# US 12,509,683 B2 · Methods and compositions for modulating gene expression

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,509,683 B2
- Document ID: US12509683B2
- Status: Granted
- Kind: B2
- Application number: 17/698,912
- Filed: 2022-03-18
- Published: 2023-06-08
- Granted: 2025-12-30
- Earliest claimed: 2016-09-07
- Assignees: Flagship Pioneering Innovations V, Inc.
- Applicants: Flagship Pioneering Innovations V, Inc.
- Inventors: David Arthur Berry; Rahul Karnik; Laura Gabriela Lande
- Web page: https://patentgrep.com/patent/US12509683B2

## CPC

- 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)}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00)
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/80: acting on amide bonds in linear amides {(3.5.1)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/85: for animal cells
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 15/907: {in mammalian cells}
- C12Y 201/01037: DNA (cytosine-5-)-methyltransferase (2.1.1.37)
- C12Y 201/01043: Histone-lysine N-methyltransferase (2.1.1.43)
- C12Y 305/01098: Histone deacetylase (3.5.1.98), i.e. sirtuin deacetylase
- A01K 2217/056: due to mutation of coding region of the transgene (dominant negative)
- A01K 2227/706: Insects, e.g. Drosophila melanogaster, medfly
- A01K 2267/0318: Animal model for neurodegenerative disease, e.g. non- Alzheimer's
- C07K 2319/00: Fusion polypeptide
- C07K 2319/85: containing an RNA binding domain
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- 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)
- 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 present disclosure provides compositions with a modulating gene expression and methods for modulating transcription.

## Claims

### Claim 1

A method of decreasing expression of a gene within a cell, the gene being within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:
contacting the cell with a composition comprising a targeting element or a nucleic acid encoding the targeting element,
wherein the targeting element comprises an enzymatically inactive Cas nuclease and a gRNA which targets the first anchor sequence, the second anchor sequence, or both of the first anchor sequence and the second anchor sequence,
wherein the first and second anchor sequences each comprise a CTCF binding site, and wherein the CTCF binding site has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 2 (depends on 1)

The method of claim 1, wherein neither the first nor second anchor sequences are located within an internal enhancing sequence or a promoter.

### Claim 3 (depends on 1)

The method of claim 1, wherein the gene is FOXJ3, MYC, SHMT2, or CDK6.

### Claim 4 (depends on 1)

The method of claim 1, wherein the gRNA comprises a sequence of any of SEQ ID NOs: 40-47, 86, or 87.

### Claim 5 (depends on 4)

The method of claim 4, wherein the cell is a human cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the Cas nuclease comprises Cas9.

### Claim 7 (depends on 1)

The method of claim 1, wherein contacting comprises transfecting the cell with the composition.

### Claim 8 (depends on 1)

The method of claim 1, wherein the gRNA comprises a sequence at least 90% complementary to the first and/or second anchor sequence.

### Claim 9 (depends on 1)

The method of claim 1, wherein the composition physically interferes with formation and/or maintenance of the anchor sequence-mediated conjunction.

### Claim 10 (depends on 1)

The method of claim 1, wherein the gene is an oncogene, a tumor suppressor, or a gene associated with a nucleotide repeat.

### Claim 11 (depends on 1)

The method of claim 1, wherein the targeting agent is linked to an effector domain with an amino acid linker.

### Claim 12 (depends on 11)

The method of claim 11, wherein the effector domain comprises a DNA methyltransferase, histone methyltransferase, acetyltransferase, or deacetylase.

### Claim 13 (depends on 1)

The method of claim 1, wherein the CTCF binding site has the sequence of SEQ ID NO: 1.

### Claim 14 (depends on 1)

The method of claim 1, wherein the CTCF binding site has the sequence of SEQ ID NO: 2.

### Claim 15 (depends on 1)

The method of claim 1, wherein the gene is separated from the first anchor sequence or the second anchor sequence by about 1 kb to about 100 Mb.

### Claim 16 (depends on 1)

The method of claim 1, wherein the gene is separated from the internal enhancing sequence by about 100 kb to about 750 kb.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first anchor sequence is located at least 400 base pairs away from any transcription start site.

### Claim 18 (depends on 1)

The method of claim 1, wherein the anchor sequences are between 20-80 nucleotides.

### Claim 19 (depends on 1)

The method of claim 1, wherein the targeting element binds the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 20 (depends on 1)

The method of claim 1, wherein the targeting element binds a sequence proximal to SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 21 (depends on 1)

The method of claim 1, which comprises contacting the cell with the nucleic acid encoding the targeting element.