# US 11,028,394 B2 · CRISPR/CAS-related methods and compositions for treating cystic fibrosis

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- Number: US 11,028,394 B2
- Document ID: US11028394B2
- Status: Granted
- Kind: B2
- Application number: 15/288,475
- Filed: 2016-10-07
- Published: 2017-01-26
- Granted: 2021-06-08
- Earliest claimed: 2014-04-09
- Assignees: Editas Medicine, Inc.
- Applicants: Editas Medicine, Inc.
- Inventors: Deepak Reyon; Morgan L. Maeder; Ari E. Friedland; G. Grant Welstead; David A. Bumcrot
- Web page: https://patentgrep.com/patent/US11028394B2

## CPC

- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/34: Allele or polymorphism specific uses
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are genome editing systems and compositions that target a cystic fibrosis transmembrane conductance regulator (CFTR) gene and a sodium channel epithelial 1 alpha (SCNN1A) gene, comprising a Cas9 molecule, and a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CFTR gene or a SCNN1A gene, and cells comprising such genome editing systems and compositions. Also provided are methods for using the genome editing systems, compositions, and cells for genome engineering (e.g., altering a CFTR gene and/or a SCNN1A gene), and for preventing or treating Cystic Fibrosis (CF) and CF-like disease.

## Claims

### Claim 1

A CRISPR/Cas system, comprising:
a) a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene or a sodium channel epithelial 1 alpha (SCNN1A) gene, wherein said target sequence of said CFTR gene is within intron 2 or intron 10 of said CFTR gene; and
b) a Cas9 molecule,
wherein said targeting domain that is complementary to said CFTR gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265 and wherein said targeting domain that is complementary to said SCNN1A gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100.

### Claim 2 (depends on 1)

The system of claim 1, wherein said targeting domain that is complementary to said CFTR gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265.

### Claim 3 (depends on 1)

The system of claim 1, wherein said targeting domain that is complementary to said SCNN1A gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100.

### Claim 4

A gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CFTR gene or a SCNN1A gene, wherein the target sequence of the CFTR gene is within intron 2 or intron 10 of the CFTR gene, wherein said targeting domain that is complementary to said CFTR gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265 and wherein said targeting domain that is complementary to said SCNN1A gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100.

### Claim 5 (depends on 4)

The gRNA molecule of claim 4, wherein said targeting domain that is complementary to said CFTR gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence set forth in SEQ ID NOs: 15101-27265.

### Claim 6 (depends on 4)

The gRNA molecule of claim 4, wherein said targeting domain that is complementary to said SCNN1A gene comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100.

### Claim 7

A CRISPR/Cas system, comprising:
a) a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a cystic fibrosis transmembrane conductance regulator (CFTR) gene or a sodium channel epithelial 1 alpha (SCNN1A) gene, wherein said target sequence of said CFTR gene is within intron 2 or intron 10 of said CFTR gene, wherein said targeting domain that is complementary to said SCNN1A gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100 and wherein said targeting domain that is complementary to said CFTR gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265;
b) a Cas9 molecule; and
c) a cDNA donor template that alters said CFTR gene or SCNN1A gene, wherein said cDNA donor template that alters said CFTR gene comprises (a) contiguous coding sequence of exon 3 through exon 27 of said CFTR gene; or (b) contiguous coding sequence of exon 11 through exon 27 of said CFTR gene.

### Claim 8

A composition comprising:
a) a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CFTR gene or a SCNN1A gene, wherein the target sequence of the CFTR gene is within intron 2 or intron 10 of the CFTR gene, wherein said targeting domain that is complementary to said SCNN1A gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100 and wherein said targeting domain that is complementary to said CFTR gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265; and
b) a cDNA donor template that alters said CFTR gene or SCNN1A gene, wherein said cDNA donor template that alters said CFTR gene comprises (a) contiguous coding sequence of exon 3 through exon 27 of said CFTR gene; or (b) contiguous coding sequence of exon 11 through exon 27 of said CFTR gene.

### Claim 9

A nucleic acid composition that comprises:
a) a first nucleotide sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CFTR gene or a SCNN1A gene, wherein the target sequence of the CFTR gene is within intron 2 or intron 10 of the CFTR gene, wherein said targeting domain that is complementary to said SCNN1A gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 497-654, 803-916, and 4176-15100 and wherein said targeting domain that is complementary to said CFTR gene comprises a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences set forth in SEQ ID NOs: 15101-27265; and
b) a template nucleic acid comprising a cDNA donor template that alters said CFTR gene or SCNN1A gene, wherein said cDNA donor template that alters said CFTR gene comprises (a) contiguous coding sequence of exon 3 through exon 27 of said CFTR gene; or (b) contiguous coding sequence of exon 11 through exon 27 of said CFTR gene.