Modified ligand-gated ion channels and methods of use

Patent metadata

Field	Value
Publication number	US-12570705-B2
Application number	US-202117397658-A
Country	US
Kind code	B2
Filing date	Aug 9, 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 method of treating neuropathic pain in a mammal, the method comprising:
administering to the mammal a nucleic acid encoding a modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising:
(a) a modified alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) having:
(i) at least 93 percent sequence identity to the sequence set forth in residues 23-224 of SEQ ID NO:1, residues 23-229 of SEQ ID NO:2, or residues 23-233 of SEQ ID NO:11; and
(ii) an L131G amino acid substitution as numbered in SEQ ID NOs: 1, 2, or 11; and

(b) a human glycine receptor (GlyR) ion pore domain (IPD),
wherein an exogenous LGIC ligand selectively binds to and activates the modified LGIC; and administering the exogenous ligand to the mammal.

2 . The method of claim 1 , wherein the modified α7-nAChR LBD further comprises an amino acid substitution at amino acid residue 139 and/or 217 as numbered in SEQ ID NOs: 1, 2, or 11.

3 . The method of claim 2 , wherein the amino acid substitution at residue 139 is a Q139L amino acid substitution and the amino acid substitution at residue 217 is a Y217F amino acid substitution.

4 . The method of claim 3 , wherein the α7-nAChR LBD comprises the L131G amino acid substitution and the Q139L amino acid substitution.

5 . The method of claim 3 , wherein the α7-nAChR LBD comprises the L131G amino acid substitution and the Y217F amino acid substitution.

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

7 . The method of claim 3 , wherein the GlyR IPD is a modified GlyR IPD comprising an A298G amino acid substitution as numbered in SEQ ID NO:7.

8 . The method of claim 1 , wherein the nucleic acid encoding the modified LGIC is delivered to the mammal in a viral vector.

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

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

11 . The method of claim 1 , wherein the nucleic acid encoding the modified LGIC is operably linked to a promoter.

12 . The method of claim 1 , wherein the mammal is a human.

13 . A method of modulating the activity of a cell in a mammal, said method comprising:
administering to the cell a nucleic acid encoding a modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising:
(a) a modified alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising:
(i) at least 93 percent sequence identity to the sequence set forth in residues 23-224 of SEQ ID NO:1, residues 23-229 of SEQ ID NO:2, or residues 23-233 of SEQ ID NO:11; and
(ii) an L131G amino acid substitution as numbered in SEQ ID NOs: 1, 2, or 11; and

(b) a human glycine receptor (GlyR) ion pore domain (IPD),
wherein an exogenous LGIC ligand selectively binds to and activates the modified LGIC; and administering the exogenous ligand to the mammal.

14 . The method of claim 13 , wherein the modified α7-nAChR LBD further comprises an amino acid substitution at amino acid residue 139 and/or 217 as numbered in SEQ ID NOs: 1, 2, or 11.

15 . The method of claim 14 , wherein the amino acid substitution at residue 139 is a Q139L amino acid substitution and the amino acid substitution at residue 217 is a Y217F amino acid substitution.

16 . The method of claim 15 , wherein the α7-nAChR LBD comprises the L131G amino acid substitution and the Q139L amino acid substitution.

17 . The method of claim 15 , wherein the α7-nAChR LBD comprises the L131G amino acid substitution and the Y217F amino acid substitution.

18 . The method of claim 15 , wherein the α7-nAChR LBD comprises the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution.

19 . The method of claim 15 , wherein the GlyR IPD is a modified GlyR IPD comprising an A298G amino acid substitution as numbered in SEQ ID NO:7.

20 . The method of claim 13 , wherein the nucleic acid encoding the modified LGIC is delivered to the mammal in a viral vector.

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

22 . The method of claim 21 , wherein the viral vector is an adeno-associated viral vector.

23 . The method of claim 13 , wherein the nucleic acid encoding the modified LGIC is operably linked to a promoter.

24 . The method of claim 13 , wherein the mammal is a human.

25 . A method of treating neuropathic pain in a mammal, the method comprising:
administering to the mammal a nucleic acid encoding a modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising:
(a) a modified alpha7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising: the amino acid sequence of SEQ ID NO:2 having (i) an L131G amino acid substitution, (ii) an L131G amino acid substitution and a Q139L amino acid substitution, (iii) an L131G amino acid substitution and a Y217F amino acid substitution, or (iv) 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),
wherein an exogenous LGIC ligand selectively binds to and activates the modified LGIC; and administering the exogenous ligand to the mammal.

26 . The method of claim 25 , wherein the neuropathic pain is trigeminal neuralgia.

27 . The method of claim 25 , wherein the modified α7-nAChR LBD comprises only the L131G amino acid substitution.

28 . The method of claim 25 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution and the Q139L amino acid substitution.

29 . The method of claim 25 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution and the Y217F amino acid substitution.

30 . The method of claim 25 , wherein the modified α7-nAChR LBD comprises the L131G amino acid substitution, the Q139L amino acid substitution, and the Y217F amino acid substitution.

31 . The method of claim 25 , wherein the GlyR IPD is a modified GlyR IPD comprising an A298G amino acid substitution as numbered in SEQ ID NO:7.

32 . The method of claim 25 , wherein the nucleic acid encoding the modified LGIC is delivered to the mammal in a viral vector.

33 . The method of claim 32 , wherein the viral vector is selected from the group consisting of an adeno-associated virus, a herpes simplex virus, and a lentivirus vector.

34 . The method of claim 33 , wherein the viral vector is an adeno-associated viral vector.

35 . The method of claim 25 , wherein the nucleic acid encoding the modified LGIC is operably linked to a promoter.

36 . The method of claim 25 , wherein the mammal is a human.

37 . A method of treating neuropathic pain in a mammal, the m

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