Mutant KLF protein, and method for producing induced pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-12559730-B2
Application number	US-202017758747-A
Country	US
Kind code	B2
Filing date	Dec 16, 2020
Priority date	Jan 16, 2020
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

Abstract

There is provided a mutant KLF protein that can induce reprogramming of a somatic cell at a higher efficiency than a KLF protein having a natural amino acid sequence. There is also provided a method for efficiently producing an iPS cell by using the mutant KLF protein. There is provided a mutant KLF protein having an amino acid substitution, or a peptide fragment thereof containing the amino acid substitution.

First claim

The invention claimed is:

1 . A mutant Krüppel-like factor (KLF) protein comprising an amino acid substitution, wherein
the amino acid substitution is a substitution of any of the following:
(a) serine at position 349 and/or leucine at position 356 in the amino acid sequence set forth in SEQ ID NO: 1;
(b) serine at position 342 and/or leucine at position 349 in the amino acid sequence set forth in SEQ ID NO: 3;
(c) serine at position 500 and/or leucine at position 507 in the amino acid sequence set forth in SEQ ID NO: 5; or
(d) serine at position 443 and/or leucine at position 450 in the amino acid sequence represented by SEQ ID NO: 7.

2 . The mutant KLF protein according to claim 1 , wherein
the substitution of (a) is S349A, and/or L356A, L356N, L356D, L356C, L356E, L356G, L356K, L356M, L356S, or L356T, the substitution of (b) is S342A, and/or L349A, L349N, L349D, L349C, L349E, L349G, L349K, L349M, L349S, or L349T, the substitution of (c) is S500A, and/or L507A, L507N, L507D, L507C, L507E, L507G, L507K, L507M, L507S, or L507T, or the substitution of (d) is S443A, and/or L450A, L450N, L450D, L450C, L450E, L450G, L450K, L450M, L450S, or L450T.

3 . A nucleic acid encoding the mutant KLF protein according to claim 1 .

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

5 . An induced pluripotent stem cell (iPS cell) inducer comprising the nucleic acid according to claim 3 .

6 . A direct reprogramming agent comprising the nucleic acid according to claim 3 .

7 . A cancer therapeutic agent comprising the nucleic acid according to claim 3 .

8 . An induced pluripotent stem cell (iPS cell) inducer comprising the gene expression vector according to claim 4 .

9 . A direct reprogramming agent comprising the gene expression vector according to claim 4 .

10 . A cancer therapeutic agent comprising the gene expression vector according to claim 4 .

11 . An induced pluripotent stem cell (iPS cell) inducer comprising the mutant KLF protein according to claim 1 .

12 . The iPS cell inducer according to claim 11 , further comprising (i) and/or (ii):
(i) any of an OCT3/4 protein, a nucleic acid encoding the protein, or a gene expression vector comprising the nucleic acid in an expressible state; (ii) any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein or a SOX17 protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state.

13 . The iPS cell inducer according to claim 12 , further comprising
(iii) any of a C-MYC protein, a T58A mutant of the C-MYC protein, an N-MYC protein or a L-MYC protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state.

14 . A direct reprogramming agent comprising a mutant KLF protein according to claim 1 .

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

16 . The production method according to claim 15 , further comprising a selection step of selecting an iPS cell induced in the cultivation step.

17 . The production method according to claim 15 , wherein the somatic cell is human-derived.

18 . A method for producing an iPS cell, comprising introducing an iPS cell inducer comprising the following (1) to (4), into a somatic cell:
(1) a mutant KLF protein according to claim 1 , (2) any of an OCT3/4 protein, a nucleic acid encoding the protein, or a gene expression vector comprising the nucleic acid in an expressible state, (3) any of a SOX1 protein, a SOX2 protein, a SOX3 protein, a SOX15 protein or a SOX17 protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state, and (4) any of a C-MYC protein, an N-MYC protein, an L-MYC protein, or a T58A mutant protein of the C-MYC protein, a nucleic acid encoding any of the proteins, or a gene expression vector comprising the nucleic acid in an expressible state; and cultivating the somatic cell after the introduction step.

19 . A cancer therapeutic agent comprising, the mutant KLF protein according to claim 1 .

Assignees

• Riken
• Univ Tsukuba

Inventors

• Hayashi Yohei
• BORISOVA Evgeniia
• Hisatake Koji
• Nishimura Ken
• Yumoto Fumiaki

Classifications

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N2501/606

  c-Myc · CPC title

• C12N2501/604

  Klf-4 · CPC title

• C12N2501/603

  Oct-3/4 · CPC title

• C12N2501/602

  Sox-2 · CPC title