TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR (TALE) - LYSINE-SPECIFIC DEMETHYLASE 1 (LSD1) FUSION PROTEINS

• US 20150267176A1
• Filed: 10/11/2013
• Published: 09/24/2015
• Est. Priority Date: 10/12/2012
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Joung, Jae Keith, Bernstein, Bradley E., Mendenhall, Eric M., Reyon, Deepak
• Application Number:
  US14/435,065
• Publication Number:
  US 20150267176A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/0026 acting on CH-NH groups of donors (1.5)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 105/00 Oxidoreductases acting on the CH-NH group of donors (1.5)
• CPC Class Codes: C12Y 114/99 Miscellaneous (1.14.99)

Abstract

Fusion proteins comprising a DNA binding domain, e.g., a TAL effector repeat array (TALE) or zinc finger array, and a catalytic domain comprising a sequence that catalyzes histone demethylation, and methods of use thereof.

Claims

1. A fusion protein comprising

an engineered DNA-binding domain that binds specifically to a preselected target sequence, and

a catalytic domain comprising a sequence that catalyzes histone demethylation.

2. The fusion protein of claim 1, further comprising a linker between the DNA binding domain and the catalytic domain.

3. The fusion protein of claim 1, wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease.

4. The fusion protein of claim 1, wherein the catalytic domain comprises full length LSD1, or a catalytic domain of LSD1.

5. The fusion protein of claim 4, wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2.

6. The fusion protein of claim 1, comprising a plurality of catalytic domains, optionally with linkers therebetween.

7. A method of reducing methylation of histones associated with a selected DNA sequence in a mammalian cell, the method comprising contacting the cell with a fusion protein comprising an engineered DNA-binding domain that binds specifically to a target sequence, wherein the target sequence is within about 10 kb, 5 kb, 2 kb, or 1 kb, 500 bp, 250 bp, 100 bp, 50 bp, 40 bp, 30 bp, or 20 bp, of the selected DNA sequence, and a catalytic domain comprising a sequence that catalyzes histone demethylation.

9. The method of claim 7, wherein the fusion protein further comprises a linker between the DNA binding domain and the catalytic domain.

10. The method of claim 7, wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease.

11. The method of claim 7, wherein the catalytic domain comprises a full-length LSD1 or a catalytic domain of LSD1.

12. The method of claim 11, wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2.

13. The method of claim 7, wherein the cell is a human cell.

14. The method of claim 7, wherein the cell is in a living mammal.

15. The method of claim 7, wherein the selected DNA sequence is a sequence of a p14^ARF gene.

23. The method of claim 7, wherein the fusion protein comprises a plurality of catalytic domains, optionally with linkers therebetween.

8. A method of reducing methylation of histones associated with a selected DNA sequence in a mammalian cell, the method comprising contacting the cell with a nucleic acid encoding a fusion protein comprising an engineered DNA-binding domain that binds specifically to a target sequence, wherein the target sequence is within about 10 kb, 5 kb, 2 kb, 1 kb, 500 bp, 250 bp, 100 bp, 50 bp, 40 bp, 30 bp, or 20 bp, of the selected DNA sequence, and a catalytic domain comprising a sequence that catalyzes histone demethylation.

16. The method of claim 8, wherein the fusion protein further comprises a linker between the DNA binding domain and the catalytic domain.

17. The method of claim 8, wherein the DNA-binding domain is or comprises an engineered transcription activator-like (TAL) effector repeat array, zinc finger, triplex-forming oligonucleotide, peptide nucleic acid, or a DNA-binding domain from a homing meganuclease (preferably a catalytically inactive homing meganuclease), or a catalytically inactive Cas9 nuclease.

18. The method of claim 8, wherein the catalytic domain comprises a full-length LSD1 or a catalytic domain of LSD1.

19. The method of claim 18, wherein the catalytic domain comprises amino acids 172-833 of the human LSD1 variant 2.

20. The method of claim 8, wherein the cell is a human cell.

21. The method of claim 8, wherein the cell is in a living mammal.

22. The method of claim 8, wherein the selected DNA sequence is a sequence of a p14^ARF gene.