Targeted Gene Therapies for Pain and Other Neuro-Related Disorders

Abstract

Provided herein are nucleic acids for expressing modified ligand-gated ion channel proteins in excitable cells or secretory cells, such as nerves and neurons and optionally including viral sequences, such as Adeno-associated virus sequences, for delivery to excitable cells or secretory cells of a patient. Also provided herein are methods of modulating cell membrane potentials in an excitable cell or secretory cell, and for treatment of a disease or disorder associated with the nervous system in a patient, such as chronic pain or itch.

What is claimed is: 
     
         1 . A nucleic acid comprising a gene for expressing a modified ligand-gated ion channel, comprising an open reading frame encoding a modified ligand-gated ion channel under transcriptional control of a regulatory element governing cell-specific expression in dorsal horn neurons, wherein the modified ligand-gated ion channel comprises a modified ligand binding domain activatable by an exogenous ligand, and an ion pore domain,
 wherein the modified ligand-gated ion channel is a modified α7 nicotinic acetylcholine ligand binding domain, and   wherein the ion pore domain is an ion pore domain of an ionotropic nicotinic acetylcholine receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, an ionotropic GABA receptor, a serotonin 3 receptor (5HT3) ion pore domain, a glycine receptor (GlyR) ion pore domain, a gamma-aminobutyric acid (GABA) receptor ion pore domain, or an α7 nicotinic acetylcholine receptor.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the regulatory element is a promoter, a transcription response element, a repressor, and/or an enhancer. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the modified ligand binding domain is a modified α7 nicotinic acetylcholine ligand binding domain. 
     
     
         4 . The nucleic acid of  claim 3 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises a sequence having at least 75 percent sequence identity to a sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         5 . The nucleic acid of  claim 3 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises an amino acid substitution at one or more of amino acid residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, or 219 of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as Q79A, Q79G, L141A, L141 F, L141 P, W77F, W77Y, or W77M. 
     
     
         6 . The nucleic acid of  claim 3 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises:
 a. a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution; 
 b. a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution; 
 c. a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution; 
 d. a Y115F amino acid substitution and a G175K amino acid substitution; 
 e. a Q79G amino acid substitution and a P2161 amino acid substitution; or 
 f. a R27D amino acid substitution and/or a E41R amino acid substitution. 
 
     
     
         7 . The nucleic acid of  claim 3 , wherein the modified α7 nicotinic acetylcholine ligand binding domain has reduced binding with endogenous acetylcholine (ACh) as compared to unmodified α7-nAChR LBD. 
     
     
         8 . The nucleic acid of  claim 1 , wherein the exogenous ligand is selected from the group consisting of a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a 1,4-diazabicyclo[3.2.2]nonane. 
     
     
         9 . The nucleic acid of  claim 1 , comprising a sequence of a packageable viral genome comprising the gene for expressing a modified ligand-gated ion channel. 
     
     
         10 . The nucleic acid of  claim 9 , comprising Adeno-associated virus ITR sequences flanking the gene for expressing a modified ligand-gated ion channel, producing a sequence of a packageable recombinant AAV genome or a self-complementary AAV genome comprising the gene for expressing a modified ligand-gated ion channel. 
     
     
         11 . A method of preparing a patient for a treatment for relieving chronic pain, comprising:
 delivering to the patient a nucleic acid comprising an open reading frame encoding a modified ligand-gated ion channel under transcriptional control of a regulatory element governing cell-specific expression in dorsal horn neurons, wherein the modified ligand-gated ion channel comprises a modified ligand binding domain activatable by an exogenous ligand, and an ion pore domain,   wherein the modified ligand-gated ion channel is a modified α7 nicotinic acetylcholine ligand binding domain, and   wherein the ion pore domain is an ion pore domain of an ionotropic nicotinic acetylcholine receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, an ionotropic GABA receptor, a serotonin 3 receptor (5HT3) ion pore domain, a glycine receptor (GlyR) ion pore domain, a gamma-aminobutyric acid (GABA) receptor ion pore domain, or an α7 nicotinic acetylcholine receptor.   
     
     
         12 . The method of  claim 11 , wherein the nucleic acid is delivered to the dorsal horn of the spinal cord of the patient. 
     
     
         13 . The method of  claim 11 , wherein the regulatory element is a promoter, a transcription response element, a repressor, and/or an enhancer. 
     
     
         14 . The method of  claim 11 , wherein the modified ligand binding domain is a modified α7 nicotinic acetylcholine ligand binding domain. 
     
     
         15 . The method of  claim 14 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises a sequence having at least 75 percent sequence identity to a sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         16 . The method of  claim 14 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises an amino acid substitution at one or more of amino acid residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, or 219 of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as Q79A, Q79G, L141A, L141 F, L141 P, W77F, W77Y, or W77M. 
     
     
         17 . The method of  claim 14 , wherein the modified α7 nicotinic acetylcholine ligand binding domain comprises:
 a. a L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution; 
 b. a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution; 
 c. a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution; 
 d. a Y115F amino acid substitution and a G175K amino acid substitution; 
 e. a Q79G amino acid substitution and a P2161 amino acid substitution; or 
 f. a R27D amino acid substitution and/or a E41R amino acid substitution. 
 
     
     
         18 . The method of  claim 14 , wherein the modified α7 nicotinic acetylcholine ligand binding domain has reduced binding with endogenous acetylcholine (ACh) as compared to unmodified α7-nAChR LBD. 
     
     
         19 . The method of  claim 11 , further comprising administering the exogenous ligand to the patient in an amount effective to activate the modified ligand gated ion channel, thereby treating the chronic pain in the patient.