# US 12,060,588 B2 · Methods of editing DNA methylation

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- Number: US 12,060,588 B2
- Document ID: US12060588B2
- Status: Granted
- Kind: B2
- Application number: 17/858,758
- Filed: 2022-07-06
- Published: 2023-03-30
- Granted: 2024-08-13
- Earliest claimed: 2016-08-19
- Assignees: Whitehead Institute for Biomedical Research
- Applicants: Whitehead Institute for Biomedical Research
- Inventors: Rudolf Jaenisch; X. Shawn Liu; Hao Wu
- Web page: https://patentgrep.com/patent/US12060588B2

## CPC

- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61K 35/33: Fibroblasts
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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)}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12Y 201/01037: DNA (cytosine-5-)-methyltransferase (2.1.1.37)
- A01K 2227/105: Murine
- A01K 2267/0393: Animal model comprising a reporter system for screening tests
- C07K 2319/00: Fusion polypeptide
- C07K 2319/09: containing a nuclear localisation signal
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2740/15031: Uses of virus other than therapeutic or vaccine, e.g. disinfectant
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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 invention relates to methods of modifying DNA methylation by contacting a cell with a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity and one or more guide sequences.

## Claims

### Claim 1

A method of modulating the methylation of one or more genomic sequences in an individual, the method comprising administering to the individual: a. a nucleic acid that encodes a polypeptide comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; b. a guide sequence or a nucleic acid that encodes a guide sequence, wherein the genomic sequence comprises a CTCF binding site or an enhancer of MyoD, and wherein the effector domain comprises Tet1 or Dnmt3a.

### Claim 2 (depends on 1)

The method of claim 1, wherein the guide sequence targets the polypeptide to the one or more genomic sequences.

### Claim 3 (depends on 1)

The method of claim 1, wherein the genomic sequence further comprises a differentially methylated region or a promoter.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method comprises modulating the methylation of at least two genomic sequences in a cell, wherein the genomic sequences are selected from differentially methylated regions, enhancers, promoters, and CTCF binding sites.

### Claim 5 (depends on 1)

The method of claim 1, wherein the genomic sequence further comprises an enhancer or a promoter.

### Claim 6 (depends on 1)

The method of claim 1, wherein the promoter is a BDNF promoter.

### Claim 7 (depends on 1)

The method of claim 1, wherein the catalytically inactive site specific nuclease is a catalytically inactive Cas protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein.

### Claim 9 (depends on 1)

The method of claim 1, wherein the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein.

### Claim 10 (depends on 1)

The method of claim 1, wherein the guide sequence is a ribonucleic acid guide sequence.

### Claim 11 (depends on 1)

The method of claim 1, wherein the guide sequence is from about 10 base pairs to about 150 base pairs in length.

### Claim 12 (depends on 1)

The method of claim 1, wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genomic sequences are modified in the individual.

### Claim 13 (depends on 1)

The method of claim 1, wherein the individual is a human.

### Claim 14 (depends on 1)

The method of claim 1, wherein the individual has a disease, disorder, or condition.