# US 11,898,179 B2 · Suppression of pain by gene editing

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- Number: US 11,898,179 B2
- Document ID: US11898179B2
- Status: Granted
- Kind: B2
- Application number: 16/492,548
- Filed: 2018-03-09
- Published: 2021-04-22
- Granted: 2024-02-13
- Earliest claimed: 2017-03-09
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Juan Pablo Maianti; David R. Liu
- Web page: https://patentgrep.com/patent/US11898179B2

## CPC

- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/1138: {against receptors or cell surface proteins}
- A61K 9/0029: {Parenteral nutrition; Parenteral nutrition compositions as drug carriers}
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 38/50: acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- 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}
- C12Y 305/04: in cyclic amidines (3.5.4)
- C12Y 305/04001: Cytosine deaminase (3.5.4.1)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 2319/00: Fusion polypeptide
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/31: Combination therapy
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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
- 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)

## Abstract

Provided herein are systems, compositions, kits, and methods for the suppression of pain (e.g., chronic pain). Genes encoding ion channels (e.g., SCN9A) responsible for the propagation pain signals in neurons (e.g., DRG neurons) may be edited using a genome editing agent (e.g., a nucleobase editor). In some embodiments, loss-of-function ion channel mutants are generated, leading to pain suppression. In some embodiments, the genome editing agent is administered locally to the site of pain or to the nerves responsible for propagation of the pain signal.

## Claims

### Claim 1

A method of editing a polynucleotide encoding an ion channel in a dorsal root ganglion (DRG) neuron, the method comprising contacting the ion channel-encoding polynucleotide with:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain, wherein the fusion protein further comprises a uracil glycosylase inhibitor (UGI) domain; and
(ii) a guide nucleic acid molecule targeting the fusion protein of (i) to a target cytosine (C) base in the ion channel-encoding polynucleotide;
whereby the contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine (C) to thymine (T) change in the ion channel-encoding polynucleotide; and
wherein the C to T change leads to a mutation in the ion channel that either introduces a premature stop codon in the ion channel-coding polynucleotide that leads to a truncated or non-functional ion channel or destabilizes ion-channel protein folding, or both.

### Claim 2 (depends on 1)

The method of claim 1, wherein the guide nucleotide sequence-programmable DNA binding protein domain is selected from the group consisting of: nuclease inactive Cas9 (dCas9) domains, nuclease inactive Cpf1 domains, nuclease inactive Argonaute domains, and variants thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cytosine deaminase domain comprises an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cytosine deaminase domain is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced deaminase (AID), and pmCDA1.

### Claim 5 (depends on 4)

The method of claim 4, wherein the cytosine deaminase domain comprises the amino acid sequence of any one of SEQ ID NOs: 271-292, 303, or 2483-2494.

### Claim 6 (depends on 1)

The method of claim 1, wherein the UGI domain comprises the amino acid sequence of SEQ ID NO: 304.

### Claim 7 (depends on 1)

The method of claim 1, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 296-302 and 2495.

### Claim 8 (depends on 1)

The method of claim 1, wherein the C to T change occurs in the coding region of the ion channel-encoding polynucleotide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mutation introduces a premature stop codon in the ion channel-coding sequence that leads to a truncated or non-functional ion channel.

### Claim 10 (depends on 1)

The method of claim 1, wherein the mutation destabilizes ion-channel protein folding.

### Claim 11 (depends on 1)

The method of claim 1, wherein the C to T change occurs at a C base-paired with the G base in a start codon (AUG).

### Claim 12 (depends on 1)

The method of claim 1, wherein the C to T change occurs in the non-coding region of the ion channel-encoding polynucleotide.

### Claim 13 (depends on 1)

The method of claim 1, wherein the ion channel is selected from the group consisting of: NaV1.7, NaV1.8, NaV1.9, NaV1.3, CaV3.2, HCN1, HCN2, and Ano1.

### Claim 14 (depends on 13)

The method of claim 13, wherein the ion channel is NaV1.7 encoded by the SCN9A gene.

### Claim 15 (depends on 1)

The method of claim 1, wherein a PAM sequence is either located 3′ of the C being changed or is located 5′ of the C being changed.

### Claim 16 (depends on 1)

The method of claim 1, wherein the DRG neuron is in a mammal.

### Claim 17 (depends on 16)

The method of claim 16, wherein the mammal is a human.

### Claim 18 (depends on 1)

The method of claim 1, wherein a nucleic acid construct encoding the fusion protein is delivered to the DRG neuron via a neurotropic viral delivery vector.

### Claim 19 (depends on 9)

The method of claim 9, wherein the mutation is installed at an amino acid position W188 of SEQ ID NO: 2434.

### Claim 20 (depends on 1)

The method of claim 1, wherein the guide nucleotide sequence-programmable DNA binding protein domain is an S. pyogenes Cas9 nickase, an S. aureus Cas9 nickase, or an S. aureus Cas9-KKH.

### Claim 21 (depends on 20)

The method of claim 20, wherein the guide nucleotide sequence-programmable DNA binding protein domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 3, 4, 268, or 2426-2428.

### Claim 22 (depends on 20)

The method of claim 20, wherein the guide nucleotide sequence-programmable DNA binding protein domain comprises the amino acid sequence of any of SEQ ID NOs: 3, 4, 268, or 2426-2428.

### Claim 23 (depends on 4)

The method of claim 4, wherein the cytosine deaminase domain comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 271-292, 303, or 2483-2494.

### Claim 24 (depends on 1)

The method of claim 1, wherein the guide nucleic acid molecule is selected from SEQ ID NOs: 834-1125 or 1273-1287.

### Claim 25

A method of editing a polynucleotide encoding an ion channel in a DRG neuron, the method comprising contacting the ion channel-encoding polynucleotide with:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain; and
(ii) a guide nucleic acid molecule targeting the fusion protein of (i) to a target C base in the ion channel-encoding polynucleotide;
whereby the contacting results in deamination of the target C base by the fusion protein, resulting in a C to T change in the ion channel-encoding polynucleotide, and
wherein the guide nucleic acid molecule is selected from SEQ ID NOs: 339-1456.

### Claim 26

A method of editing a polynucleotide encoding an ion channel in a dorsal root ganglion (DRG) neuron, the method comprising contacting the ion channel-encoding polynucleotide with:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain, wherein the fusion protein further comprises a uracil glycosylase inhibitor (UGI) domain; and
(ii) a guide nucleic acid molecule targeting the fusion protein of (i) to a target cytosine (C) base in the ion channel-encoding polynucleotide;
whereby the contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine (C) to thymine (T) change in the ion channel-encoding polynucleotide; and
wherein the ion channel-encoding polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 2435 and/or wherein the ion channel-encoding polynucleotide comprises a sequence containing the target C that comprises the nucleic acid sequence of any one of SEQ ID NOs: 1457-1503.

### Claim 27 (depends on 26)

The method of claim 26, wherein the ion channel comprises the amino acid sequence of SEQ ID NO: 2434.

### Claim 28

A method of editing a polynucleotide encoding an ion channel in a dorsal root ganglion (DRG) neuron, the method comprising contacting the ion channel-encoding polynucleotide with:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain, wherein the fusion protein further comprises a uracil glycosylase inhibitor (UGI) domain; and
(ii) a guide nucleic acid molecule targeting the fusion protein of (i) to a target cytosine (C) base in the ion channel-encoding polynucleotide, wherein the guide nucleic acid molecule is selected from SEQ ID NOs: 834-1125 or 1273-1287;
whereby the contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine (C) to thymine (T) change in the ion channel-encoding polynucleotide.