# US 10,369,232 B2 · Allele selective gene editing and uses thereof

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- Number: US 10,369,232 B2
- Document ID: US10369232B2
- Status: Granted
- Kind: B2
- Application number: 15/272,107
- Filed: 2016-09-21
- Published: 2017-03-23
- Granted: 2019-08-06
- Earliest claimed: 2015-09-21
- Assignees: Arcturus Therapeutics, Inc.
- Applicants: Arcturus Therapeutics, Inc.
- Inventors: Padmanabh Chivukula; Rachel Wilkie-Grantham; Kiyoshi Tachikawa
- Web page: https://patentgrep.com/patent/US10369232B2

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 3/06: Antihyperlipidemics
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 17/00: Drugs for dermatological disorders
- A61P 19/00: Drugs for skeletal disorders
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/16: Anti-Parkinson drugs
- A61P 27/02: Ophthalmic agents
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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 2310/323: modified ring structure
- C12N 2320/34: Allele or polymorphism specific uses
- 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

This invention encompasses compounds, structures, compositions and methods for therapeutic guide molecules that direct CRISPR gene editing. A guide molecule for directing gene editing can be allele selective, or disease allele selective, and can exhibit reduced off target activity. A guide molecule can be composed of monomers, including UNA monomers, nucleic acid monomers, and modified nucleotides, wherein the compound is targeted to a genomic DNA. The guide molecules of this invention can be used as active ingredients for editing or disrupting a gene in vitro, ex vivo, or in vivo.

## Claims

### Claim 1

A guide compound targeted to a genomic DNA, comprising a target guide chain of 14-24 contiguous monomers attached to a crRNA, wherein the guide compound directs CRISPR gene editing of the genomic DNA, wherein the monomers comprise UNA monomers and nucleic acid monomers, and wherein the guide compound comprises a sequence of bases targeted to direct CRISPR gene editing of the genomic DNA.

### Claim 2 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs double strand breaks in human gene TTR and the target guide chain comprises 16-20 contiguous monomers of 5′-UGCAUGGCCACAUUGAUGGC-3′ (SEQ ID NO:13), wherein the crRNA is attached at the 3′ end of the target guide chain.

### Claim 3 (depends on 2)

The guide compound of claim 2, wherein the guide compound comprises SEQ ID NO:32.

### Claim 4 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs double strand breaks in human gene TTR and the target guide chain comprises 16-20 contiguous monomers of 5 ′ -CACAUGCAUGGCCACAUUGA-3′ (SEQ ID NO:40), wherein the crRNA is attached at the 3′ end of the target guide chain.

### Claim 5 (depends on 4)

The guide compound of claim 4, wherein the guide compound comprises SEQ ID NO:61.

### Claim 6 (depends on 1)

The guide compound of claim 1, wherein the crRNA is 5′-GUUUUAGAGCUAUGCU-3′ (SEQ ID NO:605).

### Claim 7 (depends on 1)

The guide compound of claim 1, wherein the sequence of bases of the target guide chain has up to three mismatches from the genomic DNA.

### Claim 8 (depends on 1)

The guide compound of claim 1, wherein the guide compound contains one to five UNA monomers.

### Claim 9 (depends on 1)

The guide compound of claim 1, wherein one or more of the nucleic acid monomers is a 2′-O-methyl ribonucleotide, a 2′-O-methyl purine nucleotide, a 2′-deoxy-2′-fluoro ribonucleotide, a 2′-deoxy-2′-fluoro pyrimidine nucleotide, a 2′-deoxy ribonucleotide, a 2′-deoxy purine nucleotide, a universal base nucleotide, a 5-C-methyl-nucleotide, an inverted deoxyabasic monomer residue, a 3′-end stabilized nucleotide, a 3′-glyceryl nucleotide, a 3′-inverted abasic nucleotide, a 3′-inverted thymidine, a locked nucleic acid nucleotide (LNA), a 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotide, a 2′-methoxyethoxy (MOE) nucleotide, a 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotide, a 2′-O-methyl nucleotide, a 2′,4′-Constrained 2′-O-Methoxyethyl (cMOE), a 2′-O-Ethyl (cEt), a 2′-amino nucleotide, a 2′-O-amino nucleotide, a 2′-C-allyl nucleotides, a 2′-O-allyl nucleotide, a N6-methyladenosine nucleotide, a nucleotide with modified base 5-(3-amino)propyluridine, a nucleotide with modified base 5-(2-mercapto)ethyluridine, a nucleotide with modified base 5-bromouridine, a nucleotide with modified base 8-bromoguanosine, a nucleotide with modified base 7-deazaadenosine, a 2′-O-aminopropyl substituted nucleotide, or a nucleotide with a 2′-OH group replaced with a 2′-R, a 2′-OR, a 2′-halogen, a 2′-SR, or a 2′-amino, where R can be H, alkyl, alkenyl, or alkynyl.

### Claim 10 (depends on 1)

The guide compound of claim 1, wherein one or more of the last three monomers at each end of the guide compound is connected by a phosphorothioate, a chiral phosphorothioate, or a phosphorodithioate linkage.

### Claim 11 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs double strand breaks in a gene selected from TTR, BIRC5, CDK16, STAT3, CFTR, F9, KRAS, and CAR.

### Claim 12 (depends on 1)

The guide compound of claim 1, wherein the genomic DNA contains a target disease-related single nucleotide polymorphism.

### Claim 13 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs double strand breaks in a disease-related allele.

### Claim 14 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs double strand breaks in a disease-related allele selected from V30M TTR, G284R ColA1, L132P Keratin12, R135T Keratin12, G85R SOD1, G272V Tau, P301L Tau, V337M Tau, R406W Tau, Q39STOP beta-Globin, T8993G/C mtDNA, G719S EGFR, and G12C Kras.

### Claim 15 (depends on 1)

The guide compound of claim 1, comprising 30-300 contiguous monomers.

### Claim 16 (depends on 1)

The guide compound of claim 1, wherein the CRISPR gene editing uses Cas9.

### Claim 17 (depends on 1)

The guide compound of claim 1, wherein the guide compound directs more double strand breaks in a disease-related allele than in the same allele as a wild type.

### Claim 18 (depends on 1)

A guide compound of claim 1 annealed with a tracrRNA.

### Claim 19 (depends on 18)

The guide compound of claim 18, wherein the tracrRNA is derived from S. pneumonia, S. pyogenes, N. menigiditis, or S. thermophiles.

### Claim 20 (depends on 18)

The guide compound of claim 18, wherein the tracrRNA is SEQ ID NO:606.

### Claim 21 (depends on 18)

A pharmaceutical composition comprising one or more guide compounds of claim 18 and a pharmaceutically acceptable carrier.

### Claim 22 (depends on 21)

The composition of claim 21, wherein the pharmaceutically acceptable carrier comprises a viral vector or a non-viral vector.

### Claim 23 (depends on 21)

The composition of claim 21, wherein the pharmaceutically acceptable carrier comprises liposomes.

### Claim 24 (depends on 1)

A guide compound of claim 1 annealed with a tracrRNA and complexed with a CRISPR-associated gene editing protein.

### Claim 25 (depends on 24)

The guide compound of claim 24, wherein the CRISPR-associated gene editing protein is Cas9.