) When wild-type KLF4 was used, in the presence of Shield1, no Nanog-GFP-positive colonies were observed on day 11 after infection, and weakly fluorescent Nanog-GFP-positive colonies were first observed on day 23 after infection (Fig. 4, left column, day 23 after infection, Shield1 100 nM, arrowhead). In this case, the ratio of the number of Nanog-GFP-positive colonies to the total number of colonies on day 23 after virus infection was approximately 10%.

[0172] When the KLF4(K483A) mutant was used as a control, no Nanog-GFP-positive colonies were observed either on day 11 or day 23 after infection (FIG. 4, middle row).

[0173] On the other hand, when the KLF4(L507A) mutant was used, weakly fluorescent Nanog-GFP-positive colonies were observed from day 11 after infection in the presence of Shield1 (Fig. 4, right column, day 11 after infection, Shield1 100 nM, arrowhead), and many strongly fluorescent Nanog-GFP-positive colonies were observed by day 23 after infection (Fig. 4, right column, day 23 after infection, Shield1 100 nM, arrowhead). In this case, the ratio of the number of Nanog-GFP-positive colonies to the total number of colonies was more than 50% by day 23 after virus infection, which was significantly higher than that when wild-type KLF4 was used (approximately 10%).

[0174] In addition, when the KLF4(L507A) mutant was used, the induction of Nanog-GFP positive colonies was confirmed even in the absence of Shield1 (Figure 4, right column, 23 days after infection, Shield1 0 nM, arrowhead). The DD-tagged KLF4 protein was not completely degraded even in the absence of Shield1, with approximately 30% remaining (Nishimura K., et al., Stem Cell Reports. 2014 Nov 11;3(5):915-929.). Therefore, the Nanog-GFP positive colonies induced in the absence of Shield1 are thought to be due to the activity of the KLF4 protein that remained without being degraded. In contrast, when wild-type KLF4 or the KLF4(K483A) mutant was used, no Nanog-GFP positive colonies were observed in the absence of Shield1. Therefore, this result also showed that the KLF4(L507A) mutant has a significantly higher reprogramming induction activity than the wild-type KLF4.

[0175] These results indicate that the KLF4(L507A) mutant can increase reprogramming efficiency independent of gene delivery systems, and that it can be used in combination with the highly safe SeVdp vector.

[0176] <Example 4: Prediction of substitution mutations that stabilize the structure of KLF4 protein> (the purpose) When substituting the 507th leucine in the KLF4 protein, we will examine which amino acid residue should be substituted to stabilize the protein structure.

[0177] (Methods and Results) The free energy change (ΔΔG) when the 507th leucine (L507) in the KLF4 protein was replaced with various amino acid residues was calculated using Site Directed Mutator (http: / / marid.bioc.cam.ac.uk / sdm2 / prediction) (Worth CL, Preissner R, and Blundell TL, Nucleic Acids Res., 2011, May 18, 39:W215-22). The results are shown in Figure 5. As a result, it was predicted that the structure of the KLF4 protein would be more stabilized than that of the wild type when L507 was replaced with alanine (A), glutamic acid (E), glutamine (Q), or tryptophan (W) (Figure 5).

[0178] Among these, the KLF4 mutant having the L507A mutation increases the efficiency of somatic cell reprogramming induction as shown in Example 1. Therefore, other amino acid substitutions (L507E, L507Q, and L507W) predicted in this example to stabilize the structure of KLF4 are also likely to increase the efficiency of somatic cell reprogramming induction. Similarly, L356E, L356Q, or L356W of the KLF1 protein, L349E, L349Q, or L349W of the KLF2 protein, and L450E, L450Q, or L450W of the KLF5 protein, which correspond to L507E, L507Q, and L507 of the KLF4 protein, are also likely to increase the efficiency of somatic cell reprogramming induction.

[0179] <Example 5: Generation of iPS cells from human fibroblasts using Sendai virus vector and KLF4 mutant> (the purpose) We will generate iPS cells from human fibroblasts using Sendai virus vectors and KLF4 mutants, and evaluate the quality of the resulting iPS cells.

[0180] (method) Using the KLF4(L507A) mutant and wild-type KLF4, which were prepared in Example 3 and had a FLAG tag attached to the C-terminus and a DD tag attached to the N-terminus, four reprogramming factors (OCT3 / 4, SOX2, KLF4, and C-MYC) were introduced into human fibroblasts using the SeVdp vector, and iPS cells were generated under feeder-free culture conditions.

[0181] Normal human fibroblasts (100,000 NB1RGB cells) were infected with the SeVdp vector in a 12-well plate for 24 hours at 32°C. After infection, the cells were cultured in human ES cell medium prepared with Primate ES cell medium (Reprocell), 1× penicillin / streptomycin (Nacalai Tesque), and 10 ng / mL basic FGF (Wako) in the presence or absence of 100 nM Shield1. From day 3 to day 5 of culture, the cells were cultured and selected in the presence of 1 μg / mL Blasticidin S (Wako), and on day 11, they were replated in a 60 mm dish. Individual iPS cell colonies were cultured in 10 μM Y-27632 (Wako) and 0.25 μg / cm 2 They were cultured in StemFit AK02N medium (Ajinomoto) supplemented with iMatrix-511 (Nippi), transferred to a 24-well plate on day 22, and then transferred to a 60 mm dish on day 32. Cells were stored in STEM-CELLBANKER (Takara Bio, #CB045) until mRNA analysis and used for total RNA extraction as described below.

[0182] The expression of NANOG, residual Sendai virus NP (nucleocapsid protein), HERV-H, and lincRNA-RoR was quantified by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) for the above cells derived from individual colonies. HERV-H and lincRNA-RoR are known to be markers of iPS cells that are not easily induced to differentiate (differentiation resistance markers) (Ohnuki M., et al., Proc Natl Acad Sci US A. 2014;111(34):12426-12431.), and iPS cells with low expression levels of differentiation resistance markers are high quality iPS cells. RT-qPCR was performed as follows. Total RNA was extracted using Monarch Total RNA Miniprep Kit (New England BioLabs) or FastGene RNA premium kit (Nippon Genetics). Genomic DNA removal and reverse transcription were performed using ReverTra Ace qPCR RT kit (Toyobo). qPCR was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems) with THUNDERBIRD SYBR qPCR Mix (Toyobo) or THUNDERBIRD Probe qPCR Mix (Toyobo). All qPCRs were performed in triplicate.

[0183] (result) RNA samples were collected from individual iPS cell clones at passage 1. In the 13 iPS cell clones generated using wild-type KLF4, the NANOG expression level varied widely among the clones, with some clones expressing NANOG at less than half the level of HiPS-WTc11 (Coriell Institute, #GM25256; Kreitzer FR, et al., Am J Stem Cells, 2013, 2(2):119-31.), which is used as a standard human iPS cell line in the art (Figure 6A). In addition, 6 of the 13 clones generated using wild-type KLF4 showed abnormally high NANOG expression levels, with NP RNA levels more than 10,000 times higher than the standard human iPS cell line (HiPS-WTc11).

[0184] In contrast, 16 iPS cell clones generated using the KLF4(L507A) mutant exhibited NP RNA levels less than 1,000-fold lower than that of the standard human iPS cell line (HiPS-WTc11) and relatively homogeneous NANOG expression levels within a 2-fold range (Figure 6A).

[0185] Furthermore, some iPS cell clones generated using wild-type KLF4 showed high expression of the differentiation resistance markers HERV-H and / or lincRNA-RoR, whereas no such clones were observed among iPS cell clones generated using the KLF4(L507A) mutant (Figure 6B).

[0186] These results indicate that when the KLF4(L507A) mutant is introduced using the SeVdp vector to produce iPS cells, the amount of SeVdp vector remaining is low, and high-quality human iPS cell clones with high homogeneity can be stably produced without abnormally high expression of differentiation resistance markers.

[0187] <Example 6: Substitution of KLF4 L507 with Ala, Gln, Asp, Cys, Glu, Gly, Lys, Met, Ser, and Thr> (the purpose) We will create a KLF4 mutant in which the 507th leucine of KLF4 (KLF4 L507) is replaced with 10 types of amino acid residues including alanine, and examine the effect on somatic cell reprogramming efficiency.

[0188] (Methods and Results) KLF4 mutants (hereinafter referred to as L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, and L507T) were prepared by substituting KLF4 L507 with 10 kinds of amino acids, Ala, Gln, Asp, Cys, Glu, Gly, Lys, Met, Ser, and Thr. Retroviral vectors were prepared for each substitution mutant in the same manner as in Example 1, and introduced into Nanog-GFP mouse fetal fibroblasts together with OCT4, SOX2, and MCYCL1 in the same manner as in Example 1 to prepare iPS cells.

[0189] At an early stage after virus infection (day 15 after virus infection), more Nanog-GFP-positive iPS cell colonies were observed in L507A, L507N, L507D, L507C, L507G, L507K, L507M, and L507S than in wild-type KLF4 (Figure 7A). This result indicates that L507A, L507N, L507C, L507G, L507K, and L507S improved the reprogramming speed to iPS cells and allowed iPS cells to be obtained earlier. This effect was particularly remarkable in L507K.

[0190] On day 25 after viral infection, more GFP-positive iPS cell colonies were observed in L507A, L507N, L507D, L507C, L507G, and L507S compared to wild-type KLF4 (FIG. 7B). The most iPS cell colonies were observed in L507G. This result indicated that L507A, L507N, L507D, L507C, L507G, and L507S improved the efficiency of reprogramming into iPS cells.

[0191] Furthermore, the percentage of Nanog-GFP-positive iPS cell colonies relative to the total number of colonies was calculated on day 25 after virus infection, and the results are shown in FIG. 7C.

[0192] The above results demonstrate that L507A, L507N, L507D, L507C, L507G, and L507S improve both the reprogramming speed and efficiency into iPS cells.

[0193] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A mutant KLF protein comprising an amino acid substitution, The amino acid substitutions are (a) a serine at position 349 and / or a leucine at position 356 in the amino acid sequence shown in SEQ ID NO:1; (b) a serine at position 342 and / or a leucine at position 349 in the amino acid sequence shown in SEQ ID NO: 3; (c) serine at position 500 and / or leucine at position 507 in the amino acid sequence shown in SEQ ID NO:5, or (d) serine at position 443 and / or leucine at position 450 in the amino acid sequence shown in SEQ ID NO:

7. or the substitution in (a) is S349A, and / or L356A, L356N, L356D, L356C, L356E, L356G, L356K, L356M, L356S, or L356T; the substitution (b) is S342A, and / or L349A, L349N, L349D, L349C, L349E, L349G, L349K, L349M, L349S, or L349T; the substitution (c) is S500A, and / or L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, or L507T; the substitution in (d) is S443A, and / or L450A, L450N, L450D, L450C, L450E, L450G, L450K, L450M, L450S, or L450T; The mutant KLF protein is a polypeptide consisting of an amino acid sequence that has 90% or more amino acid identity to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 with respect to the full length sequence of the mutant KLF protein excluding the position of the amino acid substitution, and has an activity of inducing somatic cell reprogramming more efficiently than a wild-type KLF protein. The mutant KLF protein.

2. A nucleic acid encoding the mutant KLF protein of claim 1.

3. A gene expression vector comprising the nucleic acid according to claim 2 in an expressible state.

4. An induced pluripotent stem cell (iPS cell) inducer comprising any one of the mutant KLF protein according to claim 1, the nucleic acid according to claim 2, or the gene expression vector according to claim 3.

5. The iPS cell inducer according to claim 4 , further comprising the following (i) and / or (ii): (i) any one of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state; (ii) any one of SOX1 protein, SOX2 protein, SOX3 protein, SOX15 protein, and SOX17 protein, a nucleic acid encoding any one of them, and a gene expression vector containing the nucleic acid in an expressible state.

6. The iPS cell inducer according to claim 5 , further comprising the following (iii): (iii) any one of a C-MYC protein, a T58A mutant of a C-MYC protein, an N-MYC protein, or an L-MYC protein, a nucleic acid encoding any one of them, or a gene expression vector comprising the nucleic acid in an expressible state.

7. A direct reprogramming agent comprising any one of the mutant KLF protein of claim 1, the nucleic acid of claim 2, or the gene expression vector of claim 3.

8. Use of the iPS cell inducer according to any one of claims 4 to 6 for producing iPS cells from somatic cells.

9. A method for producing iPS cells, comprising the steps of: An introduction step of introducing an iPS cell-inducing agent into a somatic cell, the agent comprising the following (1) to (3): (1) Any one of the mutant KLF protein according to claim 1, the nucleic acid according to claim 2, and the gene expression vector according to claim 3. (2) Any one of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state, and (3) A nucleic acid encoding any one of SOX1 protein, SOX2 protein, SOX3 protein, SOX15 protein, or SOX17 protein, or a gene expression vector containing the nucleic acid in an expressible state; and a culturing step of culturing the somatic cells after the introduction step in the presence of any one or more of basic fibroblast growth factor, TGF-β1 protein, BMP protein, Wnt3 protein, GSK3β inhibitor, Wnt inhibitor, retinoic acid, ascorbic acid, and ROCK inhibitor. The manufacturing method comprising the steps of:

10. A method for producing iPS cells, comprising the steps of: An introduction step of introducing an iPS cell-inducing agent into a somatic cell, the agent comprising the following (1) to (4): (1) Any one of the mutant KLF protein according to claim 1, the nucleic acid according to claim 2, and the gene expression vector according to claim 3. (2) Any of an OCT3 / 4 protein, a nucleic acid encoding the same, or a gene expression vector containing the nucleic acid in an expressible state; (3) A nucleic acid encoding a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein, or a SOX17 protein, or any of them, or a gene expression vector containing the nucleic acid in an expressible state; and (4) A C-MYC protein, an N-MYC protein, an L-MYC protein, or a T58A mutant protein of a C-MYC protein, or a nucleic acid encoding any of them, or a gene expression vector containing the nucleic acid in an expressible state; and A culturing step of culturing the somatic cells after the introduction step. The manufacturing method comprising the steps of:

11. The method according to claim 9 or 10, further comprising a selection step of selecting iPS cells induced in the culture step.

12. The method according to any one of claims 9 to 11, wherein the somatic cells are of human origin.

13. 10. A cancer therapeutic agent comprising, as an active ingredient, the mutant KLF protein according to claim 1, the nucleic acid according to claim 2, or the gene expression vector according to claim 3.

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