JP6161261B2 - DNA demethylation induction method and use thereof - Google Patents

Description

  The present invention relates to a method for inducing DNA demethylation and uses thereof.

  Methylation of cytosine in the CpG sequence plays an extremely important role in gene expression control, and it is known that abnormal DNA methylation is a cause of various diseases such as cancer. Primordial germ cells that are the origin of sperm or eggs have the activity of demethylating DNA methylation of the entire genome in order to prevent totipotency acquisition and accumulation of abnormal methylation. Such genome-wide demethylation is a very unique phenomenon that occurs only in germline cells, suggesting the existence of a germline-specific DNA demethylation mechanism. However, details of such a DNA demethylation mechanism have not been clarified (Non-patent Document 1).

  In addition, a method for producing iPS cells from somatic cells using a nuclear reprogramming substance is known. Although iPS cells are expected to be applied to regenerative medicine, various problems such as instability of undifferentiated maintenance and heterogeneity of differentiation ability have been pointed out. Specifically, iPS cells are significantly different from ES cells in which methylation is normal (Non-Patent Document 2), and it is also known that this abnormality of DNA methylation adversely affects the quality of iPS cells. It has been. For example, it is known that the expression of the Dlk1-Dio3 gene cluster is suppressed due to abnormal methylation in many iPS cells. And, it has been pointed out that due to such suppression of expression, these iPS cells do not function sufficiently when a chimera or an animal derived only from iPS cells is created (Non-patent Document 3). ).

Science Vol. 324, 15 May, 2009, pp. 930-935 Nature Vol 471, 3 March, 2011, pp. 68-73 Nature Vol 465, 13 May, 2010, pp. 175-183

The subject of this invention is as follows.
(1) To provide a method for inducing DNA demethylation in cells.
(2) To provide iPS cells with improved quality.

  The present inventor has conducted extensive studies and found that DNA demethylation via TET1 activity is promoted by PRDM14 activity. Furthermore, the present inventor has found that even in a region that cannot be demethylated by TET1 activity alone, it can be demethylated by using PRDM14 activity together. Based on such new findings, the present inventor has found that DNA demethylation can be induced in cells by using a step of increasing the intracellular contents of TET1 and PRDM14.

  In addition to this, the present inventors have found that dedifferentiation into ES cells can be induced by expressing PRDM14 in the differentiated ES cells.

  Furthermore, the present inventor has found that an abnormality in DNA methylation known to adversely affect the quality of iPS cells can be normalized by the method for inducing DNA demethylation of the present invention. Based on such new findings, the present inventor provides iPS cells with improved quality by applying the DNA demethylation induction method to a method of producing iPS cells from somatic cells using a nuclear reprogramming substance. I found out that I can do it.

The present invention has been completed as a result of further studies based on such knowledge, and is described below.
[1. DNA demethylation induction method]
[1. A method of inducing DNA demethylation in a cell,
(1) A method comprising increasing the intracellular content of a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same.
[1.1.1] The cell is a human cell. The method described in 1.
[1.1.2] The cell is a mouse cell. The method described in 1.
[1.1.3] The cell is a cultured cell. -The method in any one of 1.1.2.
[1.1.3.1] The method according to 1.1.3, wherein the cultured cells are somatic cells.
[1.1.4] The cell is a cell in a living body. -The method in any one of 1.1.2.
[1.1.4.1] The method according to 1.1.4, wherein the cell in the living body is a cell having a DNA methylation abnormality that repairs an epigenome mutation.
[1.1.4.2] The method according to 1.1.4, wherein the cells in the living body are cancer cells.
[1.2.1] The step (1)
1. a step of introducing a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same into a cell; -The method in any one of 1.1.4.2.
[1.2.2] The step (1)
A step of introducing a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a polynucleotide encoding a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same into a cell; Yes, -The method in any one of 1.1.4.2.
[1.2.2.1] The step (1)
A step of overexpressing in a cell a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same. The method according to 1.2.2.
[1.3.1] A TET1 active domain variant, a fusion polypeptide comprising a TET1 active domain, and a fusion polypeptide comprising a TET1 active domain variant have TET1 activity. -The method in any one of 1.2.2.1.
[1.3.1.1] The method according to 1.3.1, wherein the TET1 activity is an activity of converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (hmC).
[1.4.1] A PRDM14 active domain variant, a fusion polypeptide comprising a PRDM14 active domain and a fusion polypeptide comprising a PRDM14 active domain variant have PRDM14 activity. -The method in any one of 1.3.1.1.
[1.4.1.1] The method according to 1.4.1, wherein the PRDM14 activity is an activity that promotes TET1 activity.
[1.5] A fusion polypeptide comprising a TET1 active domain or a variant thereof,
1. (i) a methylated cytosine binding domain; and (ii) an active domain consisting of a TET1 active domain or a variant thereof. -The method in any one of 1.4.1.1.
[1.6] A fusion polypeptide comprising PRDM14 or a variant thereof,
(I) a DNA binding domain that binds to a target gene region; and (ii) an active domain consisting of PRDM14 or a variant thereof. The method in any one of -1.5.
[1.6.1] The method according to 1.6, wherein the target gene region is a promoter region of the target gene.
[1.6.1.1] The method according to 1.6.1, wherein the target gene is a tumor suppressor gene.
[1.6.1.1.1.1] The method according to 1.6.1.1, wherein the tumor suppressor gene is p16, p53, or E-cadherin.
[1.7.1] The TET1 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. -The method in any one of 1.6.1.1.1.
[1.7.1.1] The method according to 1.7.1, wherein the variant of the TET1 active domain consists of a polypeptide having an identity of 80% or more with the polypeptide according to 1.7.1.
[1.7.1.2] The method according to 1.7.1, wherein the variant of the TET1 active domain is composed of a polypeptide having 85% or more identity with the polypeptide according to 1.7.1.
[1.7.1.3] The method according to 1.7.1, wherein the variant of the TET1 active domain consists of a polypeptide having 90% or more identity with the polypeptide according to 1.7.1.
[1.7.1.4] The method according to 1.7.1, wherein the variant of the TET1 active domain comprises a polypeptide having 95% or more identity with the polypeptide according to 1.7.1.
[1.7.1.5] A modified TET1 active domain consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.7.1. The method according to 7.1.
[1.7.2] The TET1 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. -The method in any one of 1.6.1.1.1.
[1.7.2.1] The method according to 1.7.2, wherein the modified TET1 active domain comprises a polypeptide having an identity of 80% or more with the polypeptide according to 1.7.2.
[1.7.2.2] The method according to 1.7.2, wherein the variant of the TET1 active domain is composed of a polypeptide having an identity of 85% or more with the polypeptide according to 1.7.2.
[1.7.2.3] The method according to 1.7.2, wherein the variant of the TET1 active domain comprises a polypeptide having 90% or more identity with the polypeptide according to 1.7.2.
[1.7.2.4] The method according to 1.7.2, wherein the variant of the TET1 active domain is composed of a polypeptide having an identity of 95% or more with the polypeptide according to 1.7.2.
[1.7.2.5] A variant of the TET1 active domain consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.7.2. The method according to 7.2.
[1.7.3] 1. The TET1 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3. -The method in any one of 1.6.1.1.1.
[1.7.3.1] The method according to 1.7.3, wherein the variant of the TET1 active domain is composed of a polypeptide having an identity of 80% or more with the polypeptide according to 1.7.3.
[1.7.3.2] The method according to 1.7.3, wherein the modified TET1 active domain comprises a polypeptide having an identity of 85% or more with the polypeptide according to 1.7.3.
[1.7.3.3] The method according to 1.7.3, wherein the variant of the TET1 active domain consists of a polypeptide having 90% or more identity with the polypeptide according to 1.7.3.
[1.7.3.4] The method according to 1.7.3, wherein the modified TET1 active domain is composed of a polypeptide having 95% or more identity with the polypeptide according to 1.7.3.
[1.7.3.5] A modified TET1 active domain consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.7.3. The method according to 7.3.
[1.8.1] The PRDM14 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4. -The method in any one of 1.7.3.5.
[1.8.1.1] The method according to 1.8.1, wherein the variant of the PRDM14 active domain consists of a polypeptide having an identity of 80% or more with the polypeptide according to 1.8.1.
[1.8.1.2] The method according to 1.8.1, wherein the variant of the PRDM14 active domain comprises a polypeptide having 85% or more identity with the polypeptide according to 1.8.1.
[1.8.1.3] The method according to 1.8.1, wherein the variant of the PRDM14 active domain comprises a polypeptide having 90% or more identity with the polypeptide according to 1.8.1.
[1.8.1.4] The method according to 1.8.1, wherein the variant of the PRDM14 active domain comprises a polypeptide having 95% or more identity with the polypeptide according to 1.8.1.
[1.8.1.5] A PRDM14 active domain variant consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence described in 1.8.1. The method according to 8.1.
[1.8.2] The PRDM14 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 5. -The method in any one of 1.7.3.5.
[1.8.2.1] The method according to 1.8.2, wherein the variant of the PRDM14 active domain consists of a polypeptide having an identity of 80% or more with the polypeptide according to 1.8.2.
[1.8.2.2] The method according to 1.8.2, wherein the variant of the PRDM14 active domain consists of a polypeptide having 85% or more identity with the polypeptide according to 1.8.2.
[1.8.2.3] The method according to 1.8.2, wherein the variant of the PRDM14 active domain consists of a polypeptide having 90% or more identity with the polypeptide according to 1.8.2.
[1.8.2.4] The method according to 1.8.2, wherein the variant of the PRDM14 active domain comprises a polypeptide having 95% or more identity with the polypeptide according to 1.8.2.
[1.8.2.5] A modified PRDM14 active domain consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.8.2. The method according to 8.2.
[1.8.3] The PRDM14 active domain is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6. -The method in any one of 1.7.3.5.
[1.8.3.1] The method according to 1.8.3, wherein the variant of the PRDM14 active domain comprises a polypeptide having 80% or more identity with the polypeptide according to 1.8.3.
[1.8.3.2] The method according to 1.8.3, wherein the variant of the PRDM14 active domain consists of a polypeptide having 85% or more identity with the polypeptide according to 1.8.3.
[1.8.3.3] The method according to 1.8.3, wherein the PRDM14 active domain variant comprises a polypeptide having 90% or more identity with the polypeptide according to 1.8.3.
[1.8.3.4] The method according to 1.8.3, wherein the variant of the PRDM14 active domain comprises a polypeptide having 95% or more identity with the polypeptide according to 1.8.3.
[1.8.3.5] The PRDM14 active domain variant consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.8.3. The method according to 8.3.
[1.9] Fusion polypeptides comprising a TET1 active domain or a variant thereof are TET1 and a variant thereof, respectively. The method in any one of -1.6.1.1.1 and 1.8.1-1.8.3.5.
[1.9.1] The method according to 1.9, wherein TET1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 7.
[1.9.1.1] The method according to 1.9.1, wherein the variant of TET1 is composed of a polypeptide having an identity of 80% or more with the polypeptide according to 1.9.1.
[1.9.1.2] The method according to 1.9.1, wherein the variant of TET1 is composed of a polypeptide having 85% or more identity with the polypeptide according to 1.9.1.
[1.9.1.3] The method according to 1.9.1, wherein the variant of TET1 comprises a polypeptide having 90% or more identity with the polypeptide according to 1.9.1.
[1.9.1.4] The method according to 1.9.1, wherein the variant of TET1 is composed of a polypeptide having an identity of 95% or more with the polypeptide according to 1.9.1.
[1.9.1.5] A variant of TET1 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.9.1. The method according to 1.
[1.9.2] The method according to 1.9, wherein TET1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8.
[1.9.2.1] The method according to 1.9.2, wherein the variant of TET1 is composed of a polypeptide having 80% or more identity with the polypeptide according to 1.9.2.
[1.9.2.2] The method according to 1.9.2, wherein the variant of TET1 is composed of a polypeptide having 85% or more identity with the polypeptide according to 1.9.2.
[1.9.2.3] The method according to 1.9.2, wherein the variant of TET1 is composed of a polypeptide having 90% or more identity with the polypeptide according to 1.9.2.
[1.9.2.4] The method according to 1.9.2, wherein the variant of TET1 comprises a polypeptide having 95% or more identity with the polypeptide according to 1.9.2.
[1.9.2.5] A variant of TET1 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.9.2. 2. The method according to 2.
[1.9.3] The method according to 1.9, wherein TET1 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
[1.9.3.1] The method according to 1.9.3, wherein the variant of TET1 comprises a polypeptide having 80% or more identity with the polypeptide according to 1.9.3.
[1.9.3.2] The method according to 1.9.3, wherein the variant of TET1 comprises a polypeptide having 85% or more identity with the polypeptide according to 1.9.3.
[1.9.3.3] The method according to 1.9.3, wherein the variant of TET1 is composed of a polypeptide having 90% or more identity with the polypeptide according to 1.9.3.
[1.9.3.4] The method according to 1.9.3, wherein the variant of TET1 comprises a polypeptide having 95% or more identity with the polypeptide according to 1.9.3.
[1.9.3.5] A variant of TET1 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.9.3. 3. The method according to 3.
[1.10] The fusion polypeptides comprising a PRDM14 active domain or a variant thereof are PRDM14 and a variant thereof, respectively. The method in any one of -1.7.3.5 and 1.9-1.9.3.5.
[1.10.1] The method according to 1.10, wherein PRDM14 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10.
[1.10.1.1.1] The method according to 1.10.1, wherein the variant of PRDM14 comprises a polypeptide having an identity of 80% or more with the polypeptide according to 1.10.1.
[1.10.1.2] The method according to 1.10.1, wherein the variant of PRDM14 comprises a polypeptide having 85% or more identity with the polypeptide according to 1.10.1.
[1.10.1.3] The method according to 1.10.1, wherein the variant of PRDM14 comprises a polypeptide having 90% or more identity with the polypeptide according to 1.10.1.
[1.10.1.4] The method according to 1.10.1, wherein the variant of PRDM14 comprises a polypeptide having 95% or more identity with the polypeptide according to 1.10.1.
[1.10.1.5] A variant of PRDM14 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.10.1. The method according to 1.
[1.10.2] The method according to 1.10, wherein PRDM14 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11.
[1.10.2.2.1] The method according to 1.10.2, wherein the variant of PRDM14 comprises a polypeptide having an identity of 80% or more with the polypeptide according to 1.10.2.
[1.10.2.2] The method according to 1.10.2, wherein the variant of PRDM14 comprises a polypeptide having 85% or more identity with the polypeptide according to 1.10.2.
[1.10.2.3] The method according to 1.10.2, wherein the variant of PRDM14 comprises a polypeptide having 90% or more identity with the polypeptide according to 1.10.2.
[1.10.2.4] The method according to 1.10.2, wherein the variant of PRDM14 comprises a polypeptide having 95% or more identity with the polypeptide according to 1.10.2.
[1.10.2.5] A variant of PRDM14 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.10.2. 2. The method according to 2.
[1.10.3] The method according to 1.10, wherein PRDM14 is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 12.
[1.10.3.3.1] The method according to 1.10.3, wherein the variant of PRDM14 consists of a polypeptide having an identity of 80% or more with the polypeptide according to 1.10.3.
[1.10.3.2] The method according to 1.10.3, wherein the variant of PRDM14 comprises a polypeptide having 85% or more identity with the polypeptide according to 1.10.3.
[1.10.3.3] The method according to 1.10.3, wherein the variant of PRDM14 comprises a polypeptide having 90% or more identity with the polypeptide according to 1.10.3.
[1.10.3.4] The method according to 1.10.3, wherein the variant of PRDM14 comprises a polypeptide having 95% or more identity with the polypeptide according to 1.10.3.
[1.10.3.5] A variant of PRDM14 consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in 1.10.3. 3. The method according to 3.

[2. Method of treatment]
[2. ] A method of treating a human,
(1) A method comprising increasing the intracellular content of a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same.
[2.1.1] A method for treating diseases caused by abnormal DNA methylation that repairs epigenomic mutations. The method described in 1.
[2.1.1.1] The method according to 2.1.1, which is a method for treating cancer.
[2.1.1.2] The method according to 2.1.1, which is a method for treating a neuropsychiatric disorder.
[2.1.1.2.1] The method according to 2.1.1.2, wherein the neuropsychiatric disorder is manic depression or repetitive depression.
[2.2] The step (1) is the step (1) according to any one of 1.1.1 to 1.10.3.5. -The method in any one of 2.1.1.2.1.

[3. iPS Cell Production Method]
[3A. A method for producing iPS cells from somatic cells using a nuclear reprogramming substance,
(1) A method comprising increasing the intracellular content of a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same.
[3A. 1.1] The somatic cell is a human cell, 3A. The method described in 1.
[3A. 1.2] The somatic cell is a mouse cell, 3A. The method described in 1.
[3A. 1.3] The somatic cell is a canine cell, 3A. The method described in 1.
[3A. 2] The process (1) is the process (1) according to any one of 1.1.1 to 1.10.3.5, 3A. ~ 3A. The method according to any of 1.3.
[3A. 3] The step (1) is performed on somatic cells. 3. The method according to any one of 2.
[3A. 3.1] The step (1)
(1A-1) TET1 active domain or a variant thereof, or a fusion polypeptide containing the same; and PRDM14 active domain or a variant thereof, or a fusion polypeptide containing the same, increasing the intracellular content and nuclear reprogramming A step of converting somatic cells into iPS cells by bringing a substance into contact with the somatic cells; 3A. 3. The method according to 3.
[3A. 3.2] The step (1)
(1A-1) TET1 active domain or a variant thereof, or a fusion polypeptide containing the same; and PRDM14 active domain or a variant thereof, or a fusion polypeptide containing the same, increasing the intracellular content and nuclear reprogramming A step of converting a somatic cell into an iPS cell having a high degree of completion of completion by bringing the substance into contact with the somatic cell; 3. The method according to 3.
[3A. 4] (I) A step of converting somatic cells into high-quality iPS cells, low-quality iPS cells, and pre-iPS cells by bringing a nuclear reprogramming substance into contact with the somatic cells;
(II) evaluating the cells obtained in step (I) and selecting high-quality iPS cells, low-quality iPS cells and pre-iPS cells; and (III) low-quality selected in step (II) including the step of performing step (1) on iPS cells and pre-iPS cells; ~ 3A. 3. The method according to any one of 2.
[3A. 4.1] The step (1)
(1A-2) Intracellular in low-quality iPS cells and pre-iPS cells of TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same A step of increasing the content and contacting the nuclear reprogramming substance with low-quality iPS cells and pre-iPS cells, 3A. 4. The method according to 4.
[3A. 4.2] In the step (I), 3A. The process (1A-1) described in 3.1 is 3A. 4. The method according to 4.
[3A. 4.3] selecting the low quality iPS cells by comparing the DNA expression or DNA methylation of the GTL2 gene with those of ES cells, and evaluating the endogenous NANOG gene Performed by a method comprising selecting pre-iPS cells by comparing expression with ES cells 3A. 4-3A. The method according to any one of 4.2.
[3B. ] The method of obtaining an iPS cell by performing a process (1) with respect to a pre-iPS cell.
[3B. 1] The step (1)
(1B) Increasing the pre-iPS intracellular content and nuclear reprogramming of the TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and the PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same A step of contacting the substance with pre-iPS cells, 3B. The method described in 1.
[3C. ] The method of obtaining a high quality iPS cell by performing a process (1) with respect to a low quality iPS cell.
[3C. 1] The step (1)
(1C) increasing the low-quality iPS intracellular content and nuclear reprogramming of the TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and the PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same Contacting the substance with low quality iPS cells, 3C. The method described in 1.
[3.1] The nuclear reprogramming substance is at least one selected from the group consisting of OCT4, SOX2, KLF4, and MYC. ~ 3C. The method according to any one of 1.
[3.2] The nuclear reprogramming substance is at least one selected from the group consisting of OCT4, SOX2, and KLF4. ~ 3C. The method according to any one of 1.
[3.3] The nuclear reprogramming substance is at least one selected from the group consisting of OCT4 and SOX2. ~ 3C. The method according to any one of 1.
[3.4] The nuclear reprogramming substance is OCT4. 3A. ~ 3C. The method according to any one of 1.

[4. Polypeptide-containing composition]
[4. ] (A-1) A composition comprising a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (A-2) a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same.
[4.1] The TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. A composition according to 1.
[4.2] The PRDM14 active domain or a variant thereof, or a fusion polypeptide containing the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. Or the composition according to 4.1.
[4.3.1] The composition according to any one of 4 to 4.2, which is used as a DNA demethylation inducer.
[4.3.2] The composition according to any one of 4 to 4.2, which is used as a medicine.
[4.3.2.1] The composition according to 4.3.2, which is used as a therapeutic agent for a disease caused by abnormal DNA methylation that repairs an epigenomic mutation.
[4.3.2.2] The composition according to 4.3.2, which is used as an anticancer agent.
[4.3.2.3] The composition according to 4.3.2, which is used as a therapeutic agent for a neuropsychiatric disorder.
[4.3.2.4] The composition according to 4.3.2, which is used as a therapeutic agent for manic depression or a therapeutic agent for repeated depression.
[4.3.3] The composition according to any one of 4 to 4.2, which is used as an agent for improving iPS cell quality.
[4.3.4] The composition according to any one of 4 to 4.2, which is used as an agent for improving reprogramming completion of iPS cells.

[5. Polynucleotide-containing composition]
[5. (Α-1) a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (α-2) a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same. A composition containing a polynucleotide.
[5.1] The TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. A composition according to 1.
[5.2] The PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. Or the composition according to 5.1.
[5.3.1] The composition according to any one of 5 to 5.2, wherein the polynucleotide is DNA.
[5.3.2] The composition according to any one of 5 to 5.2, wherein the polynucleotide is RNA.
[5.4.1] The composition according to any one of 5 to 5.3.2, which is used as a DNA demethylation inducer.
[5.4.2] The composition according to any one of 5 to 5.3.2, which is used as a medicine.
[5.4.2.1] The composition according to 5.4.2, which is used as a therapeutic agent for a disease caused by abnormal DNA methylation that repairs an epigenomic mutation.
[5.4.2.2] The composition according to 5.4.2, which is used as an anticancer agent.
[5.4.2.3] The composition according to 5.4.2, which is used as a therapeutic agent for neuropsychiatric disorders.
[5.4.2.4] The composition according to 5.4.2, which is used as a therapeutic agent for manic depression or a therapeutic agent for repeated depression.
[5.4.3] The composition according to any one of 5 to 5.3.2, which is used as an agent for improving iPS cell quality.
[5.4.4] The composition according to any one of 5 to 5.3.2, which is used as an agent for improving the completion degree of iPS cell reprogramming.

[6. Expression vector-containing composition]
[6. (A-1) an expression vector comprising a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (a-2) a PRDM14 active domain or a variant thereof, or a fusion comprising the same. A composition comprising an expression vector comprising a polynucleotide encoding a polypeptide.
[6.1] The TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. A composition according to 1.
[6.2] The PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.2.5. Or the composition according to 6.1.
[6.3.1] The composition according to any one of 6 to 6.2, wherein the polynucleotide is DNA.
[6.3.2] The composition according to any one of 6 to 6.2, wherein the polynucleotide is RNA.
[6.4.1] The composition according to any one of 6 to 6.3.2, which is used as a DNA demethylation inducer.
[6.4.2] The composition according to any one of 6 to 6.3.2, which is used as a medicine.
[6.4.2.1] The composition according to 6.4.2, which is used as a therapeutic agent for a disease caused by abnormal DNA methylation that repairs an epigenomic mutation.
[6.4.2.2] The composition according to 6.4.2, which is used as an anticancer agent.
[6.4.2.3] The composition according to 6.4.2, which is used as a therapeutic agent for neuropsychiatric disorders.
[6.4.2.4] The composition according to 6.4.2, which is used as a therapeutic agent for bipolar disorder (manic depression), a therapeutic agent for repeated depression, or schizophrenia.
[6.4.3] The composition according to any one of 6 to 6.3.2, which is used as an agent for improving iPS cell quality.
[6.4.4] The composition according to any one of 6 to 6.3.2, which is used as an agent for improving reprogramming completion of iPS cells.

[7. Co-expression vector]
[7. (B) a co-expression comprising a TET1 active domain or a variant thereof, or a polynucleotide encoding a fusion polypeptide comprising the same; and a polynucleotide encoding a PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same vector.
[7.1] The TET1 active domain or a variant thereof, or a fusion polypeptide comprising the TET1 active domain is the polypeptide according to any one of 1.3.1 to 1.10.3.5. A co-expression vector according to 1.
[7.2] The PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same is the polypeptide according to any one of 1.3.1 to 1.10.3.5. Or the co-expression vector according to 7.1.
[7.3.1] The co-expression vector according to any one of 7 to 7.2, wherein the polynucleotide is DNA.
[7.3.2] The co-expression vector according to any one of 7 to 7.2, wherein the polynucleotide is RNA.
[7.4.1] The co-expression vector according to any one of 7 to 7.3.2, which is used as a DNA demethylation inducer.
[7.4.2] The co-expression vector according to any one of 7 to 7.3.2, which is used as a medicine.
[7.4.2.1] The co-expression vector according to 7.4.2, which is used as a therapeutic agent for a disease caused by abnormal DNA methylation that repairs an epigenomic mutation.
[7.4.2.2] The co-expression vector according to 7.4.2, which is used as an anticancer agent.
[7.4.2.3] The co-expression vector according to 7.4.2, which is used as a therapeutic agent for neuropsychiatric disorders.
[7.4.2.4] The co-expression vector according to 7.4.2, which is used as a therapeutic agent for manic depression or a therapeutic agent for repeated depression.
[7.4.3] The co-expression vector according to any one of 7 to 7.3.2, which is used as an agent for improving iPS cell quality.
[7.4.4] The co-expression vector according to any one of 7 to 7.3.2, which is used as an agent for improving the reprogramming completion of iPS cells.

  By utilizing the method for inducing DNA demethylation of the present invention, DNA demethylation can be artificially performed. Further, by applying this method for inducing DNA demethylation to a method for producing iPS cells, iPS cells with improved quality can be obtained.

Example 1. It is drawing which shows the result. The upper diagram shows LINE-1. Example 2 It is a Venn diagram of a gene in which increased gene expression was observed in PRDM14 highly expressing ES cells and Dnmts TKO ES cells. 1 is a drawing showing a DNA demethylation pathway triggered by hydroxylation of methylated cytosine. Example 3 FIG. It is drawing which shows the result. Example 4 It is drawing which shows the result. In the process of establishing iPS cells by introducing Yamanaka Factor (OCT4, SOX2, KLF4, MYC: OSKM) into fibroblasts, pathways through which pre-iPS cells and low-quality iPS cells with incomplete reprogramming appear FIG. Example 5 FIG. It is drawing which shows the result. Example 6 It is drawing which shows the result. The vertical axis represents the methylation amount of the CCGG sequence in the LINE-1 region, and the horizontal axis represents each cell name. Example 7 It is drawing which shows the result. Example 7 It is drawing which shows the result. Example 7 It is drawing which shows the result. Example 8 FIG. It is drawing which shows the result. Example 9 It is drawing which shows the result. Example 9 It is drawing which shows the result. Example 9 It is drawing which shows the result.

[1. DNA demethylation induction method]
The method for inducing DNA demethylation of the present invention is a method for inducing DNA demethylation in a cell,
(1) a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same (hereinafter sometimes referred to as “TET1 active domain”); and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same (Hereafter, it may be called "PRDM14 active domain etc.".)
A method comprising the step of increasing the intracellular content of

  Although the origin of a cell is not specifically limited, For example, a human cell, a mouse cell, a canine cell etc. are mentioned.

  The cells may be cultured cells or in vivo cells.

  The cultured cells may be somatic cells or undifferentiated cells such as stem cells.

  The somatic cells are not particularly limited, but for example, keratinized epithelial cells, wet and multilayered epithelial cells (mucosal epithelial cells), epithelial cells specialized for exocrine glands, for hormone secretion Specialized cells, gastrointestinal tract, exocrine gland and urogenital tract resorption epithelial cells, cells specialized for metabolism and storage, mainly constituting the interface, within the lungs, gastrointestinal tract, exocrine gland and urogenital tract Epithelial cells facing the cavity, epithelial cells forming the inner surface of a closed lumen in the body, cells with cilia having a transport function, cells specialized for secretion of extracellular matrix, contractile cells, blood cells , Immune cells, cells involved in sensation, neurons of autonomic nervous system, supporting cells of sensory organs and peripheral neurons, neurons and glial cells of central nervous system, lens (lens) cells, pigment cells, and nursing cells Be

  The keratinized epithelial cells are not particularly limited, and examples thereof include epidermal keratinocytes, nail keratinocytes, hair shaft cells, and root sheath cells.

  The epithelial cells (mucosal epithelial cells) that are moist and have a multi-layer structure and constitute the boundary surface are not particularly limited. And epithelial cells such as urethra and vagina).

  Examples of epithelial cells specialized for exocrine glands include, but are not limited to, salivary gland cells, von Ebner gland cells of the tongue, mammary cells, lacrimal gland cells, otic gland cells, eccrine sweat gland cells, apocrine sweat gland cells, and the like.

  The cells specialized for hormone secretion are not particularly limited. For example, anterior pituitary cells, pituitary midlobe cells, posterior pituitary cells, gastrointestinal and respiratory tract cells, thyroid cells, parathyroid glands ( Parathyroid cells), adrenal cells, gonadal cells, kidney paraglomerular cells, and the like.

  Absorption epithelial cells of the digestive tract, exocrine glands and urogenital tract are not particularly limited, and examples thereof include intestinal brush border cells, striatal cells of exocrine glands, and gallbladder epithelial cells.

  The cells specialized for metabolism and storage are not particularly limited, and examples thereof include hepatocytes and adipocytes.

  Epithelial cells that mainly constitute the boundary surface and face the lumen of the lungs, gastrointestinal tract, exocrine gland and urogenital tract are not particularly limited, but examples include type I alveolar cells, pancreatic duct cells, acinar center Examples thereof include duct cells such as cells, sweat glands, salivary glands and mammary glands, renal glomerular wall cells, renal glomerular octopus podocytes, and the like.

  The epithelial cells forming the inner surface of the closed lumen in the body are not particularly limited, and examples thereof include endothelial cells of blood vessels and lymph vessels, synovial cells, serosa cells and the like.

  The cilia cells having a transport function are not particularly limited, and examples thereof include airway epithelial cells, oviduct and endometrial epithelial cells, and central nervous system cells.

  The cells specialized for extracellular matrix secretion are not particularly limited. For example, enamel cells, fibroblasts, pericytes of capillaries, nucleus pulposus cells of intervertebral discs, cementoblasts, odontoblasts, cartilage Examples thereof include cells, osteoblasts, and eye glass-forming cells.

  Although it does not specifically limit as a contractile cell, For example, a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell, a myoepithelial cell etc. are mentioned.

  The blood cells are not particularly limited, and examples thereof include erythrocytes, megakaryocytes, macrophages and related cells, neutrophils, eosinophils, and basophils.

  Although it does not specifically limit as an immune cell, For example, a mast cell (mast cell), T lymphocyte (T cell), B lymphocyte, K (killer) cell etc. are mentioned.

  The cells involved in the sensory are not particularly limited, but for example, rod cells, cone cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, type II taste bud cells, olfactory neurons, olfactory epithelium Basal cells, carotid body cells, epidermal Merkel cells, primary sensory neurons specialized for tactile sensation, primary sensory neurons specialized for temperature sensation, primary sensory neurons specialized for pain sensation, deep primary sensory neurons Etc.

  Supporting cells for sensory organs and peripheral neurons are not particularly limited, but include, for example, Corti organ supporting cells, vestibular supporting cells, miso supporting cells, olfactory epithelial supporting cells, Schwann cells, associated cells, intestinal glial cells Etc.

  T cells are preferably used as somatic cells. The T cell may be CD4 positive or CD8 positive, or both CD4 / CD8 positive. Although T cell is not specifically limited, For example, it can isolate by a well-known method from a spleen, a lymph node, peripheral blood, or cord blood. Although it does not specifically limit as a well-known isolation method, For example, the method etc. which isolate by flow cytometry using the antibody with respect to cell surface markers, such as CD4, CD8, and CD3, using a cell sorter etc. are mentioned.

  Somatic cells usually do not express TET1. The present inventor has found that DNA demethylation via TET1 activity is promoted by PRDM14 activity. Based on such novel findings, the present inventors have found that DNA demethylation is significantly promoted by simultaneously increasing the intracellular contents of TET1 active domain and the like and PRDM14 active domain in somatic cells.

  The cells in the living body may be normal cells or abnormal cells. Abnormal cells are not particularly limited, and examples include cells having abnormal DNA methylation. By inducing DNA demethylation by applying the present invention to such cells, DNA methylation can be brought close to a normal state. The cells having abnormal DNA methylation are not particularly limited, and examples thereof include cancer cells, cells of neuropsychiatric diseases, cells having abnormal DNA methylation for repairing epigenomic mutations, and the like.

Specific examples of the step (1) include the following steps.
(1A) a step of introducing a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same into a cell.
(1B) a polynucleotide encoding a TET1 active domain or a variant thereof or a fusion polypeptide comprising the same; and a polynucleotide encoding a PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same is introduced into the cell Process.

As a specific example of the step (1B), for example,
(1b) a TET1 active domain or a variant thereof, or a polynucleotide encoding a fusion polypeptide containing the same; and a PRDM14 active domain or a variant thereof or a polynucleotide encoding a fusion polypeptide containing the excess in the cell The step of expressing is mentioned.

In step (1A), described later. Polypeptide-containing compositions can be used.
In step (1B), 5. 5. a polynucleotide-containing composition, 6. an expression vector-containing composition, and Co-expression vectors can be used. In step (1b), 6. 6. an expression vector-containing composition, and Co-expression vectors can be used.

  The overexpression of the polynucleotide may be transient or constitutive. When transiently overexpressing, although not particularly limited, CMV promoter and CAGGS promoter can be used as an expression system. In the case of constantly overexpressing, although not particularly limited, CMV promoter and CAGGS promoter can be used as an expression system.

  TET1 is a protein discovered as a fusion partner of H3K4 methylase MLL (mixed lineage leukemia) in acute myelogenous leukemia. TET1 is not particularly limited, and examples thereof include those derived from humans, mice, dogs, and rats. Examples of human-derived TET1 include a polypeptide having the amino acid sequence represented by SEQ ID NO: 7. Examples of mouse-derived TET1 include a polypeptide having the amino acid sequence represented by SEQ ID NO: 8. Examples of dog-derived TET1 include a polypeptide having the amino acid sequence represented by SEQ ID NO: 9. Examples of rat-derived TET1 include a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13.

  The TET1 active domain refers to a partial polypeptide including a region responsible for TET1 activity among polypeptides constituting TET1. For example, in the case of human-derived TET1, the region responsible for TET1 activity refers to a region consisting of the 1418-2136th amino acid sequence in SEQ ID NO: 7. In the case of TET1 derived from mouse, the region responsible for TET1 activity refers to a region consisting of the amino acid sequence from 1367 to 2039 in SEQ ID NO: 8. In the case of dog-derived TET1, the region responsible for TET1 activity refers to the region consisting of the 1419 to 2137th amino acid sequence in SEQ ID NO: 9.

  The TET1 activity domain may be an amino acid or polypeptide surrounding a region responsible for TET1 activity in TET1, and may be added with an unrelated to TET1 activity. Such a TET1 active domain can be obtained by, for example, excising a partial polypeptide containing a region responsible for TET1 activity from TET1. The TET1 active domain is preferably a partial polypeptide of TET1 lacking a CxxC motif that recognizes CpG island. The human-derived TET1 active domain is preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. The mouse-derived TET1 active domain is preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. The canine-derived TET1 active domain is preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3.

  TET1 activity is an activity that converts 5-methylcytosine (5 mC) into 5-hydroxymethylcytosine (hmC). When HEK293 cells are used as cells and the conversion of 5 mC to hmC is promoted by introducing the analyte into the cells, it is determined that the analyte has TET1 activity. Conversion from 5 mC to 5 hmc is detected by the dot-blot method using a specific antibody against 5 hmC.

  A TET1 active domain variant, a fusion polypeptide comprising a TET1 active domain, and a fusion polypeptide comprising a TET1 active domain variant (hereinafter sometimes referred to as a “TET1 active domain variant”) have TET1 activity. Have.

  The variant of the TET1 active domain is not particularly limited, but preferably consists of a polypeptide having 80% or more, 85% or more, 90% or more, or 95% or more identity with the TET1 active domain. In the left column, the higher the identity of amino acids, the better. Alternatively, a variant of the TET1 active domain consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added (hereinafter sometimes referred to as “modification etc.”) in the amino acid sequence of the TET1 active domain.

  As amino acids whose TET1 activity is lost by modification or the like in the TET1 active domain, those at the 1671th and 1673th are known. A modified TET1 active domain having TET1 activity can be obtained, for example, by adding modifications to other amino acids while avoiding modifications to amino acids as described above.

A fusion polypeptide comprising a TET1 active domain or a variant thereof (hereinafter sometimes referred to as “a fusion polypeptide such as a TET1 active domain”) has an amino acid or polypeptide at the C-terminal, N-terminal or both ends of the TET1 active domain or the like. Each is added.
Although not particularly limited, for example, the TET1 active domain has a function unrelated to the TET1 activity, or has no function in particular, and does not inhibit or inhibit the TET1 activity such as the TET1 active domain. Or an amino acid or polypeptide that does not significantly inhibit. The function unrelated to the TET1 activity includes a function of binding to a specific base sequence or a tertiary structure of a nucleic acid. Although it does not specifically limit as a polypeptide which has such a function, For example, a methylated cytosine binding domain (MBD) etc. are mentioned. Although it does not specifically limit as MBD, For example, the partial arrangement | sequence of MECP2 is mentioned. In the case of human-derived MECP2, for example, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 14 can be exemplified. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 14 is a polypeptide consisting of the 62-169th amino acid sequence of human MECP2. By using a fusion polypeptide such as a TET1 active domain in which a polypeptide having such a function is fused, TET1 activity is selectively expressed only in a polynucleotide region having a specific base sequence or nucleic acid tertiary structure. Can do. The length of a polypeptide that has a function unrelated to TET1 activity, or has no function in particular, and does not inhibit TET1 activity such as a TET1 activity domain, or does not significantly inhibit the TET1 activity. Although not particularly limited, it may be 20% or less, preferably 10% or less, more preferably 5% or less of the TET1 active domain.

  The fusion polypeptide such as the TET1 active domain may be TET1 itself or a modified TET1. Alternatively, the fusion polypeptide such as the TET1 active domain may be a protein itself belonging to the TET family or a modified protein belonging to the TET family. Examples of proteins belonging to the TET family include TET2 and TET3 in addition to TET1. The variant herein is not particularly limited. For example, the variant preferably comprises a polypeptide having an amino acid identity of 80% or higher, 85% or higher, 90% or higher, or 95% or higher. It may be. In the left column, the higher the identity of amino acids, the better. Alternatively, the modified form herein may be an amino acid sequence in which one or several amino acids are modified in the amino acid sequence of the original polypeptide.

  PRDM14 is a protein discovered by the present inventor as a transcriptional regulator essential for the formation and early differentiation of primordial germ cells. Although PRDM14 is not specifically limited, The thing derived from a human origin, a mouse origin, a dog origin, and a rat is mentioned. Examples of human-derived PRDM14 include a polypeptide having the amino acid sequence represented by SEQ ID NO: 10. Examples of mouse-derived PRDM14 include a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11. Examples of the canine-derived PRDM14 include a polypeptide having the amino acid sequence represented by SEQ ID NO: 12.

  The PRDM14 active domain refers to a partial polypeptide including a region responsible for PRDM14 activity among polypeptides constituting PRDM14. For example, in the case of human-derived PRDM14, the region responsible for PRDM14 activity refers to a region consisting of the amino acid sequence from 254 to 571 in SEQ ID NO: 10. In the case of PRDM14 derived from mouse, the region responsible for PRDM14 activity refers to a region consisting of the amino acid sequence from 244 to 561 in SEQ ID NO: 11. In the case of canine-derived PRDM14, the region responsible for PRDM14 activity refers to a region consisting of amino acid sequences 254 to 571 in SEQ ID NO: 12.

  The PRDM14 activity domain may be an amino acid or polypeptide surrounding a region responsible for PRDM14 activity in PRDM14, and may be added with an unrelated PRDM14 activity. Such a PRDM14 active domain can be obtained by, for example, excising a partial polypeptide containing a region responsible for PRDM14 activity from PRDM14. The PRDM14 active domain is, for example, a partial polypeptide of PRDM14 lacking the Zing finger domain present at the C-terminus. When lacking the Zing finger domain, the human PRDM14 active domain preferably refers to a region consisting of the amino acid sequence of 254 to 488, more preferably 254 to 373 in SEQ ID NO: 10. When the Zing finger domain is lacking, the mouse PRDM14 active domain preferably refers to a region consisting of the amino acid sequence from 244 to 478, more preferably from 244 to 363 in SEQ ID NO: 11.

  PRDM14 activity interacts with TET1 and promotes TET1 activity. When HEK293 cells are used as cells and the subject is introduced into the cells together with TET1, DNA demethylation via TET1 activity is promoted, and it is determined that the subject has PRDM14 activity.

  A PRDM14 active domain variant, a fusion polypeptide comprising a PRDM14 active domain and a fusion polypeptide comprising a PRDM14 active domain variant (hereinafter sometimes referred to as “PRDM14 active domain variant”) have PRDM14 activity. Have.

  The variant of the PRDM14 active domain is not particularly limited, but preferably consists of a polypeptide having an amino acid identity of 80% or more, 85% or more, 90% or more, or 95% or more with the PRDM14 active domain. In the left column, the higher the identity of amino acids, the better. Alternatively, the PRDM14 active domain variant consists of an amino acid sequence in which one or several amino acids are modified in the amino acid sequence of the PRDM14 active domain.

  A variant of the PRDM14 active domain having PRDM14 activity is obtained by adding modifications to other amino acids while avoiding modifications to amino acids whose PRDM14 activity disappears by adding modifications, etc. in the PRDM14 active domain be able to.

  A fusion polypeptide comprising a PRDM14 active domain or a variant thereof (hereinafter sometimes referred to as “a fusion polypeptide such as a PRDM14 active domain”) has an amino acid or polypeptide at the C-terminal, N-terminal or both ends of the PRDM14 active domain or the like. Each is added. Although not particularly limited, for example, the PRDM14 active domain has a function unrelated to the PRDM14 activity, or has no function in particular, and does not inhibit or inhibit the PRDM14 activity such as the PRDM14 active domain. Or an amino acid or polypeptide that does not significantly inhibit. Examples of the function unrelated to PRDM14 activity include a function of binding to a specific base sequence or a tertiary structure of a nucleic acid. The polypeptide having such a function is not particularly limited, and examples thereof include a DNA binding domain that binds to a target gene region. Promoting TET1 activity selectively only in a polynucleotide region having a specific base sequence or a tertiary structure of a nucleic acid by using a fusion polypeptide such as a PRDM14 active domain in which a polypeptide having such a function is fused Can do. The length of a polypeptide that has a function unrelated to PRDM14 activity, or has no function in particular, and does not inhibit PRDM14 activity such as a PRDM14 active domain, or does not significantly inhibit it even if it is inhibited. Although not particularly limited, 20% or less, preferably 10% or less, more preferably 5% or less of the PRDM14 active domain and the like can be mentioned.

  The target gene region may be a promoter region of the target gene. The target gene is not particularly limited, and examples thereof include a tumor suppressor gene. Although it does not specifically limit as a tumor suppressor gene, For example, p16, p53, E-cadherin etc. are mentioned.

  Although it does not specifically limit as a DNA binding domain couple | bonded with a target gene area | region, For example, the DNA binding domain of a transcription factor, or the modified thing can be used. Examples of such DNA-binding domains of transcription factors include CTCFs that are known to bind to the promoter region of p16.

The fusion polypeptide such as the PRDM14 active domain may be PRDM14 itself or a variant of PRDM14. The variant of PRDM14 here is not particularly limited, but for example, it consists of a polypeptide having preferably 80% or more, 85% or more, 90% or more, or 95% or more identity with PRDM14. May be.
In the left column, the higher the identity of amino acids, the better. Alternatively, the PRDM14 variant herein may be composed of an amino acid sequence in which one or several amino acids are modified in the amino acid sequence of PRDM14.

[2. Method of treatment]
The treatment method of the present invention is a method of treating a human,
(1) A method comprising a step of increasing the intracellular content of a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same.

  Step (1) is as follows. This is the same as described in the above.

  The therapeutic method of the present invention is preferably a method for treating a disease caused by abnormal DNA methylation that repairs an epigenetic mutation. More preferred is a method for treating cancer or neuropsychiatric disorders. The neuropsychiatric disorder is not particularly limited as long as it is caused by an abnormality in DNA methylation that repairs an epigenetic mutation. Examples thereof include bipolar disorder (manic depression), repetitive depression, schizophrenia, and the like. .

  The treatment method of the present invention will be described later. 4. a polypeptide-containing composition; 5. a polynucleotide-containing composition, 6. an expression vector-containing composition and Co-expression vectors can be used. In the treatment method of the present invention, 4. ~ 7. Each of these can be used as described below as a method of use for pharmaceutical purposes.

[3. iPS Cell Production Method]
As a method for producing iPS cells of the present invention,
[3A-1. ] A method for producing iPS cells from somatic cells using a nuclear reprogramming substance, comprising: (1) a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a PRDM14 active domain or a variant thereof, or The method of including the process of increasing the intracellular content of the fusion polypeptide containing it is mentioned.

  Step (1) is as follows. This is the same as described in the above.

The method for producing iPS cells of the present invention comprises:
(I) The process of converting a somatic cell into an iPS cell by making a nuclear reprogramming substance contact a somatic cell is included. As this conversion step, a known one can be used. The known conversion step is not particularly limited, and examples thereof include infection using a retrovirus, gene introduction of an episomal vector by a liposome method, and the like.

  In the step of producing iPS cells from somatic cells using a nuclear reprogramming substance, at least one cell selected from the group consisting of high-quality iPS cells, low-quality iPS cells, and pre-iPS cells may be obtained. Are known. Here, high quality iPS cells are defined as iPS cells in which no abnormality of DNA methylation is observed. A pre-iPS cell is defined as a cell that is incompletely reprogrammed. Reprogramming failure in pre-iPS cells is thought to be due to the fact that the epigenomic information of donor cells is still maintained. More specifically, the pre-iPS cell is an iPS cell-like cell that appears in the process of creating an iPS cell, and is morphologically similar to an iPS cell, but the expression of a foreign gene is not suppressed, Moreover, it is a cell in which no increase in the expression of endogenous pluripotency-related genes (Nanog, Oct3 / 4, Rex-1, etc.) is observed. Moreover, low quality iPS cells are defined as iPS cells in which DNA methylation is abnormal. More specifically, the low-quality iPS cell is an iPS cell in which abnormal methylation is added to the GTL2 gene region and a tetraploid chimeric embryo cannot be produced. The present inventor has found that abnormalities of DNA methylation known to adversely affect the quality of iPS cells can be normalized by the method for inducing DNA demethylation of the present invention. Specifically, the present inventors have found that abnormalities of DNA methylation observed in low-quality iPS cells can be normalized by the method for inducing DNA demethylation of the present invention, and low-quality iPS cells can be improved to high-quality iPS cells. Furthermore, the present inventors have found that reprogramming in pre-iPS cells can be completed by the method for inducing DNA demethylation of the present invention. Specifically, the present inventors have found that the epigenome information of donor cells maintained in pre-iPS cells can be lost by the DNA demethylation induction method of the present invention to convert pre-iPS cells into iPS cells. Therefore, by performing step (1) on the low-quality iPS cells and pre-iPS cells, they can be converted into high-quality iPS cells and iPS cells, respectively. In order to obtain the same effect, it is not always necessary to perform the step (1) directly on the low-quality iPS cells and pre-iPS cells. For example, the same effect can be obtained by performing step (1) in combination with step (i) on somatic cells.

  As described above, in step (1), in at least one cell selected from the group consisting of somatic cells, low-quality iPS cells, and pre-iPS cells, a fusion polypeptide such as a TET1 active domain and a fusion polypeptide such as a PRDM14 active domain are used. Increase the intracellular content of the peptide.

Therefore, the conversion step (i) may be performed before the step (1), may be performed simultaneously with the step (1), or may be performed after the step (1). When step (i) is performed before step (1), step (1) is performed on at least low-quality iPS cells or pre-iPS cells. When step (i) is performed simultaneously with or after step (1), step (1) is performed on at least somatic cells. When performing the step (i) simultaneously with the step (1), the step (1) is, in other words,
(1A-1) TET1 active domain or a variant thereof, or a fusion polypeptide containing the same; and PRDM14 active domain or a variant thereof, or a fusion polypeptide containing the same, increasing the intracellular content and nuclear reprogramming It is a step of converting somatic cells into iPS cells by bringing a substance into contact with the somatic cells.

As a method for producing iPS cells of the present invention,
[3A-2. (I) A step of converting somatic cells into high-quality iPS cells, low-quality iPS cells, and pre-iPS cells by contacting a nuclear reprogramming substance with the somatic cells;
(II) evaluating the cells obtained in step (I) and selecting high-quality iPS cells, low-quality iPS cells and pre-iPS cells; and (III) low-quality selected in step (II) Examples include a method including a step of performing the step (1) on iPS cells and pre-iPS cells.

  The GTL2 gene region is a genome imprinted region, and usually only the genome derived from the father is methylated. However, in both low-quality iPS cells, both the mother-derived and father-derived genomes are methylated, and this abnormal methylation is thought to cause the quality of iPS cells to deteriorate. Further, the present inventor has found that PRDM14 has an activity of eliminating abnormal methylation in the GTL2 gene region. In the present invention, an iPS cell in which 80% or more of methylation is added to the GTL2 gene region is defined as low quality, and it is judged that the quality is improved when it is reduced to 60% or less by introducing PRDM14. Moreover, if there is abnormal methylation in GTL2, gene expression of GTL2 is suppressed. Therefore, by introducing PRDM14 into low-quality iPS cells, it is possible to quickly verify the quality improvement effect of PRDM14 depending on whether the expression of GTL2 has been recovered to the same level as wild-type ES cells.

  Accordingly, in step (II), the degree of methylation of the GTL2 gene region is examined for the cells obtained in step (I), and iPS cells to which 80% or more of methylation has been added are determined to be of low quality. Low quality iPS cells can be selected.

  As another method, in the step (II), the expression level of the GTL2 gene is examined for the cells obtained in the step (I), and compared with the expression level of the same gene in the wild type ES cell. By determining iPS cells having a lower degree as low quality, low quality iPS cells can be selected.

In step (II), the degree of expression of the endogenous NANOG gene in the cells obtained in step (I) is examined, and iPS cells in which no expression is observed are determined as pre-iPS cells. iPS cells can be sorted.
These pre-iPS cells and low-quality iPS cells, and intermediate cells thereof, such as cells in which Nanog is weakly positive and Gtl2 is hypermethylated, are all incompletely initialized iPS Although it is a cell, it is an iPS cell to be excluded when applied to regenerative medicine. In step (II), intermediate cells as described above may be further selected in addition to high-quality iPS cells, low-quality iPS cells, and pre-iPS cells. The intermediate cells thus selected are further subjected to the same treatment as in the case of low-quality iPS cells and pre-iPS cells in step (III). As a result, it is possible to reduce the number of iPS cells incompletely initialized from the iPS cell group, and to establish iPS cells having a high degree of completion of initialization such as high-quality iPS cells. It becomes.

  The evaluation and selection of the cells in the step (II) are not particularly limited, but can be performed, for example, after the cells are colonized. Specifically, some cells are sampled from individual colonies each derived from a single cell, and the colonies to which the cells belong are evaluated by evaluating the cells. You can sort by.

  In step (III), step (1) is performed on the pre-iPS cells and low-quality iPS cells selected in step (II). The low-quality iPS cells selected in step (II) have abnormal DNA methylation, and the pre-iPS cells selected in the same manner are incompletely reprogrammed. By performing step (1) on these low-quality iPS cells and pre-iPS cells in step (III), the low-quality iPS cells and pre-iPS cells are converted into high-quality iPS cells and iPS cells, respectively. be able to. Steps (II) and (III) can be repeated until the required number of high quality iPS cells is obtained. When repeated, the description of “cells obtained in step (I)” in step (II) is read as “cells obtained in step (III)”. By repeating the process, more low-quality iPS cells and pre-iPS cells can be converted into high-quality iPS cells and iPS cells, respectively, and thus more high-quality iPS cells can be obtained.

Step 3A-2. In step (3), step (1) in step (III) may be performed simultaneously with step (i). In this case, the step (1) in the step (III)
(1A-2) Intracellular in low-quality iPS cells and pre-iPS cells of TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same In other words, it is a process of increasing the content and bringing the nuclear reprogramming substance into contact with low-quality iPS cells and pre-iPS cells.

  Step 3A-2. In step 1, step (1) may be first performed on somatic cells, and then step (1) may be performed on the low-quality iPS cells and pre-iPS cells selected in step (II). In this case, the first step (1) may be performed simultaneously with step (i). Also in this case, the process (I) can be rephrased as the process (1A-1).

As a method for producing iPS cells of the present invention,
[3B. The method of obtaining an iPS cell by performing a process (1) with respect to a pre-iPS cell is mentioned. In this case, the step (1) may be performed in the presence of a nuclear reprogramming substance, and the step (1)
(1B) Increasing the pre-iPS intracellular content and nuclear reprogramming of the TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and the PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same In other words, it is a step of bringing a substance into contact with pre-iPS cells.

As a method for producing iPS cells of the present invention,
[3C. The method of obtaining a high quality iPS cell by performing a process (1) with respect to a low quality iPS cell is mentioned. In this case, the step (1) may be performed in the presence of a nuclear reprogramming substance, and the step (1)
(1C) increasing the low-quality iPS intracellular content and nuclear reprogramming of the TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and the PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same In other words, it is a process of contacting a substance with low-quality iPS cells.

  In the method for producing iPS cells of the present invention, the somatic cells are not particularly limited, and examples thereof include human cells, mouse cells, monkeys, and the like.

  In the method for producing iPS cells of the present invention, a known nuclear reprogramming substance can be used. The nuclear initialization material is not particularly limited, and examples thereof include OCT4, SOX2, KLF4, and MYC.

[4. Polypeptide-containing composition]
The polypeptide-containing composition of the present invention comprises:
(A-1) a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (A-2) a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same. .

  The TET1 active domain or a variant thereof, or a fusion polypeptide comprising it, This is the same as described in the above.

  PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same, This is the same as described in the above.

  The polypeptide-containing composition of the present invention is, for example, 1. As described above, it can be used for the purpose of inducing DNA demethylation in cells.

Specifically, the polypeptide-containing composition of the present invention can be used for the purpose of normalizing DNA methylation, for example, by applying it to cells having abnormal DNA methylation. When the abnormality of DNA methylation is associated with some kind of disease and the normalization leads to the treatment of such a disease, the polypeptide-containing composition of the present invention can be used as a medicine. More specifically, the polypeptide-containing composition of the present invention can be used, for example, as a therapeutic agent for diseases caused by abnormal DNA methylation that repairs epigenetic mutations.
It should be noted that DNA demethylating agents such as azacitidine (Bidaza (registered trademark)) currently being clinically tested as an anti-cancer agent are DNA replication-dependent (passive) by inhibiting the maintenance of methylation by DNMT1. Since it induces demethylation and DNMT1 maintains methylation of the entire genome, there is no region selectivity, and as a result of demethylating the entire genome, side effects are unavoidable. In contrast, when the polypeptide-containing composition of the present invention is used as a therapeutic agent, DNA demethylation can be induced in a region-selective manner through base excision repair, so that a therapeutic agent with few side effects is provided. obtain. In addition, the known DNA demethylating agent has only an effect on dividing cells, but when the polypeptide-containing composition of the present invention is used as a therapeutic agent, it is advantageous in that it is also effective on non-dividing cells. There is also.
Although it does not specifically limit as said therapeutic agent, For example, an anticancer agent, a neuropsychiatric disease therapeutic agent, etc. are mentioned. Although it does not specifically limit as a therapeutic agent for neuropsychiatric diseases, For example, a therapeutic agent for manic depression (bipolar disorder), a therapeutic agent for repeated depression, schizophrenia, etc. can be mentioned.

  When used as a medicine, the polypeptide-containing composition of the present invention (in this case, sometimes referred to as “a pharmaceutical-containing polypeptide-containing composition” hereinafter) comprises the polypeptides (A-1) and (A-2). Each is contained as an active ingredient. The polypeptide-containing composition of the present invention may further contain other components as necessary in addition to these active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include anti-inflammatory agents and antibacterial agents.

  The content ratio of the polypeptides (A-1) and (A-2) in the pharmaceutical-use polypeptide-containing composition of the present invention is determined according to the dosage form, dosage form, dosage, administration frequency, and the like.

  The dosage form of the polypeptide composition for pharmaceutical use of the present invention is appropriately set according to the affected area to be applied. It may be systemic administration or local administration. Systemic administration includes oral administration or parenteral administration. Further, parenteral administration includes intravenous injection, subcutaneous injection and intramuscular injection. Examples of topical administration include administration to the skin, mucous membranes, intranasal or intraocular.

  The dosage form of the polypeptide composition for pharmaceutical use of the present invention is appropriately set according to the dosage form and the like. For example, in the case of oral administration, solid preparations such as tablets, granules, powders, suppositories, and capsules, liquids and the like can be mentioned. For parenteral administration, semi-solid forms such as creams, gels and ointments, and liquids such as liquids and lotions are included. It is often preferable to administer it as an injection.

  The dosage and administration frequency of the polypeptide-containing composition for pharmaceutical use according to the present invention include the administration form, administration method and dosage form, the condition of the recipient, and the polypeptides (A-1) and (A-2). It is appropriately set depending on the degree of DNA demethylation activity and the like. Appropriate dosages and dosages can be determined according to conventional dosage determination techniques known to those skilled in the art.

  The dose per administration may be a therapeutically effective amount.

The polypeptide-containing composition of the present invention comprises: As described above, it can be used for the purpose of producing iPS cells. In other words, the polypeptide-containing composition of the present invention can be used as an agent for improving the completeness of iPS cell reprogramming or a quality improving agent. In this case, the polypeptide-containing composition of the present invention contains the polypeptides (A-1) and (A-2) as active ingredients. The polypeptide-containing composition of the present invention may further contain other components as necessary in addition to these active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include nuclear reprogramming substances.
A well-known thing can be used as a nuclear initialization substance.

[5. Polynucleotide-containing composition]
The polynucleotide-containing composition of the present invention comprises:
(Α-1) a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (α-2) a PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same. A composition comprising a polynucleotide.

  The TET1 active domain or a variant thereof, or a fusion polypeptide comprising it, This is the same as described in the above.

  PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same, This is the same as described in the above.

  The polynucleotide is DNA or RNA. The polynucleotide is preferably DNA.

  The polynucleotide-containing composition of the present invention is 4. It can be used for the same use as the polypeptide-containing composition of the present invention described in 1. above.

  When used as a medicine, the polynucleotide-containing composition of the present invention (in this case, sometimes referred to as “pharmaceutical-use polynucleotide-containing composition” hereinafter) comprises polynucleotides (α-1) and (α-2). Each is contained as an active ingredient. The polynucleotide-containing composition of the present invention may further contain other components as required in addition to these active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include anti-inflammatory agents and antibacterial agents.

  The content ratio of the polynucleotides (α-1) and (α-2) in the pharmaceutical-containing polynucleotide-containing composition of the present invention is determined according to the dosage form, dosage form, dosage, administration frequency, and the like.

  The dosage form of the polynucleotide-containing composition for pharmaceutical use of the present invention is appropriately set depending on the affected area to be applied. It may be systemic administration or local administration. Systemic administration includes oral administration or parenteral administration. Further, parenteral administration includes intravenous injection, subcutaneous injection and intramuscular injection. Examples of topical administration include administration to the skin, mucous membranes, intranasal or intraocular.

  The dosage form of the polynucleotide-containing composition for pharmaceutical use of the present invention is appropriately set according to the dosage form and the like. For example, in the case of oral administration, solid preparations such as tablets, granules, powders, suppositories, and capsules, liquids and the like can be mentioned. For parenteral administration, semi-solid forms such as creams, gels and ointments, and liquids such as liquids and lotions are included. It is often preferable to administer it as an injection.

  The dosage and administration frequency of the polynucleotide-containing composition for pharmaceutical use according to the present invention include the administration form, administration method and dosage form, the condition of the recipient, and the polynucleotides (α-1) and (α-2). It is appropriately set depending on the degree of DNA demethylation activity and the like. Appropriate dosages and dosages can be determined according to conventional dosage determination techniques known to those skilled in the art.

  The dose per administration may be a therapeutically effective amount.

  The polynucleotide-containing composition of the present invention comprises: As described above, it can be used for the purpose of producing iPS cells with improved completion of initialization, particularly high-quality iPS cells. In other words, the polynucleotide-containing composition of the present invention can be used as an agent for improving reprogramming completion of iPS cells or a quality improving agent. More specifically, as shown in the Examples, the Nanog region can be obtained by expressing PRDM14 in imperfectly reprogrammed iPS cells such as pre-iPS cells, low-quality iPS cells and intermediate cells thereof. The quality of the iPS cell initialization, such as demethylation and reduction of abnormal methylation, is improved and quality improvement is achieved. In this case, the polypeptide-containing composition of the present invention contains polynucleotides (α-1) and (α-2) as active ingredients. The polynucleotide-containing composition of the present invention may further contain other components as required in addition to these active ingredients. Examples of such components include formulation components necessary for formulation depending on the dosage form, storage-stable components necessary for storage stability, and other active ingredients. Examples of other active ingredients include nuclear reprogramming substances. A well-known thing can be used as a nuclear initialization substance.

[6. Expression vector-containing composition]
The expression vector-containing composition of the present invention comprises:
(A-1) an expression vector comprising a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and (a-2) a PRDM14 active domain or a variant thereof, or a fusion poly comprising the same A composition comprising an expression vector comprising a polynucleotide encoding a peptide.

  The TET1 active domain or a variant thereof, or a fusion polypeptide comprising it, This is the same as described in the above.

  PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same, This is the same as described in the above.

  The polynucleotide is DNA or RNA. The polynucleotide is preferably DNA.

  The expression vector-containing composition of the present invention is, for example, 1. As described above, it can be used for the purpose of inducing DNA demethylation in cells. In this case, the expression vectors (a-1) and (a-2) only have to have an effect of inducing DNA demethylation when used in combination. In other words, the expression vector (a-1) expresses a polypeptide obtained by further adding an amino acid or polypeptide that does not inhibit or significantly inhibit TET1 activity and PRDM14 activity to a fusion polypeptide such as a TET1 active domain. It may be a vector. Since such an expression vector can express the TET1 activity when used in combination with the expression vector (a-2) and does not inhibit the PRDM14 activity expressed by the expression vector (a-2), the effect of the present invention is achieved. sell. Similarly, the expression vector (a-2) expresses a polypeptide obtained by further adding an amino acid or polypeptide that does not inhibit or significantly inhibit TET1 activity and PRDM14 activity to a fusion polypeptide such as a PRDM14 active domain. It may be a vector. Since such an expression vector can express PRDM14 activity when used in combination with the expression vector (a-1) and does not inhibit the TET1 activity expressed by the expression vector (a-1), the effect of the present invention is achieved. sell. As described above, examples of the polypeptide that may be added to the fusion polypeptide such as the TET1 active domain include a signal peptide. The length of the polypeptide that may be added to the fusion polypeptide such as the TET1 active domain is not particularly limited, but is, for example, 2 to 30 amino acids, preferably 2 to 10 amino acids, more preferably 2 to 2 amino acids. 7 amino acids are mentioned. Moreover, as above-mentioned, as a polypeptide which may be added to fusion polypeptides, such as a PRDM14 active domain, a signal peptide etc. are mentioned, for example. The length of the polypeptide that may be added to the fusion polypeptide such as the PRDM14 active domain is not particularly limited, and is, for example, 2 to 30 amino acids, preferably 2 to 10 amino acids, more preferably 2 to 2 amino acids. 7 amino acids are mentioned.

  The expression vector is not particularly limited, and a known one can be used as appropriate. For example, pCAGGS-1 vector, pCDNA3.1 vector, etc. are mentioned.

  Although it does not specifically limit as a promoter, For example, CMV, CAGGS etc. are mentioned when transient expression is intended. In addition, when constitutive expression is intended, an episomal vector having a replication origin OriP derived from pstein-Barr Virus (EBV) and expressing the EBV Nuclear Antigen 1 (EBNA1) gene can be mentioned.

  The expression vector-containing composition of the present invention is 4. It can be used for the same use as the polypeptide-containing composition of the present invention described in 1. above.

  When used as a pharmaceutical, the expression vector-containing composition of the present invention (hereinafter sometimes referred to as “pharmaceutical expression vector-containing composition”) comprises the expression vectors (a-1) and (a-2). Each is contained as an active ingredient. The expression vector-containing composition of the present invention may further contain other components as needed in addition to these active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include anti-inflammatory agents and antibacterial agents.

  The content ratio of the expression vectors (a-1) and (a-2) in the expression vector-containing composition for pharmaceutical use of the present invention is determined according to the dosage form, dosage form, dosage, administration frequency and the like.

  The dosage form of the pharmaceutical use expression vector-containing composition of the present invention is appropriately set depending on the affected area to be applied. It may be systemic administration or local administration. Systemic administration includes oral administration or parenteral administration. Further, parenteral administration includes intravenous injection, subcutaneous injection and intramuscular injection. Examples of topical administration include administration to the skin, mucous membranes, intranasal or intraocular.

  The dosage form of the expression vector-containing composition for pharmaceutical use of the present invention is appropriately set depending on the administration form and the like. For example, in the case of oral administration, solid preparations such as tablets, granules, powders, suppositories, and capsules, liquids and the like can be mentioned. For parenteral administration, semi-solid forms such as creams, gels and ointments, and liquids such as liquids and lotions are included. It is often preferable to administer it as an injection.

  The dosage and frequency of administration of the expression vector-containing composition for pharmaceutical use according to the present invention include the administration form, administration method and dosage form, the condition of the recipient, and the expression vectors (a-1) and (a-2). It is appropriately set depending on the degree of DNA demethylation activity and the like. Appropriate dosages and dosages can be determined according to conventional dosage determination techniques known to those skilled in the art.

  The dose per administration may be a therapeutically effective amount.

The expression vector-containing composition of the present invention comprises: As described above, it can be used for the purpose of producing iPS cells. In other words, the expression vector-containing composition of the present invention can be used as an agent for improving the completeness of iPS cell reprogramming or a quality improving agent. In this case, the expression vector-containing composition of the present invention contains expression vectors (a-1) and (a-2) as active ingredients. The expression vector-containing composition of the present invention may further contain other components as needed in addition to these active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include nuclear reprogramming substances.
A well-known thing can be used as a nuclear initialization substance.

[7. Co-expression vector]
The co-expression vector of the present invention is
(B) a co-expression vector comprising a polynucleotide encoding a TET1 active domain or a variant thereof, or a fusion polypeptide comprising the same; and a polynucleotide encoding a PRDM14 active domain or a variant thereof or a fusion polypeptide comprising the same It is.

  The TET1 active domain or a variant thereof, or a fusion polypeptide comprising it, This is the same as described in the above.

  PRDM14 active domain or a variant thereof, or a fusion polypeptide comprising the same, This is the same as described in the above.

  The polynucleotide is DNA or RNA. The polynucleotide is preferably DNA.

  The co-expression vector-containing composition of the present invention includes, for example, 1. As described above, it can be used for the purpose of inducing DNA demethylation in cells. In this case, the co-expression vector (b) only needs to have an action of inducing DNA demethylation. In other words, the co-expression vector (b) expresses a polypeptide obtained by further adding an amino acid or polypeptide that does not inhibit or significantly inhibit TET1 activity and PRDM14 activity to a fusion polypeptide such as a TET1 activity domain, Moreover, it may be a vector expressing a polypeptide in which a fusion polypeptide such as a PRDM14 active domain is further added with an amino acid or polypeptide that does not inhibit or significantly inhibit TET1 activity and PRDM14 activity. Since such a co-expression vector can express TET1 activity and PRDM14 activity, the effects of the present invention can be achieved. As described above, examples of the polypeptide that may be added to the fusion polypeptide such as the TET1 active domain include a signal peptide. The length of the polypeptide that may be added to the fusion polypeptide such as the TET1 active domain is not particularly limited, but is, for example, 2 to 30 amino acids, preferably 2 to 10 amino acids, more preferably 2 to 2 amino acids. 7 amino acids are mentioned. Moreover, as above-mentioned, as a polypeptide which may be added to fusion polypeptides, such as a PRDM14 active domain, a signal peptide etc. are mentioned, for example. The length of the polypeptide that may be added to the fusion polypeptide such as the PRDM14 active domain is not particularly limited, and is, for example, 2 to 30 amino acids, preferably 2 to 10 amino acids, more preferably 2 to 2 amino acids. 7 amino acids are mentioned.

  The expression vector is not particularly limited, and a known one can be used as appropriate. For example, pCAGGS-1 vector, pCDNA3.1 vector, etc. are mentioned.

  Although it does not specifically limit as a promoter, For example, CMV, CAGGS etc. are mentioned when transient expression is intended. In addition, when constitutive expression is intended, an episomal vector having a replication origin OriP derived from pstein-Barr Virus (EBV) and expressing the EBV Nuclear Antigen 1 (EBNA1) gene can be mentioned.

  The co-expression vector-containing composition of the present invention is 4. It can be used for the same use as the polypeptide-containing composition of the present invention described in 1. above.

  When used as a medicine, the co-expression vector-containing composition of the present invention (hereinafter sometimes referred to as “medical use co-expression vector-containing composition”) contains the co-expression vector (b) as an active ingredient. . The co-expression vector-containing composition of the present invention may further contain other components as necessary in addition to the active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients. Examples of other active ingredients include anti-inflammatory agents and antibacterial agents.

  The content ratio of the co-expression vector (b) in the pharmaceutical use co-expression vector-containing composition of the present invention is determined according to the dosage form, dosage form, dosage, administration frequency and the like.

  The administration form of the pharmaceutical use co-expression vector-containing composition of the present invention is appropriately set according to the affected area to be applied. It may be systemic administration or local administration. Systemic administration includes oral administration or parenteral administration. Further, parenteral administration includes intravenous injection, subcutaneous injection and intramuscular injection. Examples of topical administration include administration to the skin, mucous membranes, intranasal or intraocular.

  The dosage form of the pharmaceutical use co-expression vector-containing composition of the present invention is appropriately set depending on the dosage form and the like. For example, in the case of oral administration, solid preparations such as tablets, granules, powders, suppositories, and capsules, liquids and the like can be mentioned. For parenteral administration, semi-solid forms such as creams, gels and ointments, and liquids such as liquids and lotions are included. It is often preferable to administer it as an injection.

  The dosage and administration frequency of the pharmaceutical use co-expression vector-containing composition of the present invention include the administration form, administration method and dosage form, as well as the condition of the recipient, and the DNA demethylation activity of the co-expression vector (b). It is set as appropriate according to the degree. Appropriate dosages and dosages can be determined according to conventional dosage determination techniques known to those skilled in the art.

  The dose per administration may be a therapeutically effective amount.

The co-expression vector-containing composition of the present invention comprises: As described above, it can be used for the purpose of producing iPS cells. In other words, the co-expression vector-containing composition of the present invention can be used as an agent for improving the completeness of iPS cell reprogramming or a quality improving agent. In this case, the co-expression vector-containing composition of the present invention contains the co-expression vector (b) as an active ingredient. The co-expression vector-containing composition of the present invention may further contain other components as necessary in addition to the active ingredients. Examples of such components include pharmaceutical ingredients required for formulation according to the dosage form, storage-stable ingredients required for storage stability, and other active ingredients.
Examples of other active ingredients include nuclear reprogramming substances. A well-known thing can be used as a nuclear initialization substance.

1. In order to verify whether PRDM14 is highly expressed in ES cells by deregulating DNA in the LINE-1 region by PRDM14, methylation of LINE-1 in PRDM14-highly expressing ES cells was examined. Analysis was performed.

  LINE-1 is a transposon sequence (foreign sequence) and occupies about 20% of the mouse genome (FIG. 1), but its expression is usually suppressed by DNA methylation.

  DNA methylation analysis was performed by Bisulfite-Sequencing method. When the genome is treated with Bisulfite, cytosine is converted to uracil, but methylated cytosine remains as methylated cytosine. Therefore, the level of methylated cytosine in the genome can be quantified by confirming the sequence in the sequence. In this experiment, the methylation status of nine CpG sites present in the 5′UTR of LINE-1 was analyzed. In FIG. 1, the blue bar graph represents the methylation amount of each CpG site, and the red bar graph represents the methylation rate of the entire CpG site.

Experimental methods, results and discussion are as follows.
[experimental method]
(1) Preparation of PRDM14 highly expressing ES cells A plasmid in which the Prdm14-ires-puro gene was inserted downstream of the CAGGS promoter was introduced into ES cells by lipofection. 48 hours after gene introduction, selection was performed with puromycin, and a single colony was picked up and subcultured and cultured. RNA was extracted from the picked-up cells, the expression of Prdm14 was confirmed by quantitative RT-PCR, and cells that were expressed more than 50 times compared to control (empty vector introduced) ES cells were designated as Prdm14 highly expressing ES cells.
(2) DNA methylation analysis Genomic DNA was isolated from control ES cells and ES cells highly expressing PRDM14, and non-methylated cytosine was converted to uracil by sulfite treatment. PCR was performed using this DNA as a template and a primer for the sequence of LINE-1, and the amplified product was cloned into a TA vector, and then the sequence was confirmed by sequencing. Unmethylated cytosine is finally replaced with T, but methylated cytosine remains as cytosine. Therefore, it was determined that CG site was unmethylated and CG was methylated.
[Results and discussion]
About 80% of CpG sites were methylated in wild-type ES cells, whereas in PRDM14 high-expressing ES cells (PRDM14 OE), only about 30% was methylated, and PRDM14 It was revealed that significant demethylation of the LINE-1 region can be induced by highly expressing ES in ES cells.
2. DNA demethylation of a wide range of gene regions by PRDM14 In order to verify how much gene regions are affected by DNA demethylation by PRDM14, the lack of DNA methylase in the genome has little DNA methylation Comparison of ES cell (Dnmts TKO) and gene expression profile was analyzed by microarray.

Experimental methods, results and discussion are as follows.
[experimental method]
(1) Analysis by microarray RNA was extracted from control ES cells, PRDM14 high-expression ES cells, and Dnmts TK ES cells, and analyzed using a mouse whole gene microarray platform manufactured by Agilent. In PRDM14 high-expressing ES cells and Dnmts TKO ES cells, a gene group that was expressed twice or more from wild-type ES cells was extracted, and a Venn diagram was prepared using Biovenn.
[Results and discussion]
About 35% of the genes whose expression was increased in Dnmts TKOES cells were PRDM14 O.D. E. Expression is also elevated in ES cells (FIG. 2) and this result suggests that PRDM14 induces DNA demethylation of about 35% of the gene region.
3. The DNA demethylation pathway triggered by hydroxylation of DNA demethylated methylated cytosine via PRDM14 and hydroxymethylation is shown in FIG. Methylated cytosine produced by the action of DNA methylase is hydroxylated by TET protein and converted to hydroxymethylated cytosine. Hydroxymethylated cytosine is converted to hydroxymethylated uracil by a deaminase and finally demethylated to cytosine by a base excision repair pathway. In order to verify the possibility that PRDM14 induces DNA demethylation by promoting this pathway, the amount of genome-wide methylated cytosine and hydroxymethylated cytosine in PRDM14-highly expressing ES cells was quantified. .

  Each genome was adsorbed on a nitrocellulose membrane, and then a dot blot was performed using an anti-methylated cytosine antibody or an anti-hydroxymethylated cytosine antibody.

Experimental methods, results and discussion are as follows.
[experimental method]
(1) Preparation <br/> genomic DNA genome, was prepared according to the protocol using the Promega Wizard SV Genomic DNA purification system.
(2) Dot blot
The genomic DNA was denatured at 95 ° C., adsorbed on a nitrocellulose membrane, and then subjected to dot blot using an anti-methylated cytosine antibody or an anti-hydroxymethylated cytosine antibody. A secondary antibody labeled with HRP was used for detection.
[Results and discussion]
In Dnmts KO ES cells, both methylated cytosine and hydroxymethylated cytosine decreased in the genome, whereas PRDM14 O.D. E. In ES cells, methylated cytosine was decreased, but the amount of hydroxymethylated cytosine was increased on the contrary (FIG. 4). Therefore, it is considered that PRDM14 may induce DNA demethylation by promoting hydroxymethylation of methylated cytosine.
4). Base Exclusion Repair in DNA Demethylation by PRDM14 If PRDM14 induces DNA demethylation by promoting the pathway of FIG. 3, DNA demethylation by PRDM14 is canceled by a base excision repair inhibitor. It should be. Therefore, it was verified whether or not the increase in gene expression due to DNA demethylation by PRDM14 was inhibited by administration of base excision repair inhibition. As the base excision repair inhibitor, inhibitors of PARP1 and APE1, which are main factors for base excision repair, were used (3AB and CRT, respectively).

Experimental methods, results and discussion are as follows.
[experimental method]
The Empty vector and Prdm14-expressing Vector are introduced into ES cells, Prdm14-positive cells are concentrated by drug selection, and cultured for 6 days. RNA was collected from ES cells cultured for 6 days, and the expression change of the target gene was compared between Empty vector-introduced cells and Prdm14 vector-introduced cells using quantitative RT-PCR.
[Results and discussion]
It became clear that administration of 3AB and CRT, respectively, suppressed the induction of the expression of most of the genes that were induced by PRDM14 (FIG. 5). The results of this experiment showed that PRDM14 induces DNA demethylation by promoting the pathway of FIG. On the other hand, it is known that somatic cells originally do not have TET1, but when TET1 is overexpressed in somatic cells, conversion from 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine occurs. Have been reported (Science Vol. 324, 15 May, 2009, pp. 930-935). 3. In combination with the experimental results of the above, by increasing the intracellular content of TET1 and PRDM14 in somatic cells, the conversion to 5-hydroxymethylcytosine is further promoted compared to the case where the intracellular content of TET1 alone is increased. As a result, it was shown that DNA demethylation is further promoted.
5. Induction of pluripotent stem cells through DNA demethylation by PRDM14 iPS cells are introduced by introducing Yamanaka factor (OCT4, SOX2, KLF4 and MYC; collectively referred to as “OSKM”) into fibroblasts. Can be established.
Pre-iPS cells with poor reprogramming and low-quality iPS cells appear in the process of establishment, but the reprogramming failure in these cells is due to the maintenance of epigenomic information of donor cells (fibroblasts, etc.) It is thought that there is (Fig. 6).

  In this experiment, the effect on ES cells by expressing PRDM14 in epiblast-like cells (EpiLC) produced by differentiating ES cells was examined. ES cells can self-replicate in the presence of the cytokine LIF, but in vitro epiblasts (embryonic embryonic day 6.5 embryos) are cultured in the presence of 0.1% KSR, bFGF and ACTIVINA. ) Can be induced to differentiate into EpiLCs having similar properties. When EpiLCs are cultured again in ES cell culture medium, the cells are further differentiated. In this experiment, it was confirmed what changes were observed in EpiLCs when PRDM14 was expressed in EpiLCs and then cultured in ES cell culture medium. .

Experimental methods, results and discussion are as follows.
[experimental method]
PRDM14-induced ES cells that express PRDM14 in the absence of tetracycline were used. ES cells are cultured on fibronectin-coated dishes in the presence of 0.1% KSR, bFGF and ACTIVIN A for 2 days. Thereafter, the cells were further cultured for 2 days in the presence or absence of tetracycline, suspended in ES cell culture medium (in the presence of LIF) and subcultured. RNA was collected 0, 2, and 4 days after the start of the culture, and changes in gene expression of Oct3 / 4, Sox2, and Klf2 were quantified by quantitative RT-PCR. In addition, cells passaged in ES cell medium were cultured for 3 days, fixed with 4% PFA, and then ES cells were determined by alkaline phosphatase staining.
[Results and discussion]
When PRDM14 was expressed in EpiLCs and cultured in an ES cell culture medium, ES cell-like colonies (alkaline phosphatase positive: AP positive) appeared. Moreover, it was confirmed that the expression level of Klf2 which is a pluripotency related gene increases. (FIG. 7). Therefore, it was shown that PRDM14 has an activity of imparting pluripotency to cells that have lost pluripotency.
In the process of dedifferentiation from EpiLCs to ES cells, 5hmC in the Klf2 region increased and 5mC decreased. From these results, it was suggested that PRDM14 induces dedifferentiation from EpiLCs to ES cells by performing DNA demethylation via 5hmC in cooperation with TET1.
6). Induction of DNA demethylation by co-expression of TET1 and PERDM14 in somatic cells [Experimental method]
Empty vector, Tet1-expressing vector and Prdm14-expressing vector were introduced into HEK293 cells, and Prdm14-positive cells were concentrated by drug selection and cultured for 4 days. Genomic DNA was collected from HEK293 cells cultured for 4 days, and then treated with the methylation sensitive enzyme HpaII. HpaII is a restriction enzyme that recognizes and cleaves the CCGG site, but cannot cleave when it is methylated. Quantitative PCR was performed by designing primers for the LINE-1 region using HpaII untreated and treated genomic DNA as a template. The methylation state of the LINE-1 region in each cell was quantified by subtracting Threshold Cycle (Ct) of the amplification curve of HpaII treatment from the PCR amplification curve of HpaII treatment.
[Results and discussion]
Although TET1 alone and PRDM14 alone could not induce demethylation of the LINE-1 region, significant demethylation was observed when both TET1 and PRDM14 were expressed (FIG. 8).
7). Demethylation of the LINE-1 region was observed by expressing PRDM14 in ES cells induced by DNA demethylation by co-expression of PRDM14 and TET1 . Although ES cells highly express endogenous TET1, expression of TET1 is hardly observed in HEK293 cells which are cell lines derived from human fetal kidney. Therefore, in HEK293 cells, PRDM14 or TET1 was expressed alone or PRDM14 and TET1 were coexpressed, and the methylation variation of the LINE-1 region was examined in each cell, thereby verifying the combined effect of PRDM14 and TET1. did.
[experimental method]
(1) DNA methylation analysis Genomic DNA is isolated from control cells, cells that highly express PRDM14, cells that highly express TET1, and cells that highly express PRDM14 and TET1 (all HE293 cells), and then treated with sulfite to produce non-methyl. Cytosine was converted to uracil. PCR was performed using this DNA as a template and a primer for the sequence of LINE-1, and the amplified product was cloned into a TA vector, and then the sequence was confirmed by sequencing. Unmethylated cytosine is finally replaced with T, but methylated cytosine remains as cytosine. Therefore, it was determined that CG site was unmethylated and CG was methylated. Moreover, in the above-mentioned DNA methylation analysis by sulfite treatment, 5-methylcytosine and 5-hydroxymethylcytosine cannot be distinguished. Therefore, 5-methylcytosine and 5-hydroxymethylcytosine were identified by a detection method using glucose transferase.
After collecting genomic DNA from each cell, the genomic DNA is reacted with glucosyltransferase and the substrate glucose. Glucose is not transferred to 5-methylcytosine, but glucose is transferred to 5-hydroxymethylcytosine. The CCGG sequence containing 5-hydroxymethylcytosine to which glucose has been transferred cannot be cleaved by the restriction enzyme MspI. After cleaving with HpaII and MspI, quantitative PCR was performed using primers that amplify by sandwiching the CCGG sequence, and the amounts of 5-methylcytosine and 5-hydroxymethylcytosine were quantified (hereinafter, this quantitative PCR is referred to as “ GlucMS-qPCR ").
(2) Quantitative RT-PCR
MRNA was extracted from control cells, cells highly expressing PRDM14, cells highly expressing TET1, and cells highly expressing PRDM14 and TET1 (all HE293 cells), and cDNA was prepared by reverse transcription reaction. Quantitative PCR was performed using this cDNA as a template and a primer for LINE-1, and the transcription amount of LINE-1 in each cell was measured.
[Results and discussion]
As a result of analyzing the methylation state of the LINE-1 region of control cells, cells that highly express PRDM14, cells that highly express TET1, and cells that highly express PRDM14 and TET1, the cells that highly express PRDM14 and TET1 are highly expressed. In comparison with cells that do, significant demethylation was observed in cells that highly express PRDM14 and TET1 (FIG. 9). Further, when the amounts of 5-methylcytosine and 5-hydroxymethylcytosine were compared, demethylation of only 5-methylcytosine was observed in cells that highly express PRDM14 and TET1 (FIG. 10). Furthermore, significant expression induction of LINE-1 was also observed in cells that highly express PRDM14 and TET1 (FIG. 11). From these results, it was revealed that demethylation of the LINE-1 region can be induced by combining PRDM14 and TET1.
8). Functional analysis of Tet1 and Tet2 (hereinafter referred to as Tet1 / 2) in DNA demethylation by PRDM14 Endogenous Tet1 / 2 is highly expressed in ES cells. In order to verify whether Tet1 / 2 is functionally required for DNA demethylation by PRDM14, PRDM14 is expressed in Tet1 / 2 double knockdown ES cells, and expression of a gene whose expression is increased by demethylation and a promoter The region methylation changes were analyzed.
[experimental method]
(1) Establishment of Prdm14-inducible expression ES cells Transfecting ES cells with a plasmid encoding tTA active in the presence of doxycycline and a plasmid with the Prdm14 gene inserted downstream of the tTA regulatory region, and selecting the drug. In doing so, doxycycline-inducible Prdm14-expressing ES cells were established.
(2) Establishment of Tet1 / 2 knockdown / doxycycline-inducible Prdm14-expressing ES cells The doxycycline-inducible Prdm14-expressing ES cells prepared in (1) are infected with a lentivirus expressing shRNA against Tet1 / 2, and Tet1 / 2 Knockdown / doxycycline-induced Prdm14-expressing ES cells were established.
[Results and discussion]
The results are shown in FIG. Prdm14 was inducibly expressed in control ES cells and Tet1 / 2 knockdown ES cells, and the expression changes of Slc25a31 and PiwiL2 were analyzed by quantitative RT-PCR. As a result, a marked increase in expression was observed in both Slc25a31 and Piwill2 in the control cells, whereas suppression of the increase in expression was observed in the Tet1 / 2 knockdown cells. In addition, when the amounts of 5-methylcytosine and 5-hydroxymethylcytosine in the promoter regions of Slc25a31 and PiwiL2 were analyzed, a decrease in 5-methylcytosine was observed in the control cells, but in the Tet1 / 2 knockdown ES cells. Methylation was incomplete. From these results, it was shown that the function of TET1 / 2 is essential for the induction of DNA demethylation by PRDM14, and the combination of PRDM14 and TET is necessary for efficient demethylation induction.
9. Quality improvement of low-quality iPS cells by PRDM14 The DNA demethylation-inducing activity by PRDM14 was examined for possible application to quality improvement of iPS cells. iPS cells have greatly different properties depending on the strain, and problems such as bias in the direction of differentiation and resistance to differentiation have been reported. This time, the possibility of quality improvement was verified by expressing Prdm14 in low-quality iPS cells with low expression of Nanog gene, which is a pluripotency inducer.
[experimental method]
Oct4, Sox2, Klf4, and c-myc were expressed in fetal fibroblasts established from Nanog-EGFP-Ires-Puro transgenic mice that can visualize the expression of Nanog with GFP using retrovirus. After culturing, cells that are very similar in morphology to ES cells but have low EGFP expression (low quality iPS cells) were established. Thereafter, doxycycline-induced Prdm14 high-expressing low-quality iPS cells were prepared, and EGFP expression fluctuation and gene expression fluctuation accompanying the induction of Prdm14 expression were analyzed. The methylation state of the promoter region of Nanog gene was quantified by GlucMS-qPCR.
[Results and discussion]
The results are shown in FIGS. With the induction of Prdm14 expression, increased expression of the pluripotency inducers Nanog and Klf2 was observed (FIG. 14). Further, since high expression of Tet1 / 2 was observed in low-quality iPS cells, it was considered that Prdm14 may induce DNA demethylation in cooperation with Tet1 / 2. Thus, as a result of the methylation analysis of the Nanog promoter region, a decrease in DNA methylation was observed with the induction of Prdm14 expression (FIG. 15). These results indicate that the combination of PRDM14 and TET can repair abnormal methylation of low-quality iPS cells.

Claims (9)

A method for inducing DNA demethylation in cultured cells, comprising:
(1) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having TET1 activity, or A fusion polypeptide comprising: and a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 10 to 12 and overexpressing each of the two polypeptides by introducing each into a cell. A method comprising increasing any intracellular content. A method for producing iPS cells from somatic cells using a nuclear reprogramming substance,
(1) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having TET1 activity, or A fusion polypeptide comprising: and a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 10 to 12 and overexpressing each of the two polypeptides by introducing each into a cell. A method comprising the step of increasing any intracellular content. (Α-1) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having TET1 activity Or a polynucleotide encoding a fusion polypeptide comprising the polypeptide; and (α-2) a DNA demethylation comprising a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 10-12 A composition used as an inducer. (A-1) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having TET1 activity Or an expression vector comprising a polynucleotide encoding a fusion polypeptide comprising the polypeptide; and (a-2) a polypeptide encoding a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 10 to 12 A composition comprising an expression vector comprising nucleotides. (B) a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence and having a TET1 activity A co-expression vector comprising a nucleotide or a polynucleotide encoding a fusion polypeptide containing the polypeptide; and a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 10-12. The composition according to claim 3 or 4, which is used as an agent for improving iPS cell quality. The co-expression vector according to claim 5, which is used as an agent for improving iPS cell quality. The composition according to claim 3 or 4, which is used as an agent for improving reprogramming completion of iPS cells. The co-expression vector according to claim 5, which is used as an agent for improving reprogramming completion of iPS cells.

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