# US 2019/0169264 A1 · Modified ligand-gated ion channels and methods of use

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- Number: US 2019/0169264 A1
- Document ID: US20190169264A1
- Status: Published application
- Kind: A1
- Application number: 16/186,122
- Filed: 2018-11-09
- Published: 2019-06-06
- Earliest claimed: 2017-11-10
- Assignees: Howard Hughes Medical Institute
- Applicants: Howard Hughes Medical Institute
- Inventors: Scott Sternson; Peter Lee; Christopher Magnus
- Web page: https://patentgrep.com/patent/US20190169264A1

## CPC

- C07K 14/70571: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/03: containing a transmembrane segment
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This document relates to materials and methods for modulating 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.

## Claims

### Claim 1

A modified ligand gated ion channel (LGIC) comprising at least one modified LGIC subunit, said modified LGIC subunit comprising:
a ligand binding domain (LBD) comprising an amino acid modification wherein the LBD is an alpha7 nicotinic acetylcholine receptor (α7-nAChR) LBD comprising a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and
an ion pore domain (IPD), wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

### Claim at position 2

2-24. (canceled)

### Claim 25 (depends on 1)

The modified LGIC of claim 1, wherein an exogenous LGIC ligand activates the modified LGIC, and wherein the exogenous LGIC ligand is selected from the group consisting of compound 815, compound 816, and compound 817.

### Claim at position 4

26-55. (canceled)

### Claim 56

A ligand having increased potency for a modified ligand gated ion channel (LGIC), wherein the ligand comprises Formula V:
wherein R1=H, phenyl, 2-toluyl; 3-pyridyl; 4-pyridyl; trifluoromethyl; methoxy; ethoxy;
propoxy, isopropoxy; N,N-dimethylamino; N,N-diethylamino; imidazole, pyrrole, pyrazole, triazole, or isoxazole-3-amine; and
wherein R2=H, methyl, or phenyl; and
wherein the compound is selected from the group consisting of compound 815, compound 816, and compound 817.

### Claim at position 6

57-129. (canceled)

### Claim 130 (depends on 1)

The modified LGIC of claim 1, wherein the modified LGIC further comprises an endoplasmic reticulum export sequence.

### Claim 131 (depends on 130)

The modified LGIC of claim 130, wherein the endoplasmic reticulum export sequence comprises the amino acid sequence FCYENEV (SEQ ID NO:16).

### Claim 132 (depends on 131)

The modified LGIC of claim 131, wherein the modified LGIC comprises the amino acid sequence set forth in SEQ ID NO:13.

### Claim 133 (depends on 1)

The modified LGIC of claim 1, wherein the modified LGIC further comprises a CHRNB4 signal sequence.

### Claim 134 (depends on 133)

The modified LGIC of claim 133, wherein the CHRNB4 signal sequence comprises the amino acid sequence MRRAPSLVLFFLVALCGRGNC (SEQ ID NO:17).

### Claim 135 (depends on 134)

The modified LGIC of claim 134, wherein the modified LGIC comprises the amino acid sequence set forth in SEQ ID NO:14.

### Claim 136 (depends on 1)

The modified LGIC of claim 1, wherein the modified LGIC further comprises a KCNB1 somatic targeting sequence.

### Claim 137 (depends on 136)

The modified LGIC of claim 136, wherein the KCNB1 somatic targeting sequence comprises the amino acid sequence
(SEQ ID NO: 18)
QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSID
SFISCATDFPEATRF.

### Claim 138 (depends on 137)

The modified LGIC of claim 137, wherein the modified LGIC comprises the amino acid sequence set forth in SEQ ID NO:15.

### Claim 139

A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75 percent sequence identity to the sequence set forth in SEQ ID NO:27, wherein said nucleic acid sequence comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification, and wherein said nucleic acid sequence comprises a nucleic acid sequence encoding ion pore domain (IPD).

### Claim 140

(canceled)

### Claim 141 (depends on 139)

The synthetic nucleic acid construct of claim 139, wherein the LBD is an alpha7 nicotinic acetylcholine receptor (α7-nAChR) LBD, and wherein the modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

### Claim 142

(canceled)

### Claim 143 (depends on 139)

The synthetic nucleic acid construct of claim 139, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

### Claim 144 (depends on 143)

The synthetic nucleic acid construct of claim 143, wherein the IPD is a 5HT3 IPD.

### Claim 145

A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75 percent sequence identity to the sequence set forth in SEQ ID NO:28, wherein said nucleic acid sequence having at least 75 percent sequence identity to the sequence set forth in SEQ ID NO:28 comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification, and wherein said nucleic acid sequence comprises a nucleic acid sequence encoding ion pore domain (IPD).

### Claim 146

(canceled)

### Claim 147 (depends on 145)

The synthetic nucleic acid construct of claim 145, wherein the LBD is an alphα7 nicotinic acetylcholine receptor (α7-nAChR) LBD, and wherein the modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

### Claim 148

(canceled)

### Claim 149 (depends on 145)

The synthetic nucleic acid construct of claim 145, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

### Claim 150 (depends on 149)

The synthetic nucleic acid construct of claim 149, wherein the IPD is a GlyR IPD, and wherein said GlyR IPD comprises an amino acid modification at amino acid residue 298 of the GlyR IPD.

### Claim 151

(canceled)

### Claim 152

A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75 percent sequence identity to the sequence set forth in SEQ ID NO:29, wherein said nucleic acid sequence comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification, and wherein said nucleic acid sequence comprises a nucleic acid sequence encoding ion pore domain (IPD).

### Claim 153

(canceled)

### Claim 154 (depends on 152)

The synthetic nucleic acid construct of claim 152, wherein the LBD is an alphα7 nicotinic acetylcholine receptor (α7-nAChR) LBD, and wherein the modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

### Claim 155

(canceled)

### Claim 156 (depends on 152)

The synthetic nucleic acid construct of claim 152, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

### Claim 157 (depends on 156)

The synthetic nucleic acid construct of claim 156, wherein the IPD is a GABA IPD, and wherein said GABA IPD comprises an amino acid modification at amino acid residue 298 of the GABA IPD.

### Claim 158

(canceled)

### Claim 159

A synthetic nucleic acid construct comprising the sequence set forth in SEQ ID NO: 33.

### Claim 160

A synthetic nucleic acid construct comprising the sequence set forth in SEQ ID NO: 34.