# US 11,028,388 B2 · CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa

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- Number: US 11,028,388 B2
- Document ID: US11028388B2
- Status: Granted
- Kind: B2
- Application number: 15/123,576
- Filed: 2015-03-05
- Published: 2017-03-16
- Granted: 2021-06-08
- Earliest claimed: 2014-03-05
- Assignees: Editas Medicine, Inc.
- Applicants: Editas Medicine, Inc.
- Inventors: Morgan L. Maeder; David A. Bumcrot
- Web page: https://patentgrep.com/patent/US11028388B2

## 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 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- 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 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

## Abstract

CRISPR/Cas-related compositions and methods for treatment of Usher Syndrome and/or Retinitis Pigmentosa are disclosed herein.

## Claims

### Claim 1

A method of altering a cell comprising contacting the cell with:
(a) a first gRNA comprising a first targeting domain which is complementary with a first target domain from the USH2A gene, wherein the first targeting domain is configured to provide a first cleavage event selected from a first double strand break and a first single strand break in a region of the USH2A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a nucleic acid sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401; and
(b) a Cas9 molecule,
wherein an NHEJ-mediated indel is generated by the first break, resulting in a deletion in the USH2A gene.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cell is from a subject suffering from or likely to develop Usher Syndrome or retinitis pigmentosa-39.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cell is from a subject having a mutation in the USH2A gene.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cell is a photoreceptor cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the contacting is performed ex vivo.

### Claim 6 (depends on 5)

The method of claim 5, wherein the contacted cell is returned to the subject's body.

### Claim 7 (depends on 1)

The method of claim 1, wherein the contacting is performed in vivo.

### Claim 8 (depends on 1)

The method of claim 1, wherein the contacting comprises contacting the cell with a nucleic acid comprising a sequence encoding (a) the first gRNA.

### Claim 9 (depends on 1)

The method of claim 1, further comprising contacting the cell with (c) a second gRNA.

### Claim 10 (depends on 9)

The method of claim 9, wherein the second gRNA comprises a targeting domain which is complementary with a target domain from the USH2A gene, wherein the second targeting domain is configured to provide a second cleavage event selected from a second double strand break and a second single strand break, within 200 nucleotides of the target position of the guanine deletion at nucleotide position 2299 (2299delG) in the USH2A gene.

### Claim 11 (depends on 10)

The method of claim 10, wherein the second cleavage event is in a region of the USH2A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a nucleic acid sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401.

### Claim 12

A method of altering a cell comprising contacting the cell with:
(a) a first gRNA comprising a first targeting domain which is complementary with a first target domain from the USH2A gene, the first targeting domain comprising a sequence that is the same as, or differs by no more than 3 nucleotides from, a first targeting domain sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401,
the first targeting domain configured to provide a cleavage event selected from a first double strand break and a first single strand break, in the USH2A gene; and
(b) a Cas9 molecule.

### Claim 13 (depends on 12)

The method of claim 12, wherein the cell is from a subject suffering from or likely to develop Usher Syndrome or retinitis pigmentosa-39.

### Claim 14 (depends on 12)

The method of claim 12, wherein the cell is from a subject having a mutation in the USH2A gene.

### Claim 15 (depends on 12)

The method of claim 12, wherein the cell is a photoreceptor cell.

### Claim 16 (depends on 12)

The method of claim 12, wherein the contacting is performed ex vivo.

### Claim 17 (depends on 14)

The method of claim 14, wherein the contacted cell is returned to the subject's body.

### Claim 18 (depends on 12)

The method of claim 12, wherein the contacting is performed in vivo.

### Claim 19 (depends on 12)

The method of claim 12, wherein the contacting comprises contacting the cell with a nucleic acid comprising a sequence encoding (a) the first gRNA.

### Claim 20 (depends on 12)

The method of claim 12, further comprising contacting the cell with (c) a second gRNA.

### Claim 21 (depends on 20)

The method of claim 20, wherein the second gRNA comprises a targeting domain which is complementary with a target domain from the USH2A gene, wherein the second targeting domain is configured to provide a cleavage event selected from a second double strand break and a second single strand break, within 200 nucleotides of the target position of the guanine deletion at nucleotide position 2299 (2299delG) in the USH2A gene.

### Claim 22 (depends on 20)

The method of claim 20, wherein the second targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a second targeting domain sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401.