TRANSCRIPTIONAL REGULATORY FUSION POLYPEPTIDE

• US 20190330280A1
• Filed: 12/11/2017
• Published: 10/31/2019
• Est. Priority Date: 12/12/2016
• Status: Active Grant

• Current Assignee: Astellas Pharma Incorporated, Hiroshima University, National Center for Global Health and Medicine
• Original Assignee: Astellas Pharma Incorporated
• Inventors: ENDO, Hideki, ISHIZAKA, Yukihito, YAMAMOTO, Takashi, SAKUMA, Tetsushi
• Granted Patent: US 11,390,652 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0656 Adult fibroblasts
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/506 derived from RNA viruses
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)
• CPC Class Codes: C12Y 304/22044 : Nuclear-inclusion-a endopeptidase (3.4.22.44)

Abstract

[Problem to be Solved] It is intended to provide a fusion polypeptide that regulates the transcription of a target gene.

[Solution] The present inventors have provided a fusion polypeptide comprising: a cell-penetrating peptide; a DNA-binding polypeptide; and a transcriptional regulator.

Claims

1. A fusion polypeptide comprising:

a cell-penetrating peptide;

a DNA-binding polypeptide that binds to the nucleotide sequence represented by SEQ ID NO;

49; and

a transcriptional activator.

2. The fusion polypeptide according to claim 1, wherein the transcriptional activator is VP64, VPR, p300 or GCN5.

3. The fusion polypeptide according to claim 1, wherein the cell-penetrating peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO:

53, 54, 55, 56, 57 or 60.

4. The fusion polypeptide according to claim 1, wherein the cell-penetrating peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO:

56 or 60.

5. The fusion polypeptide according to claim 1, wherein the DNA-binding polypeptide is transcription activator-like effector (TALE).

6. The fusion polypeptide according to claim 5, wherein the DNA-binding polypeptide is TALE comprising an amino acid sequence consisting of amino acid numbers 7 to 784 of SEQ ID NO:

2.

7. The fusion polypeptide according to claim 1, wherein the DNA-binding polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

2.

8. The fusion polypeptide according to claim 1, wherein the cell-penetrating peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO:

56 or 60, the DNA-binding polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO;

2, and the transcriptional activator is VPR.

9. The fusion polypeptide according to claim 1, wherein the fusion polypeptide is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

62.

10. A polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide according to claim 1.

11. An expression vector comprising a polynucleotide according to claim 10.

12. A host cell transformed with an expression vector according to claim 11.

13. A method for producing a fusion polypeptide, comprising the step of culturing a host cell according to claim 12.

14. A method for increasing the expression of human telomerase reverse transcriptase (TERT) gene in a human somatic cell, comprising the step of culturing the human somatic cell in a medium containing a fusion polypeptide according to claim 1.

15. A method for proliferating human somatic cells, comprising the step of culturing the human somatic cells in a medium containing a fusion polypeptide according to claim 1.