# US 12,215,361 B2 · Compositions and methods for making epigenetic modifications

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- Number: US 12,215,361 B2
- Document ID: US12215361B2
- Status: Granted
- Kind: B2
- Application number: 18/444,590
- Filed: 2024-02-16
- Published: 2024-08-22
- Granted: 2025-02-04
- Earliest claimed: 2023-02-17
- Assignees: Whitehead Institute for Biomedical Research
- Applicants: Whitehead Institute for Biomedical Research
- Inventors: Edwin Neumann; Tessa Bertozzi; Jonathan Weissman
- Web page: https://patentgrep.com/patent/US12215361B2

## CPC

- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/86: Viral vectors
- C12Y 201/01037: DNA (cytosine-5-)-methyltransferase (2.1.1.37)
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Y: ENZYMES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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

### Claim 1

A fusion protein comprising a H3K4me0, a DNMT3 methyltransferase-binding domain, and a DNA-binding domain.

### Claim 2 (depends on 1)

The fusion protein 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.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

The fusion protein 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.

### Claim 5 (depends on 1)

The fusion protein 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.

### Claim 6 (depends on 1)

The fusion protein 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.

### Claim 7 (depends on 1)

The fusion protein of claim 1, comprising, 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.

### Claim 8 (depends on 1)

The fusion protein of claim 1, further comprising a Krüppel-associated box (KRAB) domain.

### Claim 9 (depends on 8)

The fusion protein of claim 8, wherein the KRAB domain is a KOX1 KRAB domain or a ZIM3 KRAB domain.

### Claim 10 (depends on 9)

The fusion protein of claim 9, comprising, 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.

### Claim 11 (depends on 1)

The fusion protein 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.

### Claim 12 (depends on 1)

The fusion protein 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.

### Claim 13 (depends on 1)

The fusion protein 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.

### Claim 14 (depends on 1)

The fusion protein 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.

### Claim 15 (depends on 8)

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

### Claim 16 (depends on 8)

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

### Claim 17 (depends on 1)

The fusion protein 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.

### Claim 18 (depends on 17)

The fusion protein 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.

### Claim 19 (depends on 17)

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

### Claim 20 (depends on 8)

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

### Claim 21 (depends on 20)

The fusion protein 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.

### Claim 22 (depends on 20)

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

### Claim 23 (depends on 1)

A polynucleotide encoding the fusion protein of claim 1.

### Claim 24 (depends on 23)

The polynucleotide of claim 23, wherein the polynucleotide is less than or equal to about 4.7 kb in length.

### Claim 25 (depends on 23)

A gene delivery system comprising the polynucleotide of claim 23 and an adeno-associated viral vector.

### Claim 26 (depends on 25)

The gene delivery system of claim 25, wherein the adeno-associated viral vector comprises an AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAV10 viral vector, or a variant thereof.

### Claim 27 (depends on 23)

A gene delivery system comprising the polynucleotide of claim 23 and lipid nanoparticles.

### Claim 28 (depends on 1)

A method of epigenetically modifying a genomic locus in a cell, comprising delivering to the cell the fusion protein of claim 1 or a polynucleotide encoding said fusion protein.

### Claim 29 (depends on 1)

A method of treating a disease in a subject in need thereof, comprising administering to the subject the fusion protein of claim 1 or a polynucleotide encoding said fusion protein.

### Claim 30 (depends on 29)

The method of claim 29, wherein the disease is a genetic disorder, an infectious disease, or a neurodegenerative disease.