# US 11,896,651 B2 · Gene editing of deep intronic mutations

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- Number: US 11,896,651 B2
- Document ID: US11896651B2
- Status: Granted
- Kind: B2
- Application number: 15/574,420
- Filed: 2016-04-15
- Published: 2019-01-24
- Granted: 2024-02-13
- Earliest claimed: 2015-05-16
- Assignees: Genzyme Corporation
- Applicants: Genzyme Corporation
- Inventors: Guoxiang Ruan; Abraham Scaria
- Web page: https://patentgrep.com/patent/US11896651B2

## CPC

- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 3/12: for electrolyte homeostasis
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 13/12: of the kidneys
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- 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 27/00: Drugs for disorders of the senses
- A61P 27/02: Ophthalmic agents
- A61P 27/12: for cataracts
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are compositions, methods, kits, and viral particles for treating a disease or disorder associated with a deep intronic mutation using an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system. In some aspects, provided herein is a self-limiting CRISPER-Cas system.

## Claims

### Claim 1

A composition for cleaving a target DNA in a cell comprising:
a) a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising a first guide RNA and a second guide RNA, wherein the target DNA comprises a deep intronic mutation in a CEP290 gene and sequences flanking the mutation, wherein the first guide RNA and second guide RNA are capable of hybridizing to opposite strands of the flanking sequences; and
b) a recombinant adeno-associated virus (rAAV) vector comprising a Cas expression cassette, wherein the Cas expression cassette comprises:
i) a nucleotide sequence encoding a Cas9 protein operably linked to a promoter that is a single minimal CMV promoter consisting of nucleotides 34-216 of SEQ ID NO:56, and
ii) a target site for a single guide RNA, wherein the first guide RNA or the second guide RNA is capable of hybridizing to the target site;
wherein the Cas9 protein is capable of being expressed from the Cas expression cassette;
wherein the Cas9 protein is capable of cleaving the target DNA sequences flanking the mutation to thereby excise a portion of target DNA comprising the mutation; and
wherein the Cas9 protein is capable of cleaving the Cas expression cassette at the target site in the Cas expression cassette to thereby reduce expression of the Cas9 protein, as compared to expression of the Cas9 protein prior to cleavage of the Cas expression cassette.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the nucleic acid encoding the CRISPR-Cas system and the Cas expression cassette are located on the same or different vectors.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the vector encoding the CRISPR-Cas system is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenoviral vector, a recombinant lentiviral vector or a recombinant herpes simplex virus (HSV) vector.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the deep intronic mutation is a c.2991+1655A>G mutation.

### Claim 5

A composition for treating Leber congenital amaurosis associated with a deep intronic mutation in a target DNA acid of an individual comprising:
a) a nucleic acid encoding an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising a first guide RNA and a second guide RNA, wherein the target DNA comprises a deep intronic mutation in a CEP290 gene and sequences flanking the mutation, wherein the first guide RNA and second guide RNA are capable of hybridizing to opposite strands of the flanking sequences; and
b) a recombinant adeno-associated virus (rAAV) vector comprising a Cas expression cassette, wherein the Cas expression cassette comprises:
i) a nucleotide sequence encoding a Cas9 protein operably linked to a promoter that is a single minimal CMV promoter consisting of nucleotides 34-216 of SEQ ID NO:56, and
ii) a target site for a single guide RNA, wherein the first guide RNA or the second guide RNA is capable of hybridizing to the target site;
wherein the Cas9 protein is capable of being expressed from the Cas expression cassette;
wherein the Cas9 protein is capable of cleaving the target DNA sequences flanking the mutation to thereby excise a portion of target DNA comprising the mutation; and
wherein the Cas9 protein is capable of cleaving the Cas expression cassette at the single target site in the Cas expression cassette to thereby reduce expression of the Cas9 protein, as compared to expression of the Cas9 protein prior to cleavage of the Cas expression cassette.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the deep intronic mutation is a c.2991+1655A>G mutation.