Suppression of pain by gene editing
AssigneePresident and Fellows of Harvard College
InventorsJuan Pablo Maianti, David R. Liu
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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.
Claims28
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.
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.
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.
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.
Dependencies are inferred from the claim text.