# US 12,214,056 B2 · Therapeutic applications of CPF1-based genome editing

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

- Number: US 12,214,056 B2
- Document ID: US12214056B2
- Status: Granted
- Kind: B2
- Application number: 16/318,745
- Filed: 2017-07-19
- Published: 2019-05-23
- Granted: 2025-02-04
- Earliest claimed: 2016-07-19
- Assignees: Duke University
- Applicants: Duke University
- Inventors: Charles A. Gersbach; Sarina Madhavan; Christopher Nelson
- Web page: https://patentgrep.com/patent/US12214056B2

## CPC

- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07K 14/4708: {Duchenne dystrophy}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- 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 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/33: Alteration of splicing
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed herein are therapeutic applications of CRISPR/Cpf1-based genome editing.

## Claims

### Claim 1

A DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA selected from the group consisting of:
(i) a first Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 54 and a second Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 62;
(ii) a first Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 55 and a second Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 63; and
(iii) a first Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 56 and a second Cpf1 gRNA comprising a polynucleotide encoded by the sequence of SEQ ID NO: 61.

### Claim 2 (depends on 1)

The DNA targeting composition of claim 1, further comprising a Cpf1 endonuclease.

### Claim 3 (depends on 2)

The DNA targeting composition of claim 2, wherein the Cpf1 endonuclease recognizes a Protospacer Adjacent Motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

### Claim 4 (depends on 3)

The DNA targeting composition of claim 3, wherein the Cpf1 endonuclease is derived from a bacterial species selected from the group consisting of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_1 7, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae.

### Claim 5 (depends on 2)

The DNA targeting composition of claim 2, wherein the Cpf1 endonuclease is derived from Lachnospiraceae bacterium ND2006 (LbCpf1) or from Acidaminococcus (AsCpf1).

### Claim 6 (depends on 2)

The DNA targeting composition of claim 2, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO: 124 or SEQ ID NO: 125.

### Claim 7 (depends on 1)

A vector encoding the DNA targeting composition of claim 1.

### Claim 8 (depends on 7)

The vector of claim 7, further comprising a polynucleotide sequence encoding a Cpf1 endonuclease.

### Claim 9 (depends on 7)

The DNA targeting composition of claim 7, wherein the vector is a viral vector.

### Claim 10 (depends on 9)

The DNA targeting composition of claim 9, wherein the vector is an Adeno-associated virus (AAV) vector.

### Claim 11 (depends on 9)

The vector of claim 9, wherein the vector comprises a tissue-specific promoter operably linked to the polynucleotide sequence encoding the first Cpf1 gRNA, the second Cpf1 gRNA and/or the Cpf1 endonuclease.

### Claim 12 (depends on 11)

The DNA targeting composition of claim 11, wherein the tissue-specific promoter is a muscle specific promoter.