IP Library › Granted Patent US 12,365,884
Granted Patent B2
US 12,365,884 · App. 18/894,709 · Granted Jul 22, 2025

Compositions and methods for making epigenetic modifications

Inventors: Edwin Neumann (Somerville, MA); Tessa Bertozzi (Cambridge, MA); Jonathan Weissman (Cambridge, MA)
Assignee: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH
C12N9/1007A61K38/465C12N9/22C12N15/86C12Y201/01037C07K2319/80C12N2750/14143
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Quick Facts
Patent No.
US 12,365,884
App. No.
18/894,709
Granted
Jul 22, 2025
Kind
B2
Abstract

The disclosure provides, in various embodiments, fusion proteins comprising a DNA-binding domain, a DNMT3A-binding domain, and a H3K4me0; and polynucleotides and vectors encoding one or more of the fusion proteins. The disclosure also provides, in various embodiments, gene-delivery systems, cells, compositions (e.g., pharmaceutical compositions) and kits comprising one or more of the fusion proteins polynucleotides, or vectors; methods of epigenetically modifying a genomic locus in a cell; and methods of treating a subject (e.g., a human) in need thereof.

Claims (61)

1. A ribonucleic acid (RNA) encoding a fusion protein comprising a H3K4me0, a DNMT3 methyltransferase-binding domain, and a DNA-binding domain.

2. The RNA of claim 1 , wherein:

a) the fusion protein lacks nuclease activity;

b) the fusion protein lacks a DNA methyltransferase catalytic domain;

c) the DNMT3 methyltransferase-binding domain binds a catalytic domain of DNMT3A,

or any combination of the foregoing.

3. The RNA of claim 1 , wherein the DNMT3 methyltransferase-binding domain comprises a DNA methyltransferase 3-like protein (Dnmt3L), or a C-terminal fragment of Dnmt3L.

4. The RNA of claim 1 , wherein the DNA-binding domain comprises a DNA-binding domain of:

a) a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas);

b) a transcription activator-like effector nuclease (TALEN);

c) a zinc finger nuclease (ZFN);

d) a tetracycline-controlled repressor (tetR);

e) a meganuclease;

f) a homing (HO) endonuclease; or

g) a eukaryotic programmable RNA-guided endonuclease.

5. The RNA of claim 1 , wherein the DNA-binding domain comprises:

a) Streptococcus pyogenes dCas9;

b) Staphylococcus aureus dCas9;

c) S. aureus dCas9;

d) dCas12a; or

e) dCas12f.

6. The RNA of claim 1 , wherein the DNA-binding domain comprises a leucine zipper domain, a winged helix domain, a helix-turn-helix domain, a helix-loop-helix domain, a chromatin-associated high-mobility group (HMG)-box domain, a white-opaque regulator 3 (Wor3) domain, an oligonucleotide/oligosaccharide-binding (OB)-fold domain, an immunoglobulin domain, or a B3 DNA-binding domain.

7. The RNA of claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus:

a) the H3K4me0, the DNMT3 methyltransferase-binding domain, and the DNA-binding domain; or

b) the H3K4me0, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain.

8. The RNA of claim 1 , wherein the fusion protein further comprises a Krüppel-associated box (KRAB) domain.

9. The RNA of claim 8 , wherein the KRAB domain is a KOX1 KRAB domain or a ZIM3 KRAB domain.

10. The RNA of claim 9 , wherein the fusion protein comprises, from N-terminus to C-terminus:

a) the H3K4me0, the DNMT3 methyltransferase-binding domain, the DNA-binding domain, and the KRAB domain;

b) the H3K4me0, the KRAB domain, the DNMT3 methyltransferase-binding domain, and the DNA-binding domain;

c) the H3K4me0, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain, and the KRAB domain;

d) the H3K4me0, the KRAB domain, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain; or

e) the H3K4me0, the DNA-binding domain, the KRAB domain, and the DNMT3 methyltransferase-binding domain.

11. The RNA of claim 1 , wherein:

a) the H3K4me0 has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:87, SEQ ID NO:81, and SEQ ID NOs: 393-396;

b) the DNMT3 methyltransferase-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NOs: 31-50 and 71-75;

c) the DNA-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs: 3-15, and SEQ ID NOs: 454-469;

or any combination of the foregoing.

12. The RNA of claim 1 , wherein:

a) the H3K4me0 has 100% sequence identity to a sequence set forth in SEQ ID NO: 87, SEQ ID NO:81, and SEQ ID NOs: 393-396;

b) the DNMT3 methyltransferase-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NOs: 31-50 and 71-75;

c) the DNA-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs: 3-15, and SEQ ID NOs: 454-469;

or any combination of the foregoing.

13. The RNA of claim 1 , wherein:

a) the H3K4me0 has at least 80% sequence identity to SEQ ID NO:87, SEQ ID NO: 81, or both;

b) the DNMT3 methyltransferase-binding domain has at least 80% sequence identity to SEQ ID NO:31, SEQ ID NO:32, or both;

c) the DNA-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs: 3-9, and SEQ ID NOs: 454-461,

or any combination of the foregoing.

14. The RNA of claim 1 , wherein:

a) the H3K4me0 has 100% sequence identity to SEQ ID NO:87 or SEQ ID NO:81;

b) the DNMT3 methyltransferase-binding domain has 100% sequence identity to SEQ ID NO:31 or SEQ ID NO:32;

c) the DNA-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs: 3-9, and SEQ ID NOs: 454-461,

or any combination of the foregoing.

15. The RNA of claim 8 , wherein the KRAB domain has at least 80% sequence identity to SEQ ID NO:155.

16. The RNA of claim 8 , wherein the KRAB domain has 100% sequence identity to SEQ ID NO: 155.

17. The RNA of claim 1 , wherein the H3K4me0 is connected to the DNMT3 methyltransferase-binding domain with a linker of about 30-50 amino acids in length.

18. The RNA of claim 17 , wherein the linker has 100% sequence identity to a sequence set forth in SEQ ID NOs: 89-100, SEQ ID NOs: 113-128, SEQ ID NO: 145, SEQ ID NO:147, and SEQ ID NO:506.

19. The RNA of claim 17 , wherein the linker has 100% sequence identity to SEQ ID NO:96 or SEQ ID NO:506.

20. The RNA of claim 8 , wherein the H3K4me0 is connected to the KRAB domain with a linker of about 30-50 amino acids in length.

21. The RNA of claim 20 , wherein the linker has 100% sequence identity to a sequence set forth in SEQ ID NOs: 89-100, SEQ ID NOs: 113-128, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO:506.

22. The RNA of claim 20 , wherein the linker has 100% sequence identity to SEQ ID NO:96 or SEQ ID NO:506.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 28, 2024
From: NEUMANN, EDWIN; BERTOZZI, TESSA; WEISSMAN, JONATHAN
To: WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH
Reel/Frame 069042/0815 →
Continuity (4)
Continuation 18444590 · Feb 16, 2024
Provisional Application 63520594 · Aug 18, 2023
Provisional Application 63485860 · Feb 17, 2023
Related Publication 20250019673A1 · Jan 16, 2025
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