Modified ligand-gated ion channels and methods of use

Patent metadata

Field	Value
Publication number	US-12570706-B2
Application number	US-202117468907-A
Country	US
Kind code	B2
Filing date	Sep 8, 2021
Priority date	Jul 7, 2016
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

Abstract

This document relates to materials and methods for controlling ligand gated ion channel (LGIC) activity. For example, modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and/or a modified ion pore domain (IPD) are provided. Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC, as well as methods of modulating ion transport across the membrane of a cell of a mammal, methods of modulating the excitability of a cell in a mammal, and methods of treating a mammal having a channelopathy.

First claim

What is claimed is:

1 . A nucleic acid encoding a modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, wherein said modified LGIC subunit comprises:
(a) a modified human alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising the amino acid sequence of SEQ ID NO:2 having (a) an L131G amino acid substitution, (b) an L131G amino acid substitution and a Q139L amino acid substitution, (c) an L131G amino acid substitution and a Y217F amino acid substitution, or (d) an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and (b) a human glycine receptor (GlyR) ion pore domain (IPD).

2 . The nucleic acid of claim 1 , wherein the modified α7-nAChR LBD comprises only the L131G amino acid substitution.

3 . The nucleic acid of claim 1 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution.

4 . The nucleic acid of claim 1 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution and the Q139L amino acid substitution.

5 . The nucleic acid of claim 1 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution and the Y217F amino acid substitution.

6 . The nucleic acid of claim 1 , wherein the GlyR IPD is a modified GlyR IPD comprising an A298G amino acid substitution as numbered in SEQ ID NO:7.

7 . A vector comprising the nucleic acid of claim 1 .

8 . The vector of claim 7 , wherein the vector is a viral vector selected from the group consisting of an adeno-associated virus, a herpes simplex virus, and a lentivirus vector.

9 . The vector of claim 8 , wherein the viral vector is an adeno-associated viral vector.

10 . A nucleic acid encoding a modified ligand gated ion channel (LGIC) subunit, said modified LGIC subunit comprising:
(a) a modified human alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising the amino acid sequence of SEQ ID NO:2 having (a) an L131G amino acid substitution, (b) an L131G amino acid substitution and a Q139L amino acid substitution, (c) an L131G amino acid substitution and a Y217F amino acid substitution, or (d) an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and (b) a human glycine receptor (GlyR) ion pore domain (IPD).

11 . A vector comprising the nucleic acid of claim 10 .

12 . The vector of claim 11 , wherein the vector is a viral vector selected from the group consisting of an adeno-associated virus, a herpes simplex virus, and a lentivirus vector.

13 . The vector of claim 12 , wherein the viral vector is an adeno-associated viral vector.

14 . A nucleic acid encoding a modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising a modified alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) and a glycine receptor (GlyR) ion pore domain (α7-GlyR LGIC subunit), wherein said α7-GlyR LGIC subunit comprises the sequence set forth in SEQ ID NO: 7 having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution as numbered in SEQ ID NO: 7.

15 . A vector comprising the nucleic acid of claim 14 .

16 . The vector of claim 15 , wherein the vector is a viral vector selected from the group consisting of an adeno-associated virus, a herpes simplex virus, and a lentivirus vector.

17 . The vector of claim 16 , wherein the viral vector is an adeno-associated viral vector.

18 . A nucleic acid encoding a modified ligand gated ion channel (LGIC) subunit, said modified LGIC subunit comprising a modified alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) and a glycine receptor (GlyR) ion pore domain (α7-GlyR LGIC subunit), wherein said α7-GlyR LGIC subunit comprises the sequence set forth in SEQ ID NO: 7 having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution as numbered in SEQ ID NO:7.

19 . A vector comprising the nucleic acid of claim 18 .

20 . The vector of claim 19 , wherein the vector is a viral vector selected from the group consisting of an adeno-associated virus, a herpes simplex virus, and a lentivirus vector.

Assignees

• Hughes Howard Med Inst

Inventors

• Sternson Scott
• Lee Peter
• Magnus Christopher

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C07K14/705 Primary

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title

• A61K38/1709

  from mammals · CPC title