Ligand-gated ion channels and methods of use - Patents.com

Abstract

Compositions and methods are provided for modulating cellular activity using engineered receptors, polynucleotides encoding engineered receptors, and gene therapy vectors that include polynucleotides encoding engineered receptors.These compositions and methods find particular use in modulating neuronal activity, for example, in treating disease or studying neural circuits.In one aspect, the present disclosure provides an engineered receptor that is a chimeric ligand-gated ion channel (LGIC) receptor, comprising (a) a ligand binding domain derived from a first wild-type Cys-loop LGIC receptor, and (b) an ion pore domain derived from a second wild-type Cys-loop LGIC receptor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 311,735, filed February 18, 2022, and U.S. Provisional Application No. 63 / 348,560, filed June 3, 2022, the entire contents of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (SWCH_035_03WO_SeqList_ST26.xml; size: 183,826 bytes; and creation date: February 20, 2023) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates to engineered receptors and the use of engineered receptors and small molecule ligands to modulate cellular activity and treat disease. [Background technology]

[0004] background Intractable neurological diseases are often associated with abnormally active neurons, and attempts to develop therapies to treat these conditions have been hampered by the lack of tractable target proteins associated with the diseases.

[0005] The most commonly used treatment for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to dependency and can cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive impairment, hallucinations, and other systemic side effects.Despite the widespread use of pharmaceutical drugs, their effectiveness in pain relief has a surprisingly low success rate.More invasive options for pain treatment include nerve blocks and electrical stimulation.The most invasive and least preferred method for managing pain is the complete surgical removal of the nerve or part thereof that is causing pain.

[0006] There is a long-felt and unmet need for safe, effective, cost-effective treatments for neurological disorders. The present disclosure provides safe, effective, cost-effective treatments for neurological disorders, including pain management. Summary of the Invention

[0007] The disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided upon request to the Office and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, dimensions of the various features have been arbitrarily enlarged or reduced for clarity. Included in the drawings are the following figures: [Brief description of the drawings]

[0008] [Figure 1-1] FIG. 1 shows a heat map of percent quenching of YFP fluorescence of CODA71 or CODA1237 transfected cells following stimulation with various doses of either acetylcholine or the indicated non-natural ligands. Ligand doses are listed across the top of each chart. Numbers in boxes indicate absolute amounts of quenching observed. Dark blue indicates strong quenching of the YFP reporter. Light blue indicates moderate quenching. Orange suggests weak or minimal quenching. Negative values ​​represent non-responders with negative quenching due to stimulation artifacts. [Figure 1-2] Same as above.

[0009] [Figure 2-1]Figure 2 shows a heat map of the percent quenching of YFP fluorescence of cells transfected with chimeric LGIC receptors based on CODA1237 containing various amino acid mutations after stimulation with increasing doses of either acetylcholine or TC-5619. CODA75 is a non-responsive chimera used as a negative control. Color coding and labeling follow the same rules as in Figure 1. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0010] [Diagram 3] Figure 3 shows a heat map of the percent quenching of YFP fluorescence of cells transfected with CODA 64, CODA71, or chimeric receptors containing the ion pore domain of GlyRα3 (CODA1342, CODA1343, CODA1344, CODA1345) after stimulation with increasing doses of acetylcholine. CODA75 is a non-responsive chimera used as a negative control. Color coding and labeling follow the same rules as in Figure 1.

[0011] [Figure 4-1] Figure 4A shows the percentage of α-bungarotoxin staining positive HEK293T cells transiently transfected with the indicated receptors. Figure 4B shows the mean fluorescence intensity (MFI) of α-bungarotoxin staining positive cells from Figure 4A. Figure 4C shows the percentage of α-bungarotoxin staining positive cells transiently transfected with the indicated receptors in HEK293T cells transduced with Ric3, Nacho, and GCAMP6s. Figure 4D shows the mean fluorescence intensity of α-bungarotoxin staining positive cells from Figure 4C. Data are representative of n=2-6 replicate samples. [Figure 4-2] Same as above.

[0012] [Figure 5-1]Figure 5A shows the percentage of α-bungarotoxin staining positive HEK293T cells transiently transfected with the indicated receptors. Figure 5B shows the mean fluorescence intensity of α-bungarotoxin staining positive cells from Figure 5A. Data are representative of n=2-6 replicate samples. Figure 5C shows the dose response of CODA71 to TC-5619 and acetylcholine in HEK cells. Figure 5D shows the dose response of CODA1055 to TC-5619 and acetylcholine in HEK cells. Figure 5E shows the dose response of CODA1316 to TC-5619 in HEK cells. Figure 5F shows the peak current of the indicated receptors in HEK cells when activated by TC-5619. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0013] [Figure 6-1] 6 shows a sequence alignment of human α7-nAChR (SEQ ID NO:4), human GlyRα1 (SEQ ID NO:2), human GlyRα2 (SEQ ID NO:59), human GlyRα3 (isoform L, SEQ ID NO:61), human GABA-A ρ1 (SEQ ID NO:10), human GABA-A ρ2 (SEQ ID NO:12), and human GABA-A ρ3 (SEQ ID NO:14) protein sequences. The β1-2 loop, Cys loop, pre-M1 linker, and M2-M3 linker regions are labeled. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Abstract In one aspect, the present disclosure provides an engineered receptor that is a chimeric ligand-gated ion channel (LGIC) receptor, comprising (a) a ligand-binding domain derived from a first wild-type Cys-loop LGIC receptor, and (b) an ion pore domain derived from a second wild-type Cys-loop LGIC receptor. In some embodiments, the first wild-type Cys-loop LGIC receptor comprises a receptor of the nicotinic acetylcholine receptor family. In some embodiments, the first wild-type Cys-loop LGIC receptor is a human α7 nicotinic acetylcholine receptor (α7-nAChR, SEQ ID NO: 4). In some embodiments, the second wild-type Cys-loop LGIC receptor is a chloride-permeable Cys-loop ligand-gated ion channel receptor. In some embodiments, the second wild-type Cys-loop LGIC receptor is a glycine receptor or a GABA-A receptor. In some embodiments, the second wild-type Cys-loop LGIC receptor comprises glycine receptor α1, glycine receptor α2, glycine receptor α3, GABA-A receptor ρ1, GABA-A receptor ρ2, or GABA-A receptor ρ3. In some embodiments, the second wild-type Cys-loop LGIC receptor is not glycine receptor α1. In some embodiments, a portion or all of the Cys-loop domain of the ligand-binding domain is derived from a second wild-type Cys-loop LGIC receptor. In some embodiments, a portion or all of the β1-2 loop domain of the ligand-binding domain is derived from a second wild-type Cys-loop LGIC receptor. In some embodiments, the engineered receptor comprises a pre-M1 linker derived from a first wild-type Cys-loop LGIC receptor or a second wild-type Cys-loop LGIC receptor. In some embodiments, the engineered receptor comprises a pre-M1 linker comprising an N-terminal segment derived from a first wild-type Cys-loop LGIC receptor and a C-terminal segment derived from a second wild-type Cys-loop LGIC receptor. In some embodiments, the pre-M1 linker comprises or consists of a sequence having at least 70% identity to any one of SEQ ID NOs:72-91.In some embodiments, some or all of the M2-M3 linker of the ion pore domain is derived from the first wild-type Cys-loop LGIC receptor. In some embodiments, the M2-M3 linker of the ion pore domain comprises one or more mutations. In some embodiments, the M2-M3 linker of the ion pore domain comprises an amino acid sequence having at least 70% identity according to amino acids 283-295 of SEQ ID NO:4. In some embodiments, the engineered receptor forms a homomeric ion channel when expressed on a cell surface. In some embodiments, more than 50% of the engineered receptors expressed on a cell surface form homomeric ion channels.

[0015] In some embodiments, the second wild-type Cys loop LGIC receptor is a human glycine receptor α1 subunit (GlyRα1). In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 248-457 of SEQ ID NO:2. In some embodiments, the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 166-180 of SEQ ID NO:2. In some embodiments, the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 80-85 of SEQ ID NO:2. In some embodiments, the ion pore domain does not include an amino acid between amino acid positions corresponding to K353 and E362 of SEQ ID NO:2. In some embodiments, the ion pore domain comprises an amino acid sequence having less than 50% sequence identity to SEQ ID NO:96 between amino acid positions corresponding to K353 and E362 of SEQ ID NO:2. In some embodiments, cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor comprising the amino acid sequence set forth in SEQ ID NO:96, between amino acid positions corresponding to K353 and E362 of SEQ ID NO:2. In some embodiments, the ion pore domain comprises one or more mutations in a region corresponding to the nuclear localization signal (NLS) / ER retention signal (ERRS) sequence of human GlyRα1. In some embodiments, the engineered receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO:16 or 33.

[0016] In some embodiments, the second wild-type Cys loop LGIC receptor is a human glycine receptor α2 subunit (GlyRα2). In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 254-452 of SEQ ID NO:59. In some embodiments, the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 172-186 of SEQ ID NO:59. In some embodiments, the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 86-91 of SEQ ID NO:59. In some embodiments, the ion pore domain comprises one or more mutations in a region corresponding to the NLS / ERRS sequence of human GlyRα2. In some embodiments, the engineered receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO:62 or 65.

[0017] In some embodiments, the second wild-type Cys loop LGIC receptor is a human glycine receptor α3 subunit (GlyRα3). In some embodiments, the ionic pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 253-464 of SEQ ID NO:61 or amino acids 253-449 of SEQ ID NO:69. In some embodiments, the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 171-185 of SEQ ID NO:61 or 69. In some embodiments, the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 85-90 of SEQ ID NO:61 or 69. In some embodiments, the ionic pore domain does not comprise an amino acid residue between amino acid positions corresponding to K357 and D358 of SEQ ID NO:69. In some embodiments, the ionic pore domain comprises an amino acid sequence having less than 50% sequence identity to SEQ ID NO:95 between amino acid positions corresponding to K357 and D358 of SEQ ID NO:69. In some embodiments, cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor comprising an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO: 61) that comprises 15 amino acid residues corresponding to amino acids 358-372 of SEQ ID NO: 61. In some embodiments, the ion pore domain comprises one or more mutations in a region corresponding to the NLS / ERRS sequence of human GlyRα3. In some embodiments, the engineered receptor comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 63, 66, 70, and 71.

[0018] In some embodiments, the second wild-type Cys loop LGIC receptor is a human GABA-A receptor ρ1 subunit (GABA-A ρ1). In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 281-479 of SEQ ID NO: 10. In some embodiments, the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 198-212 of SEQ ID NO: 10. In some embodiments, the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 112-117 of SEQ ID NO: 10. In some embodiments, the engineered receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 64 or 67.

[0019] In some embodiments, the pre-M1 linker of the ligand binding domain comprises one or more mutations. In some embodiments, the ligand binding domain is derived from human α7-nAChR, and the one or more mutations in the pre-M1 linker are at one or more positions corresponding to T225, M226, and / or T230 of the human α7-nAChR. In some embodiments, the ligand binding domain is derived from human α7-nAChR, and the one or more mutations comprise a mutation corresponding to T225I of the human α7-nAChR. In some embodiments, the one or more mutations increase surface expression of the engineered receptor. In some embodiments, the surface expression is measured by an α-bungarotoxin (a-BTX) assay.

[0020] In some embodiments, the ligand binding domain comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to amino acids 23-220 of SEQ ID NO:4.

[0021] In some embodiments, the ligand binding domain comprises an amino acid substitution at one or more residues including W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, Y140, L141, Y151, S170, W171, S172, Y173, S188, Y190, Y210, C212, C213, E215, Y217, or any combination thereof, of human α7-nAChR. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of residues including R101 and L131, Y115 and Y210, or R101 and Y210 of human α7-nAChR. In some embodiments, the ligand binding domain comprises amino acid substitutions corresponding to L131N, W77F, and S172D of SEQ ID NO:4. In some embodiments, the ligand binding domain comprises amino acid substitutions corresponding to Q139W and S172D of SEQ ID NO:4. In some embodiments, the ligand binding domain comprises one or more amino acid substitutions listed in Table 12. In some embodiments, the ligand binding domain comprises amino acid substitutions corresponding to R101W, Y115E, and Y210W of human α7-nAChR. In some embodiments, the engineered receptor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:65 and comprises amino acid substitutions corresponding to R101W, Y115E, Y210W, and T225I of SEQ ID NO:65.

[0022] In some embodiments, the potency of the engineered receptor against the natural ligand of the first wild-type Cys-loop LGIC receptor is lower than the potency of the first wild-type Cys-loop LGIC receptor against the natural ligand. In some embodiments, the potency of the engineered receptor against the natural ligand is at least 2 times lower than the potency of the first wild-type Cys-loop LGIC receptor against the natural ligand. In some embodiments, the potency of the engineered receptor against the non-natural ligand is approximately the same as the potency of the first wild-type Cys-loop LGIC receptor against the non-natural ligand. In some embodiments, the potency of the engineered receptor against the non-natural ligand is higher than the potency of the first wild-type Cys-loop LGIC receptor against the non-natural ligand. In some embodiments, the potency of the engineered receptor against the non-natural ligand is at least 2 times higher than the potency of the first wild-type Cys-loop LGIC receptor against the non-natural ligand. In some embodiments, determining the potency includes determining the EC50. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the first wild-type Cys-loop LGIC receptor in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least 2-fold greater than the efficacy of the first wild-type Cys-loop LGIC receptor in the presence of the non-natural ligand. In some embodiments, determining efficacy comprises determining the amount of current passed through the engineered receptor in vitro in the presence of the non-natural ligand. In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and facinicline / RG3487. In some embodiments, the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and APN-1125. In some embodiments, the non-natural ligand is TC-5619.

[0023] In one aspect, the present disclosure provides a polynucleotide comprising a nucleic acid encoding an engineered receptor of the present disclosure. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the engineered receptor. In some embodiments, the promoter is a regulatable promoter. In some embodiments, the regulatable promoter is active in an excitable cell. In some embodiments, the excitable cell is a neuron or a muscle cell. In some embodiments, the excitable cell is a neuron.

[0024] In one aspect, the present disclosure provides a vector comprising the polynucleotide of the present disclosure.In some embodiments, the vector is a plasmid or a viral vector.In some embodiments, the vector is a viral vector selected from the group consisting of adenovirus vector, retrovirus vector, adeno-associated virus (AAV) vector, and herpes simplex 1 virus vector (HSV-1).In some embodiments, the viral vector is an AAV vector, and the AAV vector is AAV5 or its variant, AAV6 or its variant, or AAV9 or its variant.

[0025] In one aspect, the present disclosure provides a composition comprising an engineered receptor of the present disclosure, a polynucleotide of the present disclosure, or a vector of the present disclosure.

[0026] In one aspect, the present disclosure provides a pharmaceutical composition comprising an engineered receptor of the present disclosure, a polynucleotide of the present disclosure, or a vector of the present disclosure, and a pharma- ceutically acceptable carrier.

[0027] In one aspect, the present disclosure provides a method of producing an engineered receptor in a neuron, comprising contacting the neuron with a polynucleotide of the present disclosure, a vector of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an injured Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an uninjured Aβ afferent fiber. In some embodiments, the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5. In some embodiments, the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1. In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in the subject. In some embodiments, the contacting step comprises administering the polynucleotide, vector, composition, or pharmaceutical composition to the subject. In some embodiments, the contacting step is performed in vitro or ex vivo. In some embodiments, the contacting step comprises lipofection, nanoparticle delivery, biolistics, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor can be localized to the cell surface of the neuron.

[0028] In one aspect, the present disclosure provides a method of inhibiting neuronal activity, comprising: (a) contacting the neuron with an engineered receptor of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure, and (b) contacting the neuron with a non-natural ligand of the engineered receptor. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an injured Aβ afferent fiber. In some embodiments, the Aβ afferent fibers are uninjured Aβ afferent fibers. In some embodiments, the neurons express neurofilament 200 (NF200), Piezo2, and TLR-5. In some embodiments, the neurons do not express TrpV1, prostatic acid phosphatase, NaV1.1. In some embodiments, the contacting step (a) is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step (b) is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting steps (a) and / or (b) are performed in vivo in a subject. In some embodiments, the contacting step (a) comprises administering an engineered receptor, polynucleotide, vector, or pharmaceutical composition to the subject; and / or the contacting step (b) comprises administering a non-natural ligand to the subject. In some embodiments, the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, biolistics, electroporation, sonication, or microinjection.In some embodiments, the engineered receptor may be localized to the cell surface of a neuron.

[0029] In one aspect, the present disclosure provides a method of treating and / or delaying the onset of a neurological disorder in a subject in need thereof, comprising the steps of (a) administering to the subject a therapeutically effective amount of an engineered receptor of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure, and (b) administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand simultaneously with step (a). In some embodiments, the neurological disorder is a seizure disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spastic disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation. In some embodiments, the neurological disorder is allodynia. In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. In some embodiments, the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionously, intraneurally, intracranially, intraspinally, or into the cisterna magna. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally. In some embodiments, the subject suffers from trigeminal neuralgia, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. In some embodiments, the subject is a human.In some embodiments, the therapeutically effective amount reduces the severity of signs and / or symptoms of a neurological disorder. In some embodiments, the therapeutically effective amount delays the onset of signs and / or symptoms of a neurological disorder. In some embodiments, the therapeutically effective amount removes signs and / or symptoms of a neurological disorder. In some embodiments, the signs of a neurological disorder are nerve damage, nerve atrophy, and / or seizures. In some embodiments, the nerve damage is peripheral nerve damage. In some embodiments, the symptoms of a neurological disorder are pain.

[0030] In one aspect, the disclosure provides a method of treating and / or delaying the onset of pain in a subject in need of such treatment, comprising: (a) administering to the subject a therapeutically effective amount of an engineered receptor of the disclosure, a polynucleotide of the disclosure, a vector of the disclosure, a composition of the disclosure, or a pharmaceutical composition of the disclosure; and (b) administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand simultaneously with step (a). In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. In some embodiments, the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionously, intraneurally, intracranially, intraspinally, or cisternal. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally. In some embodiments, the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. In some embodiments, the subject is human. In some embodiments, the pain is neuropathic pain. In some embodiments, the pain is associated with, caused by, or results from chemotherapy. In some embodiments, the pain is associated with, caused by, or results from trauma. In some embodiments, the subject suffers from allodynia. In some embodiments, the pain is present after a medical procedure, hi some embodiments, the pain is associated with, caused by, or results from childbirth or a Caesarean section.In some embodiments, the pain is associated with, caused by, or resulting from migraine headache.In some embodiments, the therapeutically effective amount temporarily reduces the pain in the subject, permanently reduces the pain in the subject, prevents the onset of pain in the subject, and / or eliminates the pain in the subject.In some embodiments, step (a) and (b) are carried out before the onset of pain in the subject. (Mode for carrying out the invention)

[0031] Detailed Description Overview Intractable neurological diseases are often associated with abnormally active neurons. Attempts to develop therapies to treat these conditions have been hampered by the lack of tractable target proteins associated with the disease. For example, unrelieved chronic pain is a significant health problem in the United States and worldwide. A report by the Institute of Medicine estimated that 116 million Americans suffer from pain that lasts from weeks to years, resulting in annual costs of over $560 million. There is no adequate long-term treatment for chronic pain sufferers, resulting in significant costs to both society and individuals. Pain is often disabling, and even when not disabling, it has a wide-ranging effect on quality of life. Pain management frequently fails, even when the circumstances of care delivery are optimal, including attentive, well-trained physicians; easy access to opioids; use of adjuvant analgesics; availability of patient-controlled analgesics; and evidence-based use of procedures such as nerve blocks and IT pumps.

[0032] The most commonly used treatment for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to dependence and can cause side effects such as drug dependency, tolerance, respiratory depression, sedation, cognitive impairment, hallucinations, and other systemic side effects. Despite the widespread use of pharmaceutical drugs, their effectiveness in pain relief has a surprisingly low success rate. A large randomized study with various drugs found that only one in two or three patients achieved at least 50% pain relief (Finnerup et al., 2005). A follow-up study using the most developed pharmacological treatments found the same results and showed no improvement in the effectiveness of drugs for pain (Finnerup et al., Pain, 150(3):573-81, 2010).

[0033] More invasive options for treating pain include nerve blocks and electrical stimulation. Nerve blocks are local anesthetic injections, usually in the spinal cord, to interrupt pain signals to the brain, an effect that lasts only for weeks to months. Nerve blocks are not a recommended treatment option in most cases (Mailis and Taenzer, Pain Res Manag. 17(3):150-158, 2012). Electrical stimulation involves providing an electrical current to block pain signals. The effect can last longer than nerve blocks, but complications from the electrical lead itself occur: dislocation, infection, breakage, or dead batteries. One review found that 40% of patients treated with electrical stimulation for neuropathy experienced one or more of these problems with the device (Wolter, 2014).

[0034] The most invasive and least preferred method for managing pain is the complete surgical removal of the nerve or part of it that is causing pain. This option is only recommended if the patient has exhausted previous and other less invasive procedures and found them ineffective. Radiofrequency nerve ablation uses heat to destroy the problematic nerve and provides longer pain relief than nerve blocks. However, one study found no difference between control and treatment groups in partial radiofrequency destruction of the DRG for chronic lumbosacral radicular pain (Geurts et al., 2003). Other surgical methods for surgically removing pain nerves suffer from similar drawbacks and have serious long-term side effects, including sensory or motor deficits, or cause pain elsewhere.

[0035] Provided are compositions and methods for modulating cellular activity using engineered ligand-gated ion channel (LGIC) receptors, engineered LGIC receptors encoded by polynucleotides, and gene therapy vectors that include polynucleotides encoding engineered LGIC receptors.These compositions and methods find particular use in modulating neuronal activity, for example, in treating disease or studying neural circuits.Furthermore, provided are reagents, devices, and kits thereof that are used in carrying out the subject methods.

[0036] In particular, the present disclosure provides engineered receptors that bind to and signal in response to known drugs, ligands, and / or binders. In some embodiments, the engineered receptors described herein show increased affinity for agonistic or agonistic binders. In some embodiments, the engineered receptors described herein show affinity for antagonistic or modulator binders and respond to antagonistic and / or modulatory agents as if they were agonistic agents. The present disclosure further provides a method of treating a neurological disease in a subject in need of such treatment. The present disclosure increases the number of clinical indications in which known drugs can be used by using engineered receptors that respond to known drugs in a manner different from wild-type endogenous receptors.

[0037] Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0038] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which neither, either, or both of the limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any method and material similar or equivalent to those described herein can be used in the practice or testing of this disclosure, but some possible preferred methods and materials are described here.All publications cited herein are incorporated herein by reference to disclose and describe the method and / or material together with the publication cited.It is understood that this disclosure takes precedence over any disclosure of the incorporated publication to the extent that there is a contradiction.

[0040] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0041] The publications discussed throughout this disclosure are provided solely for their disclosure prior to the filing date of the present application. Nothing in this disclosure should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. definition

[0042] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, e.g., polypeptides, and the like.

[0043] As used herein, the term "and / or" is used in the present disclosure for either "and" or "or," unless otherwise indicated.

[0044] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of a stated element or integer or group of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers. Moreover, recitations of numerical ranges throughout this specification specifically include all integers and decimal points therebetween.

[0045] Throughout this specification, unless the context requires otherwise, the phrase "consisting essentially of" refers to a limitation of the scope of a described composition, method, or kit to specified materials or steps that do not materially affect the basic and novel characteristics of the subject disclosure. For example, a ligand binding domain "consisting essentially of" a disclosed sequence has an amino acid sequence that is a disclosed sequence plus or minus about 5 amino acid residues of the boundary of the sequence, e.g., about 5, 4, 3, 2, or about 1 residue less than the described boundary amino acid residue, or about 1, 2, 3, 4, or 5 residues more than the described boundary amino acid residue.

[0046] Throughout this specification, unless the context requires otherwise, the phrase "consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a ligand binding domain "consisting of" a disclosed sequence consists solely of the disclosed amino acid sequence.

[0047] As used herein, the terms "about" and "approximately" are used as equivalents. Any numerical values ​​used in this application, including or not including about / approximately, are meant to encompass any normal variation understood by those skilled in the relevant technical field. In certain embodiments, the term "about" or "approximately" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value (greater or less), unless otherwise stated or otherwise clear from the context (except where such number exceeds 100% of possible values).

[0048] As used throughout this disclosure, the term "isolated" means material that is substantially or essentially free from components that normally accompany it as found in its natural state. In some embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0049] The terms "subject", "individual" and "patient" are used interchangeably and refer to a vertebrate animal, such as a mammal. A mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., a cynomolgus monkey, a chimpanzee) or a human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject may be an adult, a teenager, a child (2-14 years), an infant (1-24 months), or a newborn (up to 1 month). In some embodiments, an adult is about 65 years of age or older, or an elderly person about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman who is contemplating pregnancy.

[0050] The term "sample" refers to a volume and / or mass of biological material that is subjected to analysis. In some embodiments, a sample includes a tissue sample, a cell sample, a fluid sample, and the like. In some embodiments, a sample is taken from or provided by a subject (e.g., a human subject). In some embodiments, a sample includes a portion of tissue taken from any internal organ, cancerous, precancerous, or noncancerous tumor, brain, skin, hair (including hair roots), eye, muscle, bone marrow, cartilage, white adipose tissue, and / or brown adipose tissue. In some embodiments, a fluid sample includes a buccal swab, blood, umbilical cord blood, saliva, semen, urine, peritoneal fluid, pleural fluid, cerebrospinal fluid, lung lavage, tears, sweat, and the like. One of skill in the art will understand that in some embodiments, a "sample" is a "primary sample" in that it is obtained directly from a source (e.g., a subject). In some embodiments, a "sample" is the result of processing a primary sample, e.g., to remove certain potentially contaminating components, to isolate certain components, and / or to purify certain components of interest. In some embodiments, the sample is a cell or a population of cells (e.g., neural cells). A cell sample may be derived directly from a subject (e.g., a primary sample) or may be a cell line. A cell line may include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., an immortalized cell line).

[0051] "Treating" or "treatment" as used throughout this disclosure refers to the delivery of a composition (e.g., engineered receptor and / or binding agent) to a subject and / or population of cells to affect a physiological outcome. In certain embodiments, treatment results in an improvement (e.g., reduction, amelioration, or correction) of one or more disease symptoms. The improvement may be an observable or measurable improvement, or may be an improvement in the subject's general sense of well-being. Treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to a reduction in the severity of disease symptoms to a level equivalent to that before the onset of the disease. In some embodiments, treatment may refer to a short-term (e.g., temporary or acute) and / or long-term (e.g., sustained or chronic) reduction of disease symptoms. In some embodiments, treatment may refer to the amelioration of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of a subject at risk of developing a particular disease to prevent the onset of the disease. Preventing the onset of a disease may refer to preventing disease symptoms altogether, delaying the onset of the disease, reducing the severity of symptoms in subsequently developed disease, or reducing the likelihood of developing the disease.

[0052] As used throughout this disclosure, the term "management" or "control" refers to the use of the compositions or methods contemplated in this disclosure to improve the quality of life of an individual suffering from a particular disease. In certain embodiments, the compositions and methods of the present disclosure provide analgesia to a subject suffering from pain.

[0053] A "therapeutically effective amount" is the amount of a composition required to achieve a desired therapeutic result. The therapeutically effective amount may vary depending on factors such as, but not limited to, the disease state and the subject's age, sex, and weight. In general, a therapeutically effective amount is also one in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes the amount of a composition that is effective for treating a subject.

[0054] "Increase" refers to an increase in value (e.g., increased binding affinity, increased physiological response, increased therapeutic effect, etc.) of at least 5% compared to a reference or control level. For example, an increase may include an increase of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. Increase also refers to an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) over a reference or control level.

[0055] "Decrease," "reduce," "reduce" or synonyms thereof refer to a decrease in value (e.g., a decrease in binding affinity, a decrease in physiological response, a decrease in therapeutic effect, a decrease in pain in a subject, etc.) of at least 5% compared to a reference or control level. For example, a decrease may include a decrease of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. A decrease also refers to a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) lower than a reference or control level.

[0056] The terms "reference" or "control" levels are used interchangeably throughout this disclosure and are intended to The reference level refers to the value of a particular physiological and / or therapeutic effect in a subject or sample that is not treated with the composition or in a subject or sample that is treated with a vehicle control. In some embodiments, the reference level refers to the value of a particular physiological and / or therapeutic effect that is a measure in a subject or sample before administration of the composition of the present disclosure (e.g., a baseline level).

[0057] As used throughout this disclosure, the term "ligand" refers to a molecule that binds to another, larger molecule. In some embodiments, a ligand binds to a receptor. In some embodiments, the binding of a ligand to a receptor alters the function of the receptor to activate or inhibit its function. In some embodiments, the binding of a ligand to a receptor, such as a ligand-gated ion channel (LGIC), results in the opening or closing of the ion channel.

[0058] "Receptor-ligand binding" and "ligand binding" are used interchangeably throughout this disclosure and refer to the physical interaction between a receptor (e.g., LGIC) and a ligand. As used throughout this disclosure, the term "ligand" may refer to an endogenous or naturally occurring ligand. For example, in some embodiments, a ligand refers to a neurotransmitter (e.g., λ-aminobutyric acid (GABA), acetylcholine, serotonin, etc.) and a signaling intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., synthetic, or non-naturally occurring ligand (e.g., a binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by various methods known in the art (e.g., detection of association with a radiolabeled ligand).

[0059] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a receptor and a ligand. Unless otherwise indicated, as used throughout this disclosure, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described in this disclosure.

[0060] The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims and refer to binding that occurs between a pair of molecular species, such as a receptor and a ligand. When the interaction of two species results in a non-covalently bound complex, the binding that occurs is typically electrostatic, hydrogen bonded, or the result of lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In one embodiment, the affinity of specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding, or more.

[0061] "Signaling" refers to the occurrence of a biochemical or physiological response as a result of ligand binding to a receptor (e.g., as a result of binding of a binding agent to an engineered receptor of the present disclosure).

[0062] The term "wild-type" or "native" is a term of the art understood by those of skill in the art and refers to the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of that protein as it occurs in nature.

[0063] The terms "non-naturally occurring," "variant," and "mutant" are used interchangeably throughout the specification and claims to refer to mutants of a naturally occurring or wild-type composition, e.g., variant polypeptides having less than 100% sequence identity with the naturally occurring or wild-type sequence.

[0064] The amino acid modification may be an amino acid substitution, an amino acid deletion, and / or an amino acid insertion. The amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, a conservative substitution, or a conservative variation) is an amino acid replacement in a protein that changes a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used throughout this disclosure, a "conservative variation" refers to the replacement of an amino acid residue with another, biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another; or the replacement of one polar residue with another, such as the replacement of arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine. Other examples of conservative substitutions include alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to praline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, and the like.

[0065] The terms "parent" or "starter" are used interchangeably throughout this specification and claims to refer to an initial composition, or a protein that is mutated, modified or derivatized to create an engineered composition with novel properties. In some embodiments, a parent protein is a chimeric protein.

[0066] The term "engineered" is used throughout this specification and claims to refer to a non-naturally occurring composition or protein that has properties that differ from the parent composition or protein from which it is derived.

[0067] Generally, "sequence identity" or "sequence homology" refers to the correspondence between nucleotides or amino acids of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity". The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, an improved version of the BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. This program can be used to determine the percent identity over the entire length of the proteins being compared. For example, default parameters are provided for optimizing searches on short query sequences using the BLAST program.The program also allows the use of SEG filters to unmask segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Desirable ranges of sequence identity are approximately 80% to 100% and intervening integer values. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0068] As used throughout this disclosure, "substantially identical" refers to having a sequence identity that is 85% or greater, e.g., 90% or greater, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, where the activity of the composition is not altered by modifications in the sequence that result in differences in sequence identity.

[0069] As used throughout this disclosure, the term "promoter" refers to one or more nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the start site of transcription, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A "constitutive" promoter is a promoter that is active under many environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0070] As used throughout this disclosure, the term "viral vector", "viral vector" or "gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors of this disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0071] As used throughout this disclosure, "neuronal activity," "neuronal activity," "neuronal firing," and variations and synonyms thereof, refer to electrical activity resulting from stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch clamp techniques. In some embodiments, determining neuronal activity includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the level of neuronal activity depends on or is influenced by the excitatory postsynaptic potential (EPSP), the inhibitory postsynaptic potential (IPSP), and / or the action potential.

[0072] As used throughout this disclosure, "neurological disease" or "neurological disorder" refers to a disease or disorder of the nervous system. In some embodiments, a neurological disease is associated with, caused by, or results from structural, biochemical, and / or electrical abnormalities in the brain, spinal cord, nerves, or any component of the nervous system.

[0073] As used throughout this disclosure, a "sign" of a disease refers to a physical or mental characteristic that is considered to be indicative of a disease state. In some embodiments, the sign is an objective sign of the disease. In some embodiments, the sign is objectively assessed, examined, observed, or measured by a person other than the patient, such as a physician.

[0074] As used throughout this disclosure, a "symptom" of a disease refers to a physical or mental feature that is considered to be indicative of a disease state, particularly one that is evident to the patient. In some embodiments, a symptom is subjectively assessed by the patient. For example, in some embodiments, a symptom is pain.

[0075] As used throughout this disclosure, "potency" refers to the amount of ligand required to produce a certain level of activity of a protein, such as LGIC. In some embodiments, the activity of a protein, such as LGIC, refers to the opening and closing of an ion channel. In some embodiments, determining the potency includes determining the half-maximal effective concentration (EC50) of a protein, such as LGIC, for a ligand under certain conditions. EC50 refers to the concentration of ligand that induces a response halfway between baseline and maximum after a certain exposure time.

[0076] As used throughout this disclosure, "efficacy" refers to a measure of the activity of a protein, such as a LGIC, in the presence of a ligand. In some embodiments, efficacy refers to the amount of current passed through the LGIC under particular conditions, such as in the presence of a particular concentration of ligand. In some embodiments, determining efficacy includes determining the amount of current passing through a receptor and / or the rheobase of the receptor.

[0077] As used throughout this disclosure, "responsiveness" refers to a measure of the overall function of a protein, such as a LGIC, in the presence of a ligand. Determining responsiveness may include determining and considering one or more factors, such as the potency, efficacy, and subcellular localization of the protein.

[0078] As used throughout this disclosure with respect to amino acid positions, the term "corresponding to" or "corresponding to" refers to an amino acid in a first polypeptide sequence that aligns with a given amino acid in a reference polypeptide sequence when the first and reference polypeptide sequences are aligned. Alignment is performed by those skilled in the art using software designed for this purpose, such as Clustal Omega version 1.2.4, with default parameters for that version. As an example, amino acid position D449 in SEQ ID NO:69 corresponds to D464 in SEQ ID NO:61.

[0079] As used throughout this disclosure, an "amino acid mutation" refers to any difference in an amino acid sequence compared to the corresponding parent sequence, such as an amino acid substitution, deletion, and / or insertion. Engineered receptors

[0080] The present disclosure is directed to engineered receptors, engineered receptor mutants, and methods for their use. The term "receptor" as used herein refers to any protein that is on the surface of a cell and can mediate signaling to and / or from a cell. The term "engineered receptor" as used herein refers to a receptor that has been experimentally altered to be physically and / or functionally different from the parent receptor to which it corresponds. In some embodiments, the parent receptor is a wild-type receptor. The term "wild-type receptor" as used herein refers to a receptor that has a polypeptide sequence that is identical to the polypeptide sequence of a protein found in nature. Wild-type receptors include receptors that naturally occur in humans as well as orthologs that naturally occur in other eukaryotes, e.g., protists, fungi, plants, or animals, e.g., yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates. In some embodiments, the parent receptor is a non-natural receptor; i.e., it is a receptor that does not occur in nature, e.g., a receptor that has been engineered from a wild-type receptor. For example, the parent receptor may be an engineered receptor that comprises one or more subunits from one wild-type receptor together with one or more subunits from a second wild-type receptor. The resulting protein thus comprises subunits from two or more wild-type receptors. Thus, in some embodiments, the parent receptor is a chimeric receptor. The engineered receptor of the present disclosure includes, for example, parent receptor mutants and switch receptors.

[0081] In some embodiments, the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding parent receptor, for example, one or more mutations in one or more domains of the wild-type receptor. In some embodiments, the engineered receptor has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity with the corresponding parent receptor (including all values ​​and subranges therebetween). In some embodiments, the engineered receptor has 85% or more sequence identity with the corresponding parent receptor, for example, 90% or more, or 95% or more, for example, about 96%, about 97%, about 98%, or about 99% identity with the corresponding parent receptor (including all values ​​and subranges therebetween). In some embodiments, the engineered receptor (e.g., parent receptor mutant) is generated by error-prone PCR.

[0082] In some embodiments, the ligand binding domain (LBD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ligand binding domain of the parent receptor, for example, one or more mutations in the ligand binding domain of the wild-type receptor. In some embodiments, the ligand binding domain of the engineered receptor shares about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity (including all values ​​and subranges therebetween) with the corresponding ligand binding domain of the parent receptor. In some embodiments, the ligand binding domain of the engineered receptor has 85% or more sequence identity with the corresponding ligand binding domain of the parent receptor, for example, 90% or more, or 95% or more, for example, about 96%, about 97%, about 98%, or about 99% identity (including all values ​​and subranges therebetween) with the corresponding ligand binding domain of the parent receptor. In some embodiments, the ligand binding domain of the engineered receptor is generated by error-prone PCR.

[0083] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ion pore domain of the parent receptor, for example, one or more mutations in the ion pore domain of the wild-type receptor. In some embodiments, the ion pore domain of the engineered receptor shares about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity (including all values ​​and subranges therebetween) with the corresponding ion pore domain of the parent receptor. In some embodiments, the ion pore domain of the engineered receptor has 85% or more sequence identity with the corresponding ion pore domain of the parent receptor, for example, 90% or more, or 95% or more, for example, about 96%, about 97%, about 98%, or about 99% identity (including all values ​​and subranges therebetween) with the corresponding ion pore domain of the parent receptor. In some embodiments, the ion pore domain of the engineered receptor is generated by error-prone PCR.

[0084] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation compared to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, substantially reduce, or abolish the function of the engineered receptor compared to the parent receptor, for example, by reducing the binding of endogenous ligands to the engineered receptor compared to the binding of endogenous ligands to the parent receptor, or by reducing the activity of downstream signaling pathways of the engineered receptor that are typically activated in response to binding of a binding agent to the corresponding parent receptor.

[0085] In some embodiments, the amino acid mutation is a gain-of-function amino acid mutation compared to the corresponding parent receptor. A "gain-of-function" amino acid mutation refers to one or more mutations that modify the function of the engineered receptor compared to the parent receptor, for example, by altering or enhancing the affinity of the engineered receptor for a binder compared to the binding of an endogenous ligand to the parent receptor, or by altering or enhancing the activity of a signaling pathway that is activated in response to binding of a binder to the engineered receptor compared to the binding of an endogenous ligand to the corresponding parent receptor. In some embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for a binder. In certain embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for an agonist binder. In some embodiments, the gain-of-function mutation results in an antagonist binder that acts as an agonist binder when bound to the engineered receptor (e.g., results in activation of an agonist signaling pathway instead of an antagonist signaling pathway). In some embodiments, the gain-of-function mutation results in a modulator binder that acts as an agonist binder when bound to the engineered receptor. In some embodiments, the engineered receptors subject to the present disclosure, or the ligand binding domains and / or ion pore domains thereof, contain one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations compared to the corresponding parent receptor. include.

[0086] In some embodiments, the loss-of-function mutation and the gain-of-function mutation are at the same residue, i.e., they are the same mutation. In other embodiments, the loss-of-function mutation and the gain-of-function mutation are mutations at different amino acid residues. In some embodiments, the engineered receptor of interest (or its ligand-binding domain and / or ion pore domain) containing the loss-of-function mutation and / or the gain-of-function mutation has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or less sequence identity (including all ranges and subranges therebetween) with the corresponding parent receptor, e.g., wild-type receptor or non-natural receptor (or its ligand-binding domain and / or ion pore domain). In some embodiments, the engineered receptor of interest (or its corresponding ligand binding domain and / or ion pore domain) has 85% or more sequence identity, e.g., 85%, 90%, or 95% or more sequence identity, and in some examples, 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity (including all values ​​and subranges therebetween), to the corresponding parent receptor (or its corresponding ligand binding domain and / or ion pore domain).

[0087] In some aspects, the engineered receptor of the present disclosure comprises a receptor that is produced by combining one or more amino acid sequences, e.g., subunits, from one wild-type receptor with one or more amino acid sequences, e.g., subunits, from a second wild-type receptor. In other words, the engineered receptor comprises amino acid sequences that are heterologous to each other, where "heterologous" means that they do not exist together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, the chimeric receptor serves as the parent receptor from which the engineered receptor of the present disclosure is generated.

[0088] In some embodiments, the parent receptor mutant exhibits increased affinity for agonist binding agents, hi some embodiments, a ligand or binding agent that functions as an antagonist or modulator when bound to the wild-type receptor functions as an agonist when bound to the parent receptor mutant.

[0089] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGICs refer to a large group of transmembrane proteins that allow the passage of ions upon activation by a specific ligand (e.g., chemical or binding agent). LGICs are composed of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Ligand binding to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding causes a dramatic change in the permeability of the channel to a specific ion or ions; when it is inactive or closed, ions cannot effectively pass through the channel, but upon ligand binding, up to 107 ions / sec can pass through. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and facilitate the influx of ions into the cytosol. In some embodiments, LGICs respond to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and facilitate the efflux of ions from the cytosol to the extracellular environment. Importantly, activation of LGIC results in the transport of ions (e.g., Ca2+, Na+, K+, Cl-, etc.) across the cell membrane and not the transport of the ligand itself.

[0090] LGIC receptors contain multiple subunits and can be homomeric or heteromeric. A homomeric receptor may be any of a number of subunits. A homomeric receptor contains subunits that are all of the same type. A heteromeric receptor contains subunits in which at least one subunit is different from at least one other subunit contained within the receptor. For example, the glycine receptor contains five subunits, of which there are two types: α-subunits of which there are four isoforms (α1-α4) and β-subunits of which there is a single known isoform. An exemplary homomeric GlyR is a GlyR that contains five α1-GlyR subunits. Similarly, a homomeric GABAA receptor may contain a β3-GABAA subunit, and a nAchR receptor may contain an α7-nAchR subunit. An exemplary heteromeric GlyR may contain one or more α-subunits and one or more β-subunits (e.g., α1β-GlyR). The subunits of an exemplary LGIC receptor are shown in Table 1. Table 1: LGIC receptors and subunits [Table 1-1] [Table 1-2]

[0091] Illustrative examples of families of LGICs suitable for use in certain embodiments include, but are not limited to, Cys-loop receptors such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), lambda-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchRs); as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaCs), ionotropic glutamate receptors, IP3 receptors, P2X receptors, ryanodine receptors, and zinc-activated channels (ZACs).

[0092] Specific non-limiting examples of LGICs suitable for use with the methods described herein include HTR3A; HTR3B; HTR3C; HTR3D; HTR3E; ASIC1; ASIC2; ASIC3; SCNN1A; SCNN1B; SCNN1D; SCNN1G; GABRA1; GABRA2; GABRA3; GABRA4; GABRA5; GABRA6; GABRB1; GABRB2; GABRB3; GABRG1; GABRG2; GABRG3; GABRD; GABRE; GABRQ; GABRP; GABRR1; GABRR2; GABRR3; GLRA1; GLRA2; GLRA3; GLRA4; GLRB; GRIA1; GRIA2; GRIA3; GRIA4;GRID1;GRID2;GRIK1;GRIK2;GRIK3;GRIK4;GRIK5;GRIN1;GRIN2A;GRIN2B;GRIN2C;GRIN2D;GRIN3A;GRIN3B;ITPR1;ITPR2;ITPR3;CHRNA1;CHRNA2;CHRNA3;CHRNA4;CHRNA5;CHRNA6;CHRNA7;CHRNA9;CHRNA10;CHRNB1;CHRNB2;CHRNB3;CHRNB4;CHRNG;CHRND;CHRNE;P2RX1;P2RX2;P2RX3;P2RX4;P2RX5;P2RX6;P2RX7;RYR1;RYR2;RYR3; and ZACN.

[0093] TRPV1, TRPM8 and P2X2 are members of the large LGIC family that share structural characteristics and opening and closing principles.For example, TRPV4, which is similar to TRPV1, is not induced by capsaicin but by heat, and P2X3 is induced by ATP, although it is desensitized more rapidly than P2X2.TRPV1, TRPM8 and P2X2 are therefore non-limiting examples of LGIC suitable for use in certain embodiments.

[0094] In one embodiment, the engineered receptor is TRPV1 or TRPM8 receptor, or its mutant protein. TRPV1 and TRPM8 are vanilloid and menthol receptors expressed by nociceptive neurons in the peripheral nervous system. Both channels appear to function as nonselective sodium and calcium permeable homotetramers. Furthermore, both channels, and their respective primary agonists capsaicin and cooling compounds, such as menthol, are almost absent in the central nervous system. Capsaicin and some cooling compounds, including menthol and icilin, contain potential acceptor sites for photolabile blocking groups. The association of photolabile blocking groups with such acceptors results in ligand-gated ion channels in which light acts as an indirect trigger by releasing active ligands.

[0095] In one embodiment, the engineered receptor is P2X2 receptor or its mutant protein.P2X2 is an ATP-dependent non-selective cation channel that is distinguished by its slow desensitization rate.P2X2 can be used as a selectively addressable source of depolarizing current, providing a platform for the generation of engineered channel-ligand combinations that completely lack natural agonists.

[0096] Non-limiting examples of wild-type LGIC receptor sequences that are useful in generating engineered receptors of the present disclosure include the following, in which the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is underlined:

[0097] In some embodiments, the wild-type LGIC receptor is the human alpha 1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 2), encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1 (SEQ ID NO: 1): [ka]

[0098] In some embodiments, the wild-type LGIC receptor is the human alpha 2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO: 59), encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1, SEQ ID NO: 58): [ka] [ka]

[0099] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO: 61), encoded by the GLRA3 gene (GenBank Accession No. NM_006529.3, SEQ ID NO: 60): [ka]

[0100] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO: 69), encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.3, SEQ ID NO: 68): [ka] [ka]

[0101] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor alpha 7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 4), encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5 (SEQ ID NO: 3)): [ka]

[0102] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3A (5HT3A, GenBank Accession No. NP_998786.2, SEQ ID NO: 6), encoded by the HTR3A gene (GenBank Accession No. NM_213621.3, SEQ ID NO: 5): [ka]

[0103] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3B (5HT3B GenBank Accession No. NP_006019.1, SEQ ID NO: 57), encoded by the HTR3B gene (GenBank Accession No. NM_006028.4, SEQ ID NO: 56): [ka]

[0104] In some embodiments, the wild-type LGIC receptor is the human gamma-aminobutyric acid receptor A (GABA-A), subunit beta-3 (GABA-A β3) (GenBank Accession No. NP_000805.1, SEQ ID NO: 8), encoded by the GABRB3 gene (GenBank Accession No. NM_000814.5, SEQ ID NO: 7): [ka]

[0105] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho1 (ρ1) (GABA-A ρ1) (GenBank Accession No. NP_002033.2, SEQ ID NO: 10), encoded by the GABRR1 gene (GenBank Accession No. NM_002042.4, SEQ ID NO: 9): [ka] [ka]

[0106] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho2 (ρ2) (GABA-A ρ2) (GenBank Accession No. NP_002034.3, SEQ ID NO: 12), encoded by the GABRR2 gene (GenBank Accession No. NM_002043.4, SEQ ID NO: 11): [ka]

[0107] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho3 (ρ3) (GABA-A ρ3) (GenBank Accession No. NP_001099050.1, SEQ ID NO: 14), encoded by the GABRR3 gene (GenBank Accession No. NM_001105580.2, SEQ ID NO: 13): [ka] [ka]

[0108] In some aspects, the engineered receptor of interest is a chimeric receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence derived from at least a first LGIC and an ion pore conducting domain sequence derived from at least a second LGIC, or more simply, an "ion pore domain sequence". In some embodiments, the derived amino acid sequence is identical to the corresponding region of the original amino acid sequence. In some embodiments, the derived amino acid sequence may contain an alteration at at least one amino acid position compared to the corresponding region of the original amino acid sequence. In some embodiments, the derived amino acid sequence differs from the corresponding region of the original amino acid sequence by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the derived amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity (including all ranges and subranges therebetween) to the corresponding region of the original amino acid sequence.

[0109] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequence and ion pore domain sequence of Cys loop receptors are well known in the art and can be easily identified from the literature by using publicly available software such as PubMed, Genbank, Uniprot, etc. In some embodiments, the ligand binding domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ligand binding domain of the first LGIC. In some embodiments, the ion pore domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is shown in bold and the ion pore domain is shown underlined.

[0110] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO:2). In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to around amino acids 29-235 of GlyRα1, e.g., amino acids 29-235, amino acids 29-240, amino acids 29-246, amino acids 29-248, amino acids 29-250, or amino acids 29-252 of SEQ ID NO:2. In certain such embodiments, the ligand binding domain consists essentially of amino acids 29-235 of SEQ ID NO: 2, consists essentially of amino acids 29-240 of SEQ ID NO: 2, consists essentially of amino acids 29-246 of SEQ ID NO: 2, consists essentially of amino acids 29-248 of SEQ ID NO: 2, consists essentially of amino acids 29-250 of SEQ ID NO: 2, or consists essentially of amino acids 29-252 of SEQ ID NO: 2. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human GlyRα1.

[0111] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises around amino acids 23-220 of the human α7-nAChR (SEQ ID NO: 4), for example, amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ligand binding domain consists essentially of amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0112] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), such as amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of SEQ ID NO: 4. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0113] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 23-247 of 5HT3A (SEQ ID NO: 6), such as amino acids 23-240, amino acids 30-245, amino acids 23-247, amino acids 23-250, and in some examples, amino acids 30-255 of SEQ ID NO: 6. In certain embodiments, the ligand-binding domain consists essentially of amino acids 23-240 of SEQ ID NO:6, consists essentially of amino acids 23-245 of SEQ ID NO:6, consists essentially of amino acids 30-247 of SEQ ID NO:6, consists essentially of amino acids 23-250 of SEQ ID NO:6, consists essentially of amino acids 23-255 of SEQ ID NO:6. In some such embodiments, the ligand-binding domain comprises around amino acids 21-239 of 5HT3B (SEQ ID NO:57), such as amino acids 21-232, 21-235, 21-240, 21-245, and in some examples, amino acids 21-247 of SEQ ID NO:57. In certain embodiments, the ligand-binding domain consists essentially of amino acids 21-239 of SEQ ID NO:57, consists essentially of amino acids 21-232 of SEQ ID NO:57, consists essentially of amino acids 21-235 of SEQ ID NO:57, consists essentially of amino acids 21-240 of SEQ ID NO:57, consists essentially of amino acids 21-245 of SEQ ID NO:57. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human 5-hydroxytryptamine receptor 3.

[0114] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-A β3. In some such embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 26-245, 26-248, 26-250, and in some examples, amino acids 26-255 of SEQ ID NO:8. In certain such embodiments, the ligand binding domain consists essentially of amino acids 26-240 of SEQ ID NO: 8, consists essentially of amino acids 26-245 of SEQ ID NO: 8, consists essentially of amino acids 26-248 of SEQ ID NO: 8, consists essentially of amino acids 26-250 of SEQ ID NO: 8, or consists essentially of amino acids 26-255 of SEQ ID NO: 8. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human GABA-A receptor.

[0115] In some embodiments, the ion pore domain to which the ligand binding domain is fused conducts anions, e.g., it comprises the ion pore domain sequence of the human glycine receptor or the human serotonin receptor. In other embodiments, the ion conducting pore domain to which the ligand binding domain is fused conducts cations, e.g., it comprises the ion pore domain sequence of the human acetylcholine receptor or the human gamma-aminobutyric acid receptor A.

[0116] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 245-457 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 240-457, amino acids 245-457, amino acids 248-457, amino acids 249-457, amino acids 250-457, amino acids 255-457, or amino acids 260-457 of SEQ ID NO:2. In some embodiments, the ion pore domain consists essentially of amino acids 245-457 of SEQ ID NO:2, consists essentially of amino acids 248-457 of SEQ ID NO:2, consists essentially of amino acids 249-457 of SEQ ID NO:2, or consists essentially of amino acids 250-457 of SEQ ID NO:2.

[0117] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO:59. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO: 59. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 258-452 of SEQ ID NO: 59. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 260-452 of SEQ ID NO: 59.

[0118] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 61). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 61). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 61). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 61). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 253-464 of SEQ ID NO: 61. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 257-464 of SEQ ID NO: 61. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 259-464 of SEQ ID NO: 61.

[0119] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 253-449 of SEQ ID NO: 69. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 257-449 of SEQ ID NO: 69. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 259-449 of SEQ ID NO: 69.

[0120] In some embodiments, the ion pore domain is derived from an ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 230-502, e.g., amino acids 227-502, amino acids 230-502, amino acids 231-502, amino acids 232-502, or amino acids 235-502 of α7-nAChR (SEQ ID NO: 4). In certain such embodiments, the ion pore domain consists essentially of amino acids 227-502 of SEQ ID NO:4, consists essentially of amino acids 230-502 of SEQ ID NO:4, consists essentially of amino acids 231-502 of SEQ ID NO:4, consists essentially of amino acids 232-502 of SEQ ID NO:4, or consists essentially of amino acids 235-502 of SEQ ID NO:4.

[0121] In some embodiments, the ion pore domain is derived from an ion pore domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 248-516 of 5HT3A (SEQ ID NO:6), e.g., amino acids 240-516, amino acids 245-516, amino acids 248-516, amino acids 250-516, or amino acids 255-516 of SEQ ID NO:6. In certain such embodiments, the ion pore domain consists essentially of amino acids 240-516 of SEQ ID NO:6, consists essentially of amino acids 245-516 of SEQ ID NO:6, consists essentially of amino acids 248-516 of SEQ ID NO:6, consists essentially of amino acids 250-516 of SEQ ID NO:6, or consists essentially of amino acids 253-516. In some such embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 240-441 of 5HT3B (SEQ ID NO:57), e.g., amino acids 230-441, amino acids 235-441, amino acids 240-441, amino acids 245-441, or amino acids 250-441 of SEQ ID NO:57. In certain such embodiments, the ion pore domain consists essentially of amino acids 230 to 441 of SEQ ID NO:57, consists essentially of amino acids 235 to 441 of SEQ ID NO:57, consists essentially of amino acids 240 to 441 of SEQ ID NO:57, consists essentially of amino acids 245 to 441 of SEQ ID NO:57, or consists essentially of amino acids 250 to 441 of SEQ ID NO:57.

[0122] In some embodiments, the ion pore domain is derived from an ion pore domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-A β3. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 246-473 of GABA-A β3 (SEQ ID NO:8), e.g., amino acids 240-473, amino acids 245-473, amino acids 247-473, amino acids 250-473, or amino acids 253-473 of SEQ ID NO:8. In certain such embodiments, the ion pore domain consists essentially of amino acids 240 to 473 of SEQ ID NO:8, amino acids 245 to 473 of SEQ ID NO:8, amino acids 247 to 473 of SEQ ID NO:8, amino acids 250 to 473 of SEQ ID NO:8, or amino acids 253 to 473 of SEQ ID NO:8.

[0123] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-A ρ1 (GABRR1, SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-A ρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-A ρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GABA-A ρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain sequence of human GABA-A ρ1 comprises, consists essentially of, or consists of amino acids 284-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-A ρ1 comprises, consists essentially of, or consists of amino acids 288-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-A ρ1 comprises, consists essentially of, or consists of amino acids 290-479 of SEQ ID NO: 10.

[0124] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-A ρ2 (GABRR2, SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-A ρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-A ρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GABA-A ρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain sequence of human GABA-A ρ2 comprises, consists essentially of, or consists of amino acids 265-466 of SEQ ID NO: 12. In some embodiments, the ion pore domain sequence of human GABA-A ρ2 comprises, consists essentially of, or consists of amino acids 269-466 of SEQ ID NO: 12. In some embodiments, the ion pore domain sequence of human GABA-A ρ2 comprises, consists essentially of, or consists of amino acids 271-466 of SEQ ID NO: 12.

[0125] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-A ρ3 (GABRR3, SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-A ρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-A ρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GABA-A ρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain sequence of human GABA-A ρ3 comprises, consists essentially of, or consists of amino acids 271-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-A ρ3 comprises, consists essentially of, or consists of amino acids 275-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-A ρ3 comprises, consists essentially of, or consists of amino acids 277-467 of SEQ ID NO: 14.

[0126] The terms "pre-M1 linker", "pre-M1 linker" and "pre-M1 linker region" refer to a sequence within the LGIC receptor that is flanked at its amino (N) terminus by the C-terminus of the β10 region of the receptor and at its carboxy (C) terminus by the N-terminus of the transmembrane region 1 (M1) of the receptor. The pre-M1 linker of an LGIC can be readily determined from the art and / or by using any publicly available protein analysis tool, e.g., Expasy, uniProt, etc. In some embodiments, the pre-M1 linker of an LGIC receptor connects the ligand binding domain and the ion pore domain. In some embodiments, the pre-M1 linker of a human α7-nAchR corresponds to amino acids 224-233 of SEQ ID NO:4. In some embodiments, the pre-M1 linker of a human GlyR receptor corresponds to amino acids 242-251 of SEQ ID NO:2, amino acids 248-257 of SEQ ID NO:59, or amino acids 247-256 of SEQ ID NO:61 or 69. In some embodiments, the human GlyR receptor is GlyRα1, GlyRα2, or GlyRα3. In some embodiments, the pre-M1 linker of the GABA-A receptor corresponds to amino acids 275-284 of SEQ ID NO: 10. In some embodiments, the GABA-A receptor is GABA-A receptor ρ1, GABA-A receptor ρ2, or GABA-A receptor ρ3.

[0127] In some embodiments, the pre-M1 linker of the chimeric LGIC receptor comprises or consists of one pre-M1 linker sequence of the wild-type LGIC receptor from which the ligand binding domain or ion pore domain of the chimeric LGIC receptor is derived. In some embodiments, the chimeric LGIC receptor comprises a pre-M1 linker sequence that is different from both the pre-M1 linker sequence of the wild-type LGIC receptor from which the ligand binding domain and ion pore domain of the chimeric LGIC receptor are derived. In some embodiments, the pre-M1 linker of the chimeric LGIC receptor comprises or consists of a pre-M1 linker that is a chimeric sequence based on that of the wild-type LGIC receptor.

[0128] In some embodiments, the chimeric LGIC receptor comprises a ligand binding domain sequence derived from a first LGIC and an ion pore domain sequence derived from a second LGIC, and the pre-M1 linker of the chimeric LGIC receptor comprises or consists of an N-terminal portion derived from the corresponding region of the first LGIC and a C-terminal portion derived from the corresponding region of the second LGIC.

[0129] In some embodiments, the chimeric LGIC receptor comprises a ligand binding domain sequence derived from an α7-nAchR and an ion pore domain sequence derived from a GlyRα family receptor (e.g., GlyRα1, GlyRα2, or GlyRα3), and the pre-M1 linker of the chimeric LGIC receptor comprises or consists of any one of the sequences in Table 10 below. In some embodiments, the pre-M1 linker comprises or consists of a sequence having at least 70%, at least 80%, or at least 90% identity to any one of the sequences in Table 10 below. Table 10: List of pre-M1 linkers [Table 10]

[0130] In some embodiments, the chimeric LGIC receptor comprises a ligand binding domain sequence derived from an α7-nAchR and an ion pore domain sequence derived from a GABA-A family receptor selected from a GABA-A ρ1 receptor, a GABA-A ρ2 receptor, and a GABA-A ρ3 receptor, and the pre-M1 linker of the chimeric LGIC receptor comprises or consists of any one of the sequences in Table 11 below. In some embodiments, the pre-M1 linker comprises or consists of a sequence having at least 70%, at least 80%, or at least 90% identity to any one of the sequences in Table 11 below. Table 11: List of pre-M1 linkers [Table 11]

[0131] In some embodiments, the ion pore domain of a subject chimeric ligand-gated ion channel comprises an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ion pore domain. By "M2-M3 linker domain" or "M2-M3 linker" is meant a sequence within the ion pore domain of the LGIC that is flanked at the amino (N)-terminus by the C-terminus of transmembrane domain 2 (M2) of the receptor and at the carboxy (C)-terminus by the N-terminus of transmembrane domain 3 (M3) of the receptor. The M2-M3 linker of the LGIC is defined as a sequence that is heterologous to the M2-M3 linker domain of the ion pore domain of the LGIC. It can be readily determined from the field and / or by using any publicly available protein analysis tool, e.g., Expasy, uniProt, etc. In some embodiments, when the ion pore domain of the chimeric receptor comprises a heterologous M2-M3 linker, the M2-M3 linker is derived from the same receptor as the ligand binding domain of the chimeric receptor. For example, when a ligand-gated ion channel of interest comprises a ligand binding domain derived from AChR and an ion pore domain derived from GlyR, the ion pore domain sequence may comprise an M2-M3 linker sequence derived from AChR. In some embodiments, the ion pore domain is derived from GlyRα1 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the native M2-M3 linker sequence removed from the ion pore domain corresponds to around amino acids 302-313 of GlyRα1 (SEQ ID NO:2). In some embodiments, the ion pore domain is derived from GlyRα2 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the M2-M3 linker sequence removed from the ion pore domain corresponds to about amino acids 308-319 of GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain is derived from GlyRα3 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the M2-M3 linker sequence removed from the ion pore domain corresponds to about amino acids 307-318 of GlyRα3 (SEQ ID NO:61 or 69). In some embodiments, the ion pore domain is derived from GABA-A ρ1 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the M2-M3 linker sequence removed from the ion pore domain corresponds to about amino acids 335-346 of GABA-A ρ1 (SEQ ID NO:10). In some embodiments, the ion pore domain is derived from GABA-A ρ2 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the M2-M3 linker sequence that is removed from the ion pore domain corresponds to about amino acids 315-326 of GABA-A ρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain is from GABA-A ρ3 and the M2-M3 linker is from the α7-nAChR.In some embodiments, the M2-M3 linker sequence removed from the ion pore domain corresponds to around amino acids 321-332 of GABA-A ρ3 (SEQ ID NO: 14). In some embodiments, the M2-M3 linker inserted is derived from around amino acids 283-295 of the α7-nAChR (SEQ ID NO: 4), e.g., amino acids 290-295, 283-290, 283-295, 287-292, etc., or a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to amino acids 283-295 of the α7-nAChR (including all ranges and subranges therebetween). In some embodiments, the inserted M2-M3 linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the inserted M2-M3 linker is 9-16 amino acids, 10-15 amino acids, 11-14 amino acids, or 12-13 amino acids in length (including all ranges and subranges therebetween).

[0132] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a Cys loop domain sequence that is heterologous to the Cys loop sequence of the ligand-binding domain. By "Cys loop domain sequence" or "Cys loop sequence" is meant a domain in the ligand-binding domain of the Cys loop LGIC that forms a loop structure flanked by cysteines at the N-terminus and C-terminus. Without wishing to be bound by theory, upon binding of a ligand to the ligand-binding domain, the Cys loop is structurally shifted to be close to the M2-M3 loop, and this shift is believed to mediate the biophysical translation of ligand binding in the extracellular domain to signal transduction in the ion pore domain (reviewed in Miller and Smart, Trends in Pharmacological Sci 2009:31(4)). The replacement of endogenous Cys loop sequence with a heterologous Cys loop sequence can increase the conductivity of LGIC by 1.5-fold or more, for example, at least 2-fold, 3-fold or 4-fold, in some cases at least 5-fold or 6-fold, and at least 7-fold, 8-fold, 9-fold or 10-fold at a particular dose. The Cys loop domain of a Cys loop receptor can be easily determined by using the art and / or any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand binding domain of a chimeric receptor comprises a heterologous Cys loop sequence, the Cys loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when a chimeric ligand-gated ion channel of interest comprises a ligand binding domain derived from AChR and an ion pore domain derived from GlyR, the ligand-gated ion channel of interest may comprise a ligand binding domain sequence derived from AChR, except for the sequence of the Cys loop domain derived from GlyR. In some embodiments, the ligand binding domain is derived from α7-nAChR and the Cys loop sequence is derived from GLyRα1, GLyRα2 or GLyRα3.In some embodiments, the Cys loop sequence removed from the ligand binding domain corresponds to around amino acids 150-164 of α7-nAChR (SEQ ID NO:4), e.g., amino acids 150-157 of α7-nAChR. In some embodiments, the Cys loop sequence inserted is from around amino acids 166-180 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 166-172 of GlyRα1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 166-180 of GlyRα1. In some embodiments, the Cys loop sequence inserted is from around amino acids 172-186 of GlyRα2 (SEQ ID NO:59), e.g., amino acids 172-178 of GlyRα2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 172-186 of GlyRα2. In some embodiments, the inserted Cys loop sequence is derived from around amino acids 171-185 of GlyRα3 (SEQ ID NO:61 or 69), e.g., amino acids 171-177 of GlyRα3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 171-185 of GlyRα3. In some embodiments, the inserted Cys loop sequence is derived from around amino acids 198-212 of GABA-A ρ1 (SEQ ID NO:10), e.g., amino acids 198-204 of GABA-A ρ1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 198-212 of GABA-A ρ1. In some embodiments, the inserted Cys loop sequence is derived from around amino acids 178-192 of GABA-A ρ2 (SEQ ID NO:12), e.g., amino acids 178-184 of GABA-A ρ2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 178-192 of GABA-A ρ2.In some embodiments, the inserted Cys loop sequence is derived from around amino acids 184-198 of GABA-A ρ3 (SEQ ID NO:14), e.g., amino acids 184-190 of GABA-A ρ3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 184-198 of GABA-A ρ3.

[0133] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a β1-2 loop domain sequence that is heterologous to the β1-2 loop domain sequence of the ligand-binding domain. By "β1-2 loop domain sequence" or "β1-2 loop or β1-β2 loop" is meant a domain in the ligand-binding domain of the Cys loop LGIC that is flanked at its N-terminus by the C-terminus of the β1 sheet and at its C-terminus by the N-terminus of the β2 sheet. Without wishing to be bound by theory, it is believed that the β1-2 loop helps mediate the biophysical translation of ligand binding in the extracellular domain to the ion pore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). Upon ligand binding, it is believed that the β1-2 loop, together with the Cys loop, comes close to the M2-M3 loop and mediates the biophysical translation of ligand binding in the extracellular domain to signal transduction in the ion pore domain where the M2-M3 loop resides (reviewed in Miller and Smart, supra). The replacement of the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of LGIC by 1.5-fold or more, for example, at least 2-fold, 3-fold or 4-fold, in some cases at least 5-fold or 6-fold, and at least 7-fold, 8-fold, 9-fold or 10-fold at certain doses. The β1-2 loop of a Cys-loop receptor can be easily determined by using the art and / or any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand-binding domain of a chimeric receptor comprises a heterologous β1-2 loop sequence, the β1-2 loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the chimeric ligand-gated ion channel of interest comprises a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the sequence of the β1-2 loop domain of the ligand-binding domain may be derived from GlyR. In some embodiments, the ligand-binding domain is derived from α7-nAChR.In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to around amino acids 67-70 of α7-nAChR (SEQ ID NO: 4), e.g., amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to around amino acids 66-71 of α7-nAChR (SEQ ID NO: 4), and in some embodiments, the ion pore domain is derived from GlyRα1 and the inserted β1-2 loop corresponds to around amino acids 80-85 of GlyRα1 (SEQ ID NO: 2) and contains at most 3, at most 2, at most 1 amino acid mutation, or no amino acid mutation. In some embodiments, the ion pore domain is derived from GlyRα2 and the inserted β1-2 loop corresponds to around amino acids 86-91 of GlyRα2 (SEQ ID NO: 59) and contains at most 3, at most 2, at most 1 amino acid mutation, or no amino acid mutation. In some embodiments, the ion pore domain is derived from GlyRα3 and the inserted β1-2 loop corresponds to around amino acids 85-90 of GlyRα3 (SEQ ID NO:61 or 69) and contains no more than three, no more than two, no more than one amino acid mutation. In some embodiments, the ion pore domain is derived from GABA-A ρ1 and the inserted β1-2 loop corresponds to around amino acids 112-117 of GABA-A ρ1 (SEQ ID NO:10) and contains no more than three, no more than two, no more than one amino acid mutation. In some embodiments, the ion pore domain is derived from GABA-A ρ2 and the inserted β1-2 loop corresponds to around amino acids 92-97 of GABA-A ρ2 (SEQ ID NO:12) and contains no more than three, no more than two, no more than one amino acid mutation. In some embodiments, the ion pore domain is derived from GABA-A ρ3 and the inserted β1-2 loop corresponds to about amino acids 98-103 of GABA-A ρ3 (SEQ ID NO:14) and contains at most 3, at most 2, at most 1 amino acid mutations, or no amino acid mutations.

[0134] Non-limiting examples of sequences of chimeric LGIC receptors of the present disclosure include the sequences disclosed herein as SEQ ID NO: 15-52. In some embodiments, the chimeric LGIC receptor or a polynucleotide encoding same has 85% or higher sequence identity to the sequences provided herein in SEQ ID NO: 15-52, e.g., 90% or higher, 93% or higher, or 95% or higher, i.e., about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences provided herein in SEQ ID NO: 15-52. In the sequences, the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is shown underlined.

[0135] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 attachment point) that contains the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0136] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (R228 attachment point) chimera that contains the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka] [ka]

[0137] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (V224 binding site) chimera, comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0138] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (Y233 attachment point) chimera, comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0139] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 attachment point) that contains the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined) containing the α7-nAChR M2-M3 linker (lowercase): (a) [ka] (b) [ka] (c) [ka] [ka] (d) [ka] (e) [ka] or (f) [ka] [ka]

[0140] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα1 Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 33: [ka] (a) [ka] [ka]

[0141] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that contains a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα1 β1-2 loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined). (a) [ka]

[0142] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, comprising the human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising the GlyRα1 β1-2 loop sequence (lowercase) and Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined): (a) [ka] (b) [ka] (c) [ka] (d) [ka]

[0143] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a ligand binding domain (bold) comprising the human α7-nAChR signal peptide (italics) and the GlyRα1 β1-2 loop sequence (lowercase) fused to a human GlyRα1 ion pore domain (underlined) comprising the human α7-nAChR M2-M3 linker (lowercase). (a) [ka]

[0144] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a ligand binding domain (bold) comprising the human α7-nAChR signal peptide (italics) and the GlyRα1 Cys loop sequence (lowercase) fused to a human GlyRα1 ion pore domain (underlined) comprising the human α7-nAChR M2-M3 linker (lowercase). (a) [ka]

[0145] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (R241 attachment point) that contains the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): (a) [ka]

[0146] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (V236 junction) that contains the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): (a) [ka]

[0147] In some embodiments, the chimeric LGIC receptor is a GABRB3 / GLRA1 chimera (Y245 attachment point) that contains the human GABA-A β3 signal peptide (italics) and the ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): (a) [ka]

[0148] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA2 (R229 attachment point) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GlyRα2 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:62: (a) [ka]

[0149] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 attachment point) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GlyRα3 isoform L ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:63: (a) [ka]

[0150] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 attachment point) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GlyRα3 isoform K ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:70: (a) [ka]

[0151] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GABRR1 (R229 attachment point) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GABA-A ρ1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:64: (a) [ka]

[0152] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA2 (R229 attachment point) chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα2 Cys loop sequence (lowercase) fused to the human GlyRα2 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:65: (a) [ka] [ka]

[0153] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 attachment point) chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα3 Cys loop sequence (lowercase) fused to the human GlyRα3 isoform L ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:66: (a) [ka]

[0154] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 attachment point) chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα3 Cys loop sequence (lowercase) fused to the human GlyRα3 isoform K ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:71: (a) [ka] [ka]

[0155] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GABRR1 (R229 attachment point) chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GABA-A ρ1 Cys loop sequence (lowercase) fused to the human GABA-A ρ1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO:67: (a) [ka] Cell surface expression

[0156] In some embodiments, the engineered receptor comprises one or more amino acid mutations, which alter the expression level of the engineered receptor on the cell surface, compared to the corresponding region of the wild-type receptor. In some embodiments, the one or more amino acid mutations increase the expression level of the engineered receptor on the cell surface, compared to the corresponding engineered receptor without such one or more mutations. In some embodiments, the one or more amino acid mutations that alter the surface expression of the engineered receptor do not negatively affect the potency, efficacy, and / or responsiveness of the engineered receptor.

[0157] In some embodiments, the one or more amino acid mutations are in the pre-M1 linker of the engineered receptor. In some embodiments, the one or more amino acid mutations are 1, 2, 3, 4, 5, or more than 5 amino acid mutations. In some embodiments, the one or more amino acid mutations are a single amino acid mutation.

[0158] In some embodiments, the pre-M1 linker of the engineered receptor comprises an amino acid mutation at one or more positions including positions corresponding to T225, M226, and / or T230 of the human α7-nAChR (SEQ ID NO: 4). In some embodiments, the pre-M1 linker of the engineered receptor comprises an amino acid mutation corresponding to one or more of T225I, M226I, and / or T230P of the human α7-nAChR (SEQ ID NO: 4).

[0159] In some embodiments, the ligand binding domain of the engineered receptor is derived from human a7-nAChR (SEQ ID NO: 4) and comprises a mutation at an amino acid position corresponding to T225 of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation at a position corresponding to T225 of human a7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human a7-nAChR (SEQ ID NO: 4). In some embodiments, the mutation corresponds to T225A, T225C, T225D, T225E, T225F, T225G, T225H, T225I, T225K, T225L, T225M, T225N, T225P, T225Q, T225R, T225S, T225V, T225W, or T225Y of SEQ ID NO:4.

[0160] In some embodiments, the ligand binding domain of the engineered receptor is derived from human a7-nAChR (SEQ ID NO: 4) and comprises a mutation at an amino acid position corresponding to M226 of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation at a position corresponding to M226 of human a7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human a7-nAChR (SEQ ID NO: 4). In some embodiments, the mutation corresponds to M226A, M226C, M226D, M226E, M226F, M226G, M226H, M226I, M226K, M226L, M226N, M226P, M226Q, M226R, M226S, M226T, M226V, M226W, or M226Y of SEQ ID NO:4.

[0161] In some embodiments, the ligand binding domain of the engineered receptor is derived from human a7-nAChR (SEQ ID NO: 4) and comprises a mutation at an amino acid position corresponding to T230 of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation at a position corresponding to T230 of human a7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human a7-nAChR (SEQ ID NO: 4). In some embodiments, the mutation corresponds to T230A, T230C, T230D, T230E, T230F, T230G, T230H, T230I, T230K, T230L, T230M, T230N, T230P, T230Q, T230R, T230S, T230V, T230W, or T230Y of SEQ ID NO:4.

[0162] In some embodiments, the ligand binding domain of the engineered receptor is derived from human α7-nAChR (SEQ ID NO: 4) and comprises a mutation corresponding to T225I in SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the T225I mutation in human α7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human α7-nAChR (SEQ ID NO: 4).

[0163] In some embodiments, the ligand binding domain of the engineered receptor is derived from human a7-nAChR (SEQ ID NO: 4) and comprises a mutation corresponding to M226I in SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the M226I mutation in human a7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human a7-nAChR (SEQ ID NO: 4).

[0164] In some embodiments, the ligand binding domain of the engineered receptor is derived from human a7-nAChR (SEQ ID NO: 4) and comprises a mutation corresponding to T230P in SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the T230P mutation in human a7-nAChR (SEQ ID NO: 4), and the ligand binding domain of the engineered receptor has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity (including all ranges and subranges therebetween) to the ligand binding domain of human a7-nAChR (SEQ ID NO: 4).

[0165] In some embodiments, the cell surface expression of an engineered receptor comprising one or more mutations is increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more (including all ranges and subranges therebetween) compared to the cell surface expression of the corresponding engineered receptor without such one or more mutations. In some embodiments, the cell surface expression of an engineered receptor comprising one or more mutations is increased by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, or at least 100-fold (including all ranges and subranges therebetween) compared to the cell surface expression of the corresponding engineered receptor without such one or more mutations.

[0166] In some embodiments, the ion pore domain of the engineered receptor is derived from human GlyRα2 (SEQ ID NO:59), human GlyRα3 (SEQ ID NO:61 or 69), human GABA-A ρ1 (SEQ ID NO:10), human GABA-A ρ2 (SEQ ID NO:12), or human GABA-A ρ3 (SEQ ID NO:14). In some embodiments, such engineered receptors have higher surface expression compared to a corresponding engineered receptor having an ion pore domain derived from human GlyRα1 (SEQ ID NO:2). In some embodiments, the ion pore domain of such engineered receptors is derived from human GlyRα2 (SEQ ID NO:59). In some embodiments, the ion pore domain of such engineered receptors is derived from human GlyRα3 (SEQ ID NO:61 or 69). In some embodiments, the ion pore domain of such engineered receptors is derived from human GABA-A ρ1 (SEQ ID NO:10).

[0167] In some embodiments, cell surface expression of engineered receptors can be measured using an α-bungarotoxin (a-BTX) binding assay. For example, relative cell surface expression can be assessed using fluorescently labeled α-bungarotoxin according to the fluorescence intensity of α-bungarotoxin staining positive cells.

[0168] Without being bound by any particular theory, the increased cell surface expression of the engineered receptor may be due to one or more of the following factors: (a) increased trafficking of the engineered receptor to the cell surface; (b) reduced trafficking of the engineered receptor to non-cell surface destinations; (c) longer retention of engineered receptors on the cell surface; (d) reduced internalization of the engineered receptor from the cell surface; (e) reducing degradation of the engineered receptor; and / or (f) Increase in total expression of engineered receptors. In some embodiments, increasing cell surface expression of the engineered receptor reduces stress on the endoplasmic reticulum (ER). In some embodiments, increasing cell surface expression of the engineered receptor reduces stress on the cellular degradation machinery. In some embodiments, increasing cell surface expression of the engineered receptor reduces stress on the cellular degradation machinery. In some embodiments, increasing cell surface expression of the engineered receptor increases survival (e.g., long-term survival) of cells expressing the engineered receptor. In some embodiments, increasing cell surface expression of the engineered receptor increases the sensitivity of cells expressing the engineered receptor to one or more ligands (e.g., non-natural ligands). In some embodiments, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100% (including all ranges and subranges therebetween) of the total engineered receptors in the cell are expressed on the cell surface.

[0169] In some embodiments, the engineered receptor forms a homomeric receptor on the cell surface. In some embodiments, the engineered receptor forms a pentameric channel containing homomeric subunits. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα1. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα2. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα3. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GABA-A ρ1. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GABA-A ρ2. In some embodiments, the engineered receptor forms a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GABA-A ρ3. In some embodiments, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or 100% (including all ranges and subranges therebetween) of the engineered receptors expressed on the cell surface form homomeric ion channels. Engineered receptor mutations

[0170] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO:2), wherein eight amino acids corresponding to amino acids 354-361 of SEQ ID NO:2 ("SPMLNLFQ", SEQ ID NO:96) are removed from the ion pore domain. In some embodiments, the engineered receptor in which these eight amino acids have been removed exhibits higher cell surface expression, enhanced efflux to the cell surface, slower internalization from the cell surface and / or slower degradation compared to the corresponding engineered receptor containing these eight amino acids. In some embodiments, the engineered receptor in which these eight amino acids have been removed exhibits higher potency, higher efficacy and / or higher responsiveness compared to the corresponding engineered receptor containing these eight amino acids. In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1, wherein the ion pore domain does not include amino acids between the amino acid positions corresponding to K353 and E362 of SEQ ID NO:2. In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1, wherein the ion pore domain comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, or at most 7 amino acids between amino acid positions corresponding to K353 and E362 of SEQ ID NO: 2. In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1, wherein the ion pore domain comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, or at most 7 amino acids between amino acid positions corresponding to K353 and E362 of SEQ ID NO: 2 that has less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90% sequence identity to SEQ ID NO: 96 (including all ranges and subranges therebetween). In some embodiments, such engineered receptors comprise a ligand binding domain derived from human α7-nAChR, with the Cys loop domain optionally replaced by a sequence derived from the corresponding Cys loop domain of GlyRα1.In some embodiments, such engineered receptors exhibit higher cell surface expression compared to the corresponding engineered receptors that contain an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO:2) and contain these eight amino acids. In some embodiments, the cell surface expression of such engineered receptors is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, or at least 100-fold (including all ranges and subranges therebetween) compared to the corresponding engineered receptors that contain an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO:2) and contain these eight amino acids. In some embodiments, the cell surface expression of such engineered receptors comprises an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO:2) and is increased by at least 50% compared to the corresponding engineered receptor containing these eight amino acids.

[0171] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO: 61). The difference between these two GlyRα3 isoforms is that human GlyRα3 isoform L (SEQ ID NO: 61) comprises an extra 15 amino acid residues corresponding to amino acids 358-372 of SEQ ID NO: 61 ("TEAFALEKFYRFSDM", SEQ ID NO: 95) in the ion pore domain compared to human GlyRα3 isoform K (SEQ ID NO: 69). In some embodiments, engineered receptors comprising an IPD derived from the IPD of GlyRα3 isoform K (i.e., with the 15 amino acid segment removed) exhibit higher cell surface expression, enhanced shedding to the cell surface, slower internalization and / or slower degradation from the cell surface compared to a corresponding engineered receptor comprising an IPD derived from the IPD of GlyRα3 isoform L (i.e., with the 15 amino acid segment). In some embodiments, engineered receptors with the 15 amino acid segment removed exhibit higher potency, higher efficacy and / or higher responsiveness compared to a corresponding engineered receptor comprising the 15 amino acid segment.

[0172] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 isoform K (SEQ ID NO: 69), wherein the ion pore domain does not comprise an amino acid residue between the amino acid positions corresponding to K357 and D358 of SEQ ID NO: 69. In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 isoform K (SEQ ID NO: 69), wherein the ion pore domain comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, or at most 14 amino acids between the amino acid positions corresponding to K357 and D358 of SEQ ID NO: 69. In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 isoform K (SEQ ID NO: 69), the ion pore domain comprises an amino acid sequence between amino acid positions corresponding to K357 and D358 of SEQ ID NO: 69 that has less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90% sequence identity to SEQ ID NO: 95 (including all ranges and subranges therebetween). In some embodiments, such engineered receptors comprise a ligand binding domain derived from human α7-nAChR, the Cys loop domain is optionally replaced by a sequence derived from the corresponding Cys loop domain of GlyRα3. In some embodiments, such engineered receptors exhibit higher cell surface expression compared to a corresponding engineered receptor comprising an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO: 61).In some embodiments, cell surface expression of such engineered receptors is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, or at least 100-fold (including all ranges and subranges therebetween) compared to a corresponding engineered receptor that comprises an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO:61) that comprises the 15 amino acid residues corresponding to amino acids 358-372 of SEQ ID NO:61. In some embodiments, cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor that comprises an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO:61).

[0173] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO:2), and the nuclear localization signal (NLS) / ER retention signal (ERRS) of the ion pore domain comprises one or more mutations. In some embodiments, the NLS / ERRS sequence comprises, consists of, or is within six amino acids corresponding to amino acids 346-351 of SEQ ID NO:2 ("RRKRRH", SEQ ID NO:97). In some embodiments, the mutation comprises one, two, three, four, five, or more than five non-lysine, non-arginine substitutions. In some embodiments, the mutation comprises a deletion of one, two, three, four, five, or more than five amino acids within the NLS / ERRS sequence. In some embodiments, the NLS / ERRS sequence of the engineered receptor corresponding to amino acids 346-351 of SEQ ID NO:2 is replaced by an amino acid sequence of SEQ ID NO:98 ("RRRQKR"), or an amino acid sequence having at most one or at most two mutations according to SEQ ID NO:98. In some embodiments, the NLS / ERRS sequence of the engineered receptor corresponding to amino acids 346-350 of SEQ ID NO:2 ("RRKRR") is replaced by the first five amino acids of SEQ ID NO:98 ("RRRQK"). In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit higher cell surface expression, enhanced efflux to the cell surface, slower internalization and / or slower degradation from the cell surface compared to corresponding engineered receptors containing intact NLS / ERRS sequences. In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit higher potency, higher efficacy and / or higher responsiveness compared to corresponding engineered receptors containing intact NLS / ERRS sequences.

[0174] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα2 (SEQ ID NO:59), and the nuclear localization signal (NLS) / ER retention signal (ERRS) of the ion pore domain comprises one or more mutations. In some embodiments, the NLS / ERRS sequence comprises, consists of, or is within six amino acids corresponding to amino acids 352-357 of SEQ ID NO:59 ("RRRQKR", SEQ ID NO:98). In some embodiments, the mutation comprises one, two, three, four, five, or more than five non-lysine, non-arginine substitutions. In some embodiments, the mutation comprises a deletion of one, two, three, four, five, or more than five amino acids within the NLS / ERRS sequence. In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit higher cell surface expression, enhanced efflux to the cell surface, slower internalization from the cell surface, and / or slower degradation compared to a corresponding engineered receptor containing an intact NLS / ERRS sequence. In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit greater potency, greater efficacy and / or greater responsiveness compared to corresponding engineered receptors containing an intact NLS / ERRS sequence.

[0175] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα3 (SEQ ID NO:61 or 69), and the nuclear localization signal (NLS) / ER retention signal (ERRS) of the ion pore domain comprises one or more mutations. In some embodiments, the NLS / ERRS sequence comprises, consists of, or is within six amino acids corresponding to amino acids 351-356 of SEQ ID NO:61 or 69 ("RRKRKN", SEQ ID NO:99). In some embodiments, the mutation comprises one, two, three, four, five, or more than five non-lysine, non-arginine substitutions. In some embodiments, the mutation comprises a deletion of one, two, three, four, five, or more than five amino acids within the NLS / ERRS sequence. In some embodiments, the NLS / ERRS sequence of the engineered receptor corresponding to amino acids 351-356 of SEQ ID NO:61 or 69 is replaced by an amino acid sequence of SEQ ID NO:98 ("RRRQKR"), or an amino acid sequence having at most one or at most two mutations according to SEQ ID NO:98. In some embodiments, the NLS / ERRS sequence of an engineered receptor corresponding to amino acids 351-355 of SEQ ID NO: 61 or 69 ("RRKRK") is replaced by the first five amino acids of SEQ ID NO: 98 ("RRRQK"). In some embodiments, an engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher cell surface expression, enhanced efflux to the cell surface, slower internalization and / or slower degradation from the cell surface compared to a corresponding engineered receptor containing an intact NLS / ERRS sequence. In some embodiments, an engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher potency, higher efficacy and / or higher responsiveness compared to a corresponding engineered receptor containing an intact NLS / ERRS sequence. Amino acid mutations

[0176] As discussed above, in some aspects, the engineered receptor of interest comprises at least one amino acid mutation that changes the efficacy of ligand to engineered receptor compared to its efficacy to non-mutated parent receptor.In other words, one or more amino acid mutations, such as loss-of-function mutations or gain-of-function mutations, shift the responsiveness of engineered receptor to ligand compared to the responsiveness of non-mutated parent receptor.In some such embodiments, one or more mutations are in the ligand-binding domain of engineered receptor. In some embodiments, when the ligand-binding domain of engineered receptor is a Cys-loop receptor protein, one or more amino acid mutations are substitutions with the residues corresponding to the residues of α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213 and Y217.In some embodiments, one residue is substituted.In some embodiments, 2, 3, 4, or 5 or more residues are substituted, for example, 6, 7, 8, 9 or 10 residues are substituted. In certain embodiments, the residue corresponds to a residue of α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence.

[0177] In some embodiments, the one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine and the non-natural ligand, e.g., 2-fold or less, 3-fold or less, 4-fold or less, 5-fold or less, 10-fold or less, 20-fold or less, 30-fold or less, 50-fold or less, or 100-fold. In certain embodiments, the one or more substitutions correspond to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of α7-nAChR. In other examples, the one or more substitutions selectively reduce the potency of acetylcholine to the engineered receptor. In other words, the one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine while essentially maintaining the responsiveness to the non-natural ligand, or reduce the responsiveness of the engineered receptor to acetylcholine by 2 or less, for example, 3, 4, 5 or less, and in some cases, 10, 20, 50, or 100 or less, than it would otherwise reduce the responsiveness of the engineered receptor to the non-natural ligand. Exemplary substitutions, i.e., substitutions corresponding to L131E, L131S, L131T, L131D, or S172D of α7-nAChR. In yet other embodiments, the one or more substitutions selectively reduce the responsiveness of the engineered receptor to the non-natural ligand.In other words, the one or more substitutions reduce the responsiveness of the engineered receptor to the non-natural ligand while essentially maintaining the responsiveness to acetylcholine, or reduce the responsiveness of the engineered receptor to the non-natural ligand by 2-fold or less, for example, 3-fold, 5-fold or less, and in some cases, 10-fold, 20-fold or 50-fold or less than it would otherwise reduce the responsiveness of the engineered receptor to acetylcholine. Exemplary substitutions include those corresponding to W77M, Y115W, S172T, or S172C of α7-nAChR. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126, and facinicline / RG3487.

[0178] In other embodiments, the one or more substitutions increase the responsiveness of the engineered receptor to acetylcholine and / or the non-natural ligand, for example, by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. Exemplary substitutions include those corresponding to L131N, L141W, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S172C, S188I, S188V, S188F, S188M, S188Q, S188T, S188P, or S188W. In some examples, one or more substitutions, such as those corresponding to L131N, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q, and S188T of the α7-nAChR, increase the potency of both acetylcholine and the non-natural ligand. In other examples, the one or more substitutions selectively increase the potency of acetylcholine for the engineered receptor. In other words, one or more substitutions, such as those corresponding to L141W, S172T, S172C, S188P or S188W of α7-nAChR, increase the responsiveness of the engineered receptor to acetylcholine by 2-fold or more, such as 3-fold, 4-fold, or 5-fold or more, and in some cases 10-fold, 20-fold, 50-fold, or 100-fold, more than it increases the responsiveness of the engineered receptor to a non-natural ligand. In certain embodiments, the one or more substitutions are in the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126 and facinicline / RG3487. In yet other examples, the one or more substitutions selectively increase the potency of the non-natural ligand to the engineered receptor.In other words, the one or more substitutions increase the responsiveness of the engineered receptor to a non-natural ligand by 2-fold or more, e.g., 3-fold, 5-fold or more, and in some cases, 10-fold, 20-fold or 50-fold or more, than it increases the responsiveness of the engineered receptor to acetylcholine.

[0179] In some embodiments, the amino acid residue mutated in the engineered receptor of interest is not an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7 nAChR (SEQ ID NO: 4). In some embodiments, the amino acid residue mutated in the engineered receptor of interest is an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7 nAChR (SEQ ID NO: 4). In some embodiments, the substitution is not a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution corresponds to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in wild-type α7 nAChR. In some embodiments, when such a substitution is present in an engineered receptor, it is present in combination with one or more amino acid mutations described herein.

[0180] For example, we found that residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 4) mediate the binding of natural ligand acetylcholine.Mutations at these residues reduce the binding of acetylcholine, and therefore are loss-of-function mutations.In contrast, residues W77, Y115, N129, V130, L131, Q139, L141, S170, Y210, C212, C213, and Y217 of α7-nAChR mediate the binding of non-natural ligand AZD0328 to this receptor, and mutations at these residues increase the affinity of AZD0328 and / or other ligands to this receptor, and therefore may be gain-of-function mutations. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues in the ligand binding domain region of the α7-nAChR (SEQ ID NO: 4) or a ligand binding domain of a chimeric receptor comprising the ligand binding domain region of the α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217. In certain embodiments, the mutation in one or more amino acid residues in the ligand binding domain region of α7-nAChR (SEQ ID NO: 4) or the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of α7-nAChR is a substitution with one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217.

[0181] As another example, we have discovered that residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of the α7-nAChR (SEQ ID NO: 4) mediate the binding of acetylcholine and / or nicotine, and that mutations at one or more of these residues reduce the binding of acetylcholine and / or nicotine. R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of the α7-nAChR mediate the binding of the non-natural ligand ABT126, and mutations at one or more of these residues are predicted to increase the affinity of ABT126 and / or other ligands for the α7-nAChR. R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213 and Y217 of the α7-nAChR mediate binding of the non-natural ligand TC6987, and mutation of one or more of these residues is predicted to increase the affinity of TC6987 and / or other ligands for the α7-nAChR. R101, N120, L131, L141, S170, W171, S172, Y210 and Y217 of the α7-nAChR mediate binding of the non-natural ligand facinicline / RG3487, and mutation of one or more of these residues is predicted to increase the affinity of facinicline / RG3487 and / or other ligands for the α7-nAChR. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues in the ligand binding domain region of an α7-nAChR or a ligand binding domain of a chimeric receptor comprising the ligand binding domain region of an α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N120, N129, L131, L141, S170, W171, S172, S188, Y210, C212, C213, and Y217. In some embodiments, the one or more amino acid residues alter the binding of acetylcholine and / or nicotine to the α7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212, and Y217 of the α7-nAChR.In certain such embodiments, the amino acid is selected from C212 and S170. In some embodiments, the mutation in one or more amino acid residues alters the binding of ABT126 to α7-nAChR, and wherein one or more amino acid residues are selected from the group consisting of R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from R101, S188, and Y210. In some embodiments, the mutation in one or more amino acid residues alters the binding of TC6987 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210, and C213. In some embodiments, the mutation in one or more amino acid residues alters the binding of fasiniline / RG3487 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR. In certain such embodiments, the amino acids are selected from Y210, R101, and N129.

[0182] As another example, it has been found that residues W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3 (SEQ ID NO: 6) mediate the binding of serotonin, and mutations at one or more of these residues reduce the binding of serotonin to 5HT3.D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3 mediate the binding of the non-natural ligand silansetron, and mutations at one or more of these residues are predicted to increase the affinity of silansetron and / or other ligands for 5HT3. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues of a ligand binding domain of a chimeric receptor comprising a ligand binding domain region of 5HT3A or 5HT3, where the one or more amino acid residues are selected from the group consisting of D64, I66, W85, R87, Y89, N123, Y136, Y138, G146, N147, Y148, K149, T176, S177, S178, L179, W190, R191, F221, E224, Y228, Y229, and E231. In some embodiments, the mutation in one or more amino acid residues alters the binding of serotonin to 5HT3, wherein the amino acid is selected from the group consisting of W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3A.In certain such embodiments, the amino acid is selected from Y136, Y138, N147, K149, and L179. In some embodiments, a mutation in one or more amino acid residues alters the binding of cilansetron to 5HT3, wherein the one or more amino acid residues are selected from the group consisting of D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229 and E231 of 5HT3A.In certain such embodiments, the amino acids are selected from D64, I66, Y89, N123, T176, W190, R191, F221, E224, and E231.

[0183] In some embodiments, the one or more mutations that affect the ability of the ligand to modulate the activity of LGIC are located in the ion pore domain of LGIC. For example, residue T279 of serotonin receptor 5HT3A mediates the way that the ligand modulates the activity of the channel, such that mutation of this residue, for example to serine (T279S), converts the effect from antagonistic (i.e., reducing the activity of LGIC) to agonistic (i.e., promoting the activity of the channel). In some embodiments, the ligand-gated ion channel of interest comprises a mutation in one or more amino acid residues of the ion pore domain of human 5HT3A (SEQ ID NO: 6) or the ion pore domain of a chimeric LGIC receptor that comprises the ion pore domain of 5HT3A, where the substitution is at the amino acid corresponding to 279 of SEQ ID NO: 6. In certain embodiments, the substitution is a T279S substitution with respect to SEQ ID NO: 6.

[0184] The present disclosure provides engineered receptors that have two or more mutations, such as amino acid substitutions, compared to parent receptors. In some embodiments, the parent receptor is a chimeric receptor. In some embodiments, the parent receptor comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the engineered receptor comprises two amino acid substitutions compared to the parent receptor that comprises the amino acid sequence of SEQ ID NO: 33.

[0185] In some embodiments, the two amino acid substitutions are at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of L131S and S172D, L131T and S172D, L131D and S172D, Y115D and S170T, Y115D and L131Q, and Y115D and L131E. In some embodiments, the ligand binding domain comprises an amino acid substitution of L131E.

[0186] The present disclosure provides an engineered receptor that is a chimeric ligand-gated ion channel (LGIC) receptor, comprising: (a) a ligand-binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprising a Cys loop domain derived from a human glycine receptor α1 subunit; and (b) an ion pore domain derived from a human glycine receptor α1 subunit, wherein the ligand-binding domain comprises one or more amino acid substitutions at amino acid residues selected from the group consisting of Y140, R101, L131, Y115, and Y210, and wherein the amino acid residues correspond to the amino acid residues of the α7-nAChR. In some embodiments, the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, wherein the amino acid sequence further comprises one or more amino acid substitutions at amino acid residues selected from the group consisting of Y140, R101, L131, Y115, and Y210, and wherein the amino acid residues correspond to the amino acid residues of the α7-nAChR.

[0187] In some embodiments, the ligand-binding domain comprises an amino acid substitution at amino acid residue Y140. In some embodiments, the amino acid substitution is Y140I, or Y140C.

[0188] In some embodiments, the ligand binding domain comprises an amino acid substitution of R101W and / or Y210V. In some embodiments, the ligand binding domain comprises two or more amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of R101 and L131, Y115 and Y210, and R101 and Y210. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of R101F and L131G, R101F and L131D, Y115E and Y210W, R101W and Y210V, R101F and Y210V, R101F and Y210F, R101M and L131A, and R101M and L131F. In some embodiments, the ligand binding domain comprises three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand binding domain comprises amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0189] In some embodiments, the potency of the engineered receptor for acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is at least about 1.5 times (e.g., about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 6, about 1 / 7, about 1 / 8, about 1 / 9, about 1 / 10, about 1 / 12, about 1 / 15, about 1 / 20, about 1 / 30, about 1 / 40, about 1 / 50, about 1 / 60, about 1 / 70, about 1 / 80, about 1 / 90, or about 1 / 100 (including all subranges and values ​​therebetween)) than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine.

[0190] In some embodiments, the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is greater than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least about 1.5-fold (e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold (including all subranges and values ​​therebetween)) higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, determining the potency includes determining the EC50.

[0191] In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least about 1.5 times (e.g., about 1 / 2, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, determining efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand.

[0192] In some embodiments, when the ligand-gated ion channel of interest contains one or more non-desensitizing mutations, "desensitization" refers to the progressive reduction of ion influx in the prolonged presence of agonist. This results in the progressive loss of neuronal responsiveness to ligand. Non-desensitizing mutation refers to an amino acid mutation that prevents the LGIC from becoming desensitized to the ligand, thereby preventing the neuron from becoming less responsive or unresponsive to the ligand. Non-desensitizing mutations can be easily identified by introducing a LGIC carrying the mutation into a neuron and analyzing the current flow over time during prolonged exposure to the ligand. If the LGIC does not contain a non-desensitizing mutation, the current will recover from the peak to a plateau during prolonged exposure, whereas if the LGIC contains a non-desensitizing mutation, the current will remain at the peak flow for the duration of exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (post-processing of the proprotein at V294L to remove the signal peptide) and the L321V mutation in human GABA-A receptor GABRB3 (post-processing of the proprotein at L296V to remove the signal peptide). In some embodiments, the desensitizing mutation is replacement of amino acid residues at or near the C-terminus of the LGIC with a sequence having 90% or higher identity to a desensitizing sequence, e.g., IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53) from the C-terminus of the protein encoded by GABAR1, e.g., replacement of residues 455-479 in GABRR1 with IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53).LGIC desensitization, methods for measuring LGIC desensitization, and non-desensitizing mutations are well known in the art; see, e.g., Gielen et al. Nat Commun 2015 Apr 20, 6:6829, and Keramidas et al. Cell Mol Life Sci. 2013 Apr;70(7):1241-53, the entire disclosures of which are incorporated herein by reference.

[0193] In some embodiments, the ligand-gated ion channel of interest is a fusion protein that is capable of regulating one or more A converting mutation refers to a mutation that changes the permeability of the ion pore domain of the LGIC such that it becomes permissive for the conduction of non-native ions, i.e., ions that cannot be naturally passed. In some cases, a mutation, e.g., replacement of amino acid residues 260-281 in human α7-nAChR (CHRNA7) (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 54) or the corresponding amino acid in another cation-permeable LGIC, with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 55), converts the permeability from cations to anions. In some cases, the mutation converts the permeability from anion to cation, for example, substitution of amino acid residue 279 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, with glutamic acid (E) (which, as an A293E substitution in GLRA1, converts the LGIC from anion tolerant to calcium tolerant), or deletion of amino acid residue 278 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, substitution of amino acid residue 279 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, with glutamic acid (E), and substitution of amino acid residue 293 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, with valine (V) (which, as an P278Δ, A279E, T293V in GLRA1, converts the LGIC from anion tolerant to cation tolerant).

[0194] Further engineered receptors beyond those described herein can be easily identified by in vitro screening and validation methods. In some embodiments, a library of parent receptor mutants is generated from a limited number of parent receptors. The parent receptors can be mutated using methods known in the art, including error-prone PCR. In some embodiments, the library of parent receptor mutants is then transfected into yeast or mammalian cells and screened at high efficiency to identify functional receptors (e.g., to identify parent receptor mutants that can signal in response to binding agents or ligands). In some embodiments, the functional parent receptor mutants identified in this primary screen are then expressed in mammalian cells and screened for responsiveness to binding agents or ligands, for example, by plate readers and / or electrophysiological assays described herein. Parent receptor mutants that show increased binding affinity to agonist binding agents or that allow the use of antagonist or modulator binding agents as agonists in secondary screens can then be selected and further in vitro and / or in vivo validation and characterization assays can be performed.Such screening assays are known in the art and are described, for example, in Armbruster, BN et al. (2007) PNAS, 104, 5163-5168; Nichols, CD and Roth, BL (2009) Front. Mol. Neurosci. 2, 16; Dong, S. et al. (2010) Nat. Protoc. 5, 561-573; Alexander, GM et al. (2009) Neuron 63, 27-39; Guettier, JM et al. (2009) PNAS 106, 19197-19202; Ellefson JW et al. (2014) Nat Biotechnol. 32 (1): 97-101; Maranhao AC and Ellington AD. (2017) ACS Synth Biol. 20; 6 (1): 108-119; Talwar S et al. (2013) PLoS One; 8 (3): e58479; Gilbert DF et al. (2009) Front Mol Neurosci. 30; 2: 17; Lynagh and Lynch, (2010), Biol Chem. 14: 285 (20), 14890-14897; Islam R. et al. (2016) ACS Chem Neurosci. 21; 7 (12): 1647-1657; and Myers et al. (2008) Neuron. 8: 58 (3): 362-373. Exemplary LGIC Receptors

[0195] Exemplary LGIC receptors of the present disclosure are provided in Table 12 below. Chimeric LGIC receptors are described by their ligand binding domain (LBD) and ion pore domain (IPD) separated by a colon mark. "Gly-cys" indicates that the Cys loop of the ligand binding domain is replaced with the corresponding Cys loop of the glycine receptor from which the ion pore domain is derived. Some LGIC receptors contain one or more amino acid mutations as shown in the table.

[0196] For example, CODA1298 in Table 12 comprises a ligand binding domain derived from the human α7-nAChR receptor and an ion pore domain derived from GlyRα2, where the Cys loop of the ligand binding domain is replaced by the Cys loop of GlyRα2, and the receptor comprises a T225I mutation at the amino acid position corresponding to T225 in the human α7-nAChR receptor (SEQ ID NO:4). Table 12: Exemplary LGIC Receptors [Table 12-1] [Table 12-2] [Table 12-3] Binder

[0197] The terms "binding agent" or "drug" are used interchangeably herein and refer to an exogenous drug or compound that has a known mechanism of action on mammalian cells (e.g., known to act as an agonist, antagonist, or modulator of a receptor). Binding agents may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the binding agent includes a drug or compound approved by the U.S. Food and Drug Administration (FDA) for clinical use in the treatment of a particular disease (e.g., a neurological disease). In some embodiments, the binding agent includes a drug or compound that has not been approved by the FDA for clinical use but has been tested in one or more clinical trials, is currently being tested in one or more clinical trials, and / or is expected to be tested in one or more clinical trials. In some embodiments, the binding agent includes a drug or compound that has not been approved by the FDA for clinical use but is routinely used in laboratory research. In some embodiments, the binding agent is an analog of one of the above ligands. In certain embodiments, the binding agent is selected from any one of the agents in Tables 2-9. In some embodiments, the binding agent is selected from the group consisting of AZD0328, ABT-126, AQW-051, cannabidiol, cilansetron, PH-399733, facinicline / RG3487 / MEM-3454, TC-6987, APN-1125, and TC-5619 / AT-101. In some embodiments, the binding agent is selected from the group consisting of ABT-126, AZD-0328, APN-1125, RG3487, TC-6987, and TC-5619.

[0198] In certain embodiments, the binding agent is an analog of cilansetron, for example, as described by one of the following compound formulas 2-7 in either its R or S enantiomer. [ka]

[0199] In some embodiments, the binding agent acts as an agonist. As used herein, the term "agonist" refers to a ligand or binding agent that induces a signaling response. In some embodiments, the binding agent acts as an antagonist. As used herein, the term antagonist refers to an agent that inhibits a signaling response.

[0200] In some embodiments, the binding agent is an anxiolytic, anticonvulsant, antidepressant, antipsychotic, antiemetic, nootropic, antibiotic, antifungal, antiviral, or antiparasitic agent. Table 2: Binding agents for glycine receptor (GlyR) [Table 2] Table 3: Binding agents for λ-aminobutyric acid A receptors (GABA-A) [Table 3-1] [Table 3-2] [Table 3-3] Table 4: Binding agents for the 5-hydroxytryptamine receptor (5-HT3) [Table 4-1] [Table 4-2] Table 5: Binding agents for nicotinic acetylcholine receptors (nAchRs) [Table 5-1] [Table 5-2] Table 6: Binding agents for ATP-dependent P2X receptor cation channels (P2X) [Table 6] Table 7: Binding agents for inward rectifier potassium channels (Kir) [Table 7] Table 8: Binding agents for voltage-gated potassium channels (KCNQ / Kv7) [Table 8] Table 9: Binding agents for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) [Table 9] Polynucleotides

[0201] In various exemplary embodiments, the present disclosure contemplates, in part, polynucleotides encoding engineered receptor polypeptides comprising LGICs, as well as subunits and mutant proteins thereof, as well as fusion polypeptides, viral vector polynucleotides, and compositions comprising same.

[0202] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence" or "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example by conjugation with a labeling component. Polynucleotides may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.Polynucleotide refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 (including all ranges and subranges therebetween) or more nucleotides in length, as well as intermediate lengths.It will be readily understood that "intermediate lengths" in this context means any length between the enclosed values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In certain embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (including all ranges and subranges therebetween) sequence identity to a reference sequence described herein or known in the art, and typically the variants maintain at least one biological activity of the reference sequence, unless otherwise stated.

[0203] As used herein, the term "gene" can refer to a polynucleotide sequence that includes enhancers, promoters, introns, exons, etc. In certain embodiments, the term "gene" refers to a polynucleotide sequence that encodes a polypeptide, regardless of whether the polynucleotide sequence is identical to a genomic sequence that encodes the polypeptide.

[0204] As used herein, "cis-acting sequence", "cis-acting regulatory sequence" or "cis-acting nucleotide sequence" or equivalents refer to a polynucleotide sequence associated with the expression, e.g., transcription and / or translation, of a gene. In one embodiment, a cis-acting sequence regulates transcription because it is a binding site for a polypeptide that represses or reduces the transcript or a polynucleotide sequence associated with a transcription factor binding site that contributes to transcriptional repression. Examples of cis-acting sequences that can be operably linked to the polynucleotides of the present disclosure to regulate the expression of a polynucleotide sequence and regulate the expression of an engineered receptor of interest are well known in the art and include elements such as promoter sequences (e.g., CAG, CMV, SYN, CamKII, TRPV1), Kozak sequences, enhancers, post-transcriptional regulatory elements, miRNA binding elements, and polyadenylation sequences.

[0205] As one non-limiting example, a promoter sequence is a DNA regulatory region that can bind RNA polymerase in a cell and initiate transcription of a downstream (3' direction) coding sequence. For the purposes of defining the present invention, a promoter sequence is bound at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at a level detectable above background. Within the promoter sequence, a transcription initiation site and a protein binding domain responsible for the binding of RNA polymerase are found. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. A variety of promoters can be used to drive the various vectors of the present invention. For example, the promoter can be a constitutively active promoter, i.e., a promoter that is active in the absence of an exogenously applied agent, such as the CMV IE1 promoter, the SV40 promoter, the GAPDH promoter, the actin promoter. The promoter may be an inducible promoter, i.e., a promoter whose activity is regulated upon application of an agent to a cell, such as doxycycline, tet-on or tet-off promoters, estrogen receptor promoters, etc. The promoter may be a tissue-specific promoter, i.e., a promoter that is active in certain types of cells.

[0206] In some embodiments, the promoter is active in excitable cells. "Excitable cells" refers to cells that are activated by changes in membrane potential, such as neurons or muscle cells, such as dorsal root ganglion neurons, motor neurons, excitatory neurons, inhibitory neurons, or sensory neurons. The promoters active in excitable cells used in the polynucleotide compositions include neuronal promoters, such as synapsin (SYN), TRPV1, Na v 1.7, Na v 1.8, Na v1.9, CamKII, NSE, and advillin promoters; muscle cell promoters such as desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC) promoters; and ubiquitously acting promoters such as CAG, CBA, E1Fa, Ubc, CMV, and SV40 promoters.

[0207] As used herein, "regulatory element for inducible expression" refers to a polynucleotide sequence that is a promoter, enhancer, or functional fragment thereof, that is operably linked to a polynucleotide to be expressed and responds to the presence or absence of a molecule that binds to the element to increase (turn on) or decrease (turn off) the expression of the polynucleotide operably linked thereto. Exemplary regulatory elements for inducible expression include, but are not limited to, tetracycline-responsive promoter, ecdysone-responsive promoter, cumate-responsive promoter, glucocorticoid-responsive promoter, estrogen-responsive promoter, RU-486-responsive promoter, PPAR-gamma promoter, and peroxide-inducible promoter.

[0208] "Regulatory elements for transient expression" refers to polynucleotide sequences that can be used to express the nucleotide sequence of a polynucleotide in a short period or temporarily. In certain embodiments, one or more regulatory elements for transient expression can be used to limit the duration of the polynucleotide. In certain embodiments, the preferred duration of polynucleotide expression is in the order of minutes, hours, or days. Exemplary regulatory elements for transient expression include, but are not limited to, nuclease target sites, recombinase recognition sites, and inhibitory RNA target sites. Furthermore, to some extent, in certain embodiments, regulatory elements for inducible expression can also contribute to controlling the duration of polynucleotide expression.

[0209] As used herein, the term "polynucleotide variant" and "variant" and the like refer to a polynucleotide that shows substantial sequence identity with a reference polynucleotide sequence or a polynucleotide that hybridizes with a reference sequence under stringent conditions as defined below.These terms also encompass polynucleotides that are differentiated from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide.Thus, the term "polynucleotide variant" and "variant" includes polynucleotides in which one or more nucleotides are added or deleted, or modified, or replaced with different nucleotides.In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, whereby the modified polynucleotide retains the biological function or activity of the reference polynucleotide. In certain embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (including all ranges and subranges therebetween) sequence identity to a reference sequence described herein or known in the art, and typically the variants maintain at least one biological activity of the reference sequence, unless otherwise stated.

[0210] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. Hybridization under "stringent conditions" describes a hybridization protocol in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration). The target sequence is generally present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0211] As used herein, the phrase "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison, including, for example, "a sequence 50% identical to." Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12, but frequently 15-18, and often at least 25 monomer units, including nucleotides and amino acid residues in length. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) sequences that differ from each other between the two polynucleotides, and sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, generally about 50 to about 100, more generally about 100 to about 150, where a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may include about 20% or less additions or deletions (ie, gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA), or by inspection and best alignment (i.e., resulting in the highest homology percentage over the comparison window) generated by any of the various methods selected. For example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389 can also be referenced. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc,1994-1998, Chapter 15.

[0212] An "isolated polynucleotide," as used herein, refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotide that does not occur in nature and has been created by the hand of man.

[0213] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence can be noted in the 5' to 3' direction or the 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus" or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated the "template", "antisense", "minus" or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0214] The term "flanked" refers to a polynucleotide sequence that is between upstream and / or downstream polynucleotide sequences, i.e., 5' and / or 3' to a sequence. For example, a sequence "flanked" by two other elements (e.g., ITRs) indicates that one element is located 5' to the sequence and the other is located 3' to the sequence, however, there may be intervening sequences between them.

[0215] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complement of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often written as a reverse complement, with 5'CATGACT3' at the 5' end on the left and 3' end on the right. A sequence equivalent to its reverse complement is said to be a palindrome. Complementarity can be "partial," where only a portion of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids.

[0216] The term "nucleic acid cassette" or "expression cassette" as used herein refers to a polynucleotide sequence within a larger polynucleotide, such as a vector, that is sufficient to express one or more RNAs from the polynucleotide. The expressed RNA may be translated into a protein or may function as a guide or inhibitory RNA that targets other polynucleotide sequences for cleavage and / or degradation. In one embodiment, the nucleic acid cassette contains one or more polynucleotides of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences operably linked to one or more polynucleotides of interest. The polynucleotide comprises a polynucleotide of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or a polynucleotide that serves as a template for transcription of an inhibitory polynucleotide, such as LGIC, and its subunits and mutant proteins, as contemplated herein. In certain embodiments, the polynucleotide of interest encodes a polypeptide or fusion polypeptide that has one or more enzymatic activities, such as nuclease activity and / or chromatin remodeling or epigenetic modification activity.

[0217] The vector may contain one, two, three, four, five, six, seven, eight, nine, or ten or more nucleic acid cassettes. In a preferred embodiment of the present disclosure, the nucleic acid cassette comprises one or more expression control sequences (e.g., promoters or enhancers operable in neuronal cells) operably linked to a polynucleotide encoding an engineered receptor, e.g., LGIC, or a subunit or mutant protein thereof. The cassette may be removed from or inserted into another polynucleotide sequence, e.g., a plasmid or viral vector, as a single unit.

[0218] In one embodiment, a polynucleotide contemplated herein comprises one, two, three, four, five, six, seven, eight, nine, or more nucleic acid cassettes, any number or combination of which may be in the same or opposite orientation.

[0219] Also, as contemplated herein, it will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can code for a polypeptide, or a fragment or variant thereof. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage, e.g., polynucleotides optimized for human and / or primate codon preferences, are specifically contemplated by the present disclosure. In one embodiment, a polynucleotide is provided that comprises a particular allele sequence. An allele is an endogenous polynucleotide sequence that is altered as a result of one or more mutations, such as deletion, addition and / or substitution of nucleotides. vector

[0220] In some aspects of the disclosure, a nucleic acid molecule, i.e., a polynucleotide, encoding an engineered receptor is delivered to a subject. In some cases, the nucleic acid molecule encoding an engineered receptor is delivered to a subject by a vector. In various embodiments, the vector comprises one or more polynucleotide sequences contemplated herein. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The polynucleotide to be transferred is usually linked to, e.g., inserted into, a vector nucleic acid molecule. The vector may contain sequences directing autonomous replication in a cell or may contain sequences sufficient to allow integration into a host cell DNA. The vector can deliver a target polynucleotide to an organism, cell, or cellular component. In some cases, the vector is an expression vector. "Expression vector" as used herein refers to a vector, e.g., a plasmid, that can facilitate the replication as well as expression of a polynucleotide incorporated therein. Typically, the nucleic acid sequence to be expressed is operably linked to a cis-acting regulatory sequence, e.g., a promoter and / or enhancer sequence, and is subject to transcriptional regulatory control by the promoter and / or enhancer. In certain cases, a vector is used to deliver a nucleic acid molecule encoding an engineered receptor of the present disclosure to a subject.

[0221] In certain embodiments, any vector suitable for introducing an expression cassette or polynucleotide encoding an engineered receptor into a neuronal cell can be used. Illustrative examples of suitable vectors include plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some cases, the vector is a circular nucleic acid, e.g., for plasmids, BACs, PACs, YACs, cosmids, fosmids, and the like. In some cases, a circular nucleic acid molecule can be used to deliver a nucleic acid molecule encoding an engineered receptor to a subject. For example, a plasmid DNA molecule encoding an engineered receptor can be introduced into a cell of a subject, whereby the DNA sequence encoding the engineered receptor is transcribed into mRNA, and the mRNA "message" is translated into a protein product. A circular nucleic acid vector generally includes regulatory elements that regulate the expression of a target protein. For example, a circular nucleic acid vector can include any number of promoters, enhancers, terminators, splice signals, origins of replication, initiation signals, and the like.

[0222] In some cases, vector can contain replicon.Replicon can be any nucleic acid molecule capable of self-replication.In some cases, replicon is RNA replicon derived from virus.A variety of suitable viruses (e.g., RNA viruses) are available, including but not limited to alphavirus, picornavirus, flavivirus, coronavirus, pestivirus, rubivirus, calcivirus, and hepacivirus.

[0223] In some embodiments, the vector is a non-viral vector. "Non-viral vector" refers to any delivery vehicle that does not contain a viral capsid or envelope, such as lipid nanoparticles (anionic (negatively charged), neutral, or cationic (positively charged)), heavy metal nanoparticles, polymer-based particles, plasmid DNA, minicircle DNA, minivector DNA, ccDNA, synthetic RNA, exosomes, etc. Non-viral vectors can be delivered by any suitable method well understood in the art, including, for example, nanoparticle delivery, biolistics, electroporation, sonication, or microinjection. See, for example, Chen et al. Mol. Therapy, Methods and Clinical Development. 2016Jan; Vol 3, issue 1; and Hardy, CE et al. Genes (Basel). 2017 Feb; 8(2): 6.5.

[0224] In other embodiments, the vector is a viral vector. "Viral vector" refers to a delivery vehicle that includes a viral capsid or envelope that surrounds a polynucleotide that encodes an RNA or polypeptide of interest. In some cases, the viral vector is derived from a replication-defective virus. Non-limiting examples of viral vectors suitable for delivering the nucleic acid molecule of the present disclosure to a subject include those derived from adenovirus, retrovirus (e.g., lentivirus), adeno-associated virus (AAV), and herpes simplex 1 (HSV-1). Examples of suitable viral vectors include, but are not limited to, retrovirus vectors (e.g., lentivirus vectors), herpes virus-based vectors, and parvovirus-based vectors (e.g., adeno-associated virus (AAV)-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors).

[0225] The term "parvovirus" as used herein encompasses all parvoviruses, including self-replicating parvoviruses and dependoviruses. Self-parvoviruses include members of the Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus genera. Exemplary self-parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other self-parvoviruses are known to those skilled in the art. See, for example, Fields et al., 1996 Virology, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

[0226] The Dependovirus genus contains adeno-associated viruses (AAV), including, but not limited to, AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type rhlO, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

[0227] In a preferred embodiment, the vector is an AAV vector. In certain cases, the viral vector is an AAV-5, AAV-6 or AAV-9 vector.

[0228] The genomic organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) long. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences of the nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, 2 and 3) form the capsid and contribute to the tropism of the virus. The terminal 145 nt ITRs are self-complementary and organized such that energetically stable intramolecular duplexes that form T-shaped hairpins can form. These hairpin structures serve as origins for viral DNA replication and as primers for the cellular DNA polymerase complex. After wild-type (wt) AAV infection in mammalian cells, the Rep genes are expressed and function in the replication of the viral genome.

[0229] In some cases, the envelope protein "capsid" of the viral vector is naturally occurring, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In certain cases, the capsid is synthetically engineered (e.g., by directed evolution or rational design) to have certain unique features that do not exist in nature, such as altered tropism, increased transduction efficiency, or immune evasion. One example of a rationally designed capsid is the mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid protein. Non-limiting examples of viral vectors in which the VP3 capsid protein has been synthetically engineered and suitable for use with the compositions and methods provided herein include AAV1 (Y705+731F+T492V), AAV2 (Y444+500+730F+T491V), AAV3 (Y705+731F), AAV5 (Y436+693+719F), AAV6 (Y705+731F+T492V), AAV8 (Y733F), AAV9 (Y731F), and AAV10 (Y733F). Non-limiting examples of viral vectors in which the VP3 capsid protein has been synthetically engineered and suitable for use with the compositions and methods provided herein include AAV-7m8 and AAV-ShH10. In some embodiments, the viral vector comprises an AAV capsid protein that includes an amino acid mutation at one or more positions corresponding to T492, Y705, Y731, or any combination thereof, of the AAV6 capsid protein, wherein the AAV capsid protein is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or another AAV serotype. In some embodiments, the one or more positions are two or more positions, two positions, or three positions.In some embodiments, the viral vector comprises an AAV capsid protein that includes one or more amino acid substitutions corresponding to T492V, Y705F, or Y731F, or any combination thereof, in the AAV6 capsid protein, where the AAV capsid protein is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or another AAV serotype. In some embodiments, the one or more substitutions are two or more substitutions, two substitutions, or three substitutions.

[0230] In some embodiments, the viral vector comprises an AAV6 capsid protein comprising a mutation at one or more amino acid positions selected from T492, Y705 and Y731. In some embodiments, the viral vector comprises an AAV6 capsid protein comprising one or more mutations selected from T492V, Y705F and Y731F. In some embodiments, the viral vector comprises an AAV6 capsid protein comprising the amino acid mutations T492V+Y705F+Y731F. In some embodiments, the AAV6 capsid protein comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:93.

[0231] In some embodiments, the viral vector comprises an AAV9 capsid protein comprising a mutation at one or more amino acid positions selected from T492, Y705 and Y731. In some embodiments, the viral vector comprises an AAV9 capsid protein comprising one or more mutations selected from T492V, Y705F and Y731F. In some embodiments, the viral vector comprises an AAV9 capsid protein comprising the amino acid mutations T492V+Y705F+Y731F. In some embodiments, the AAV9 capsid protein comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:94.

[0232] In some embodiments, the viral vector comprises an AAV5 capsid protein comprising a mutation at one or more amino acid positions selected from Y693 and Y719. In some embodiments, the viral vector comprises an AAV5 capsid protein comprising one or more mutations selected from Y693F and Y719F. In some embodiments, the viral vector comprises an AAV5 capsid protein comprising the amino acid mutations Y693F+Y719F. In some embodiments, the AAV5 capsid protein comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:92.

[0233] In some embodiments, a viral vector comprising an AAV capsid protein (or a mutant of the present disclosure) contributes to targeted expression of an engineered receptor to a subpopulation of cells or neurons of a subject. In some embodiments, the neurons are nociceptors.

[0234] A "recombinant parvovirus or AAV vector" (or "rAAV vector") herein refers to a vector that comprises one or more polynucleotides contemplated herein flanked by one or more AAV ITRs. Such polynucleotides are said to be "heterologous" to the ITRs because such a combination does not naturally occur in nature. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger nucleic acid construct (e.g., in a chromosome or into another vector, e.g., a plasmid or baculovirus, used for cloning or transfection), the rAAV vector is typically referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

[0235] In certain embodiments, any AAV ITRs may be used in the AAV vector, including ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. In a preferred embodiment, the AAV vectors contemplated herein contain one or more AAV2 ITRs.

[0236] The rAAV vector that contains two ITRs has a payload capacity of about 4.4 kB. The self-complementary rAAV vector contains a third ITR and packages two strands of the recombinant part of the vector, leaving only about 2.1 kB for the polynucleotide contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0237] Expanded packaging capacity, roughly double that of rAAV (about 9 kB), has been achieved using a dual rAAV vector strategy. Dual vector strategies useful for producing rAAV contemplated herein include, but are not limited to, splicing (trans-splicing), homologous recombination (overlap), or a combination of the two (hybrid). In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' end of the 5' half of the vector, and a splice acceptor (SA) signal is placed at the 5' end of the 3' half of the vector. Upon co-infection of the same cell with dual AAV vectors and two halves of inverted terminal repeat (ITR)-mediated head-to-tail concatenation, trans-splicing leads to the production of mature mRNA and full-size protein (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the two halves of a large transgene expression cassette contained in a dual AAV vector may contain homologous overlapping sequences (the 3' end of the 5' half of the vector and the 5' end of the 3' half of the vector overlapping dual AAV), which will mediate the reconstitution of a single large genome by homologous recombination (Duanet al., 2001). This strategy relies on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region derived from an exogenous gene (i.e., alkaline phosphatase; Ghoshet al., 2008, Ghosh et al., 2011) to the trans-splicing vector. The added region is placed downstream of the SD signal in the 5' half of the vector and upstream of the SA signal in the 3' half of the vector to increase recombination between the dual AAVs.

[0238] "Hybrid AAV" or "hybrid rAAV" refers to a rAAV genome packaged in a capsid of a different AAV serotype (and preferably a different serotype derived from one or more AAV ITRs), which may alternatively be referred to as a pseudotyped rAAV. For example, a rAAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 genome may be encapsidated within an AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 capsid or a variant thereof, provided that the AAV capsid and genome (and preferably one or more AAV ITRs) are of different serotypes. In certain embodiments, pseudotyped rAAV particles can be referred to as being of the "x / y" type, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid; for example, a 2 / 5 rAAV particle has ITRs derived from AAV2 and a capsid derived from AAV6.

[0239] "Host cells" include cells that are transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present disclosure. Host cells may include virus-producing cells and cells infected with viral vectors. In certain embodiments, in vivo host cells are infected with viral vectors contemplated herein. In certain embodiments, the term "target cell" is used interchangeably with host cell and refers to an infected cell of a desired cell type.

[0240] High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. Nos. 5,658,776; 6,566,118; 6,989,264; and 6,995,006; US2006 / 0188484; WO98 / 22607; WO2005 / 072364; and WO / 1999 / 011764; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003; Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; lnoue et al., (1998) J. Virol. 72, 7024. Methods for producing pseudotyped AAV vectors (e.g., WO00 / 28004) have also been reported, as well as various modifications or formulations of AAV vectors to reduce their immunogenicity upon administration in vivo (see, e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562). Pharmaceutical Compositions

[0241] Pharmaceutical preparations are also provided, including pharmaceutical preparations of vectors and pharmaceutical preparations of binding agents. The pharmaceutical preparations include the polynucleotide (RNA or DNA) of interest encoding the engineered receptor, the vector carrying the polynucleotide (RNA or DNA) encoding the engineered receptor of interest, or the binding agent present in a pharmaceutically acceptable vehicle. A "pharmaceutically acceptable vehicle" may be a vehicle approved by a federal or state regulatory agency for use in mammals, such as humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compounds of the present disclosure are formulated for administration to a mammal. Such pharmaceutical vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical vehicle may be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants and coloring agents can be used.When administered to mammals, the compounds and compositions of the present disclosure and pharma-ceutically acceptable vehicles, excipients or diluents can be sterile.In some cases, when the compounds of the present disclosure are administered intravenously, aqueous media such as water, saline, and aqueous dextrose and glycerol solutions are used as vehicles.

[0242] The pharmaceutical composition may take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained release formulations thereof, or any other form suitable for administration to mammals.In some cases, the pharmaceutical composition is formulated for administration by routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans.Examples of suitable pharmaceutical vehicles and methods for their formulation are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, which are incorporated herein by reference.

[0243] The choice of excipient will be determined in part by the particular vector, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present disclosure.

[0244] For example, the vectors can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, together with conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives, as necessary.

[0245] As another example, the vector can be formulated into a preparation suitable for oral administration, including: (a) a liquid solution, such as an effective amount of the compound dissolved in a diluent, such as water or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as a solid or granules; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings, and pharmacologically compatible excipients. Lozenge forms may include flavored tablets containing the active ingredient in a flavored, usually sucrose and acacia or tragacanth, and an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like, containing, in addition to the active ingredient, such excipients as described herein.

[0246] As another example, the subject formulations of the present disclosure can be made into aerosol formulations that are administered via inhalation.These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. They can also be formulated as pharmaceuticals for non-pressurized preparations, such as for use in nebulizers or atomizers.

[0247] In some embodiments, the preparation suitable for parenteral administration includes aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The preparations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, which only requires the addition of a sterile liquid excipient, such as water, for injection immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type previously described.

[0248] Preparations suitable for topical administration may be provided as creams, gels, pastes, or foams, containing such carriers in addition to the active ingredient, if appropriate. In some embodiments, topical formulations contain one or more components selected from structuring agents, thickening or gelling agents, and emollients or lubricants. Frequently used structuring agents include long-chain alcohols, such as stearyl alcohol, and glyceryl ethers or esters, and their oligo(ethylene oxide) ethers or esters. Thickening and gelling agents include, for example, polymers of acrylic or methacrylic acid and their esters, polyacrylamides, and naturally occurring thickeners, such as agar, carrageenan, gelatin, and guar gum. Examples of emollients include triglyceride esters, fatty acid esters and amides, waxes, such as beeswax, spermaceti, or carnauba wax, phospholipids, such as lecithin, and sterols and their fatty acid esters. Topical formulations may further include other ingredients such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (ie, sunscreens), and the like.

[0249] The compounds of the present disclosure can be formulated for topical administration. Topical application vehicles can be in various forms, such as lotions, creams, gels, ointments, sticks, sprays, or pastes. They can contain various types of carriers, including but not limited to solutions, aerosols, emulsions, gels, and liposomes. Carriers can be formulated, for example, as emulsions, with oil-in-water or water-in-oil bases. Suitable hydrophobic (oily) components used in emulsions include, for example, vegetable oils, animal fats and oils, synthetic hydrocarbons, and their esters, including polyesters, and alcohols, as well as organopolysiloxane oils. Such emulsions also include emulsifiers and / or surfactants, such as nonionic surfactants, to disperse or suspend the discontinuous phase within the continuous phase.

[0250] Suppository formulations are also provided by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, or foams.

[0251] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., a teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor in a composition as a solution in sterile water, saline, or another pharma- ceutically acceptable carrier.

[0252] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of the compound of the present disclosure calculated in an amount sufficient to produce a desired effect, together with a pharma- ceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0253] Dosage levels may vary as a function of the particular compound, the nature of the delivery vehicle, etc. The desired dosage for a given compound is readily determined by a variety of means.

[0254] In the context of this disclosure, the dose administered to an animal, particularly a human, must be sufficient to produce a preventive or therapeutic response in the animal over a reasonable time frame, for example, as described in more detail below. The amount administered depends on a variety of factors, including the strength of the particular compound used, the condition of the animal, and the weight of the animal, as well as the severity and stage of the disease. The size of the dose is also determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular compound.

[0255] In pharmaceutical dosage forms, the ASC inducer compounds can be administered in the form of a free base, in the form of their pharma- ceutically acceptable salts, or they can be used alone or in appropriate association and combination with other pharma- ceutically active compounds. Clinical Applications and Methods of Treatment

[0256] The compositions and methods disclosed herein can be used to treat neurological disease or disorder.In some aspects of the present disclosure, a method for treating neurological disease or disorder in a subject is provided, comprising introducing engineered receptor into neuronal cells, and providing an effective amount of ligand that activates engineered receptor to control the activity of cells, thereby relieving pain in the subject.In some aspects, the vector or composition disclosed herein is used in the manufacture of medicine for treating neurological disease or disorder.

[0257] In some cases, the disclosed methods and compositions are used to treat epilepsy. The compositions described herein can be used to prevent or control epileptic seizures. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonus, absence or atonic seizures. In some cases, the disclosed compositions and methods can prevent or reduce the number of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% (including all ranges and subranges therebetween).

[0258] In some cases, the disclosed methods and compositions are used to treat eating disorders. The eating disorder may be a mental disorder defined by abnormal eating behavior that negatively impacts the subject's physical or mental health. In some cases, the eating disorder is anorexia nervosa. In other cases, the eating disorder is bulimia nervosa. In some cases, the eating disorder is pica, rumination disorder, avoidant / restrictive food intake disorder, binge eating disorder (BED), other specific eating and eating disorders (OSFED), obsessive-compulsive eating disorder, diabetic bulimia, orthorexia nervosa, selective eating disorder, drunkorexia, anorexia during pregnancy, or gourmet syndrome. In some cases, the composition comprises a G protein-coupled receptor that increases or decreases the production of one or more molecules associated with an eating disorder. In other cases, the composition comprises a ligand-gated ion channel that alters the production of one or more molecules associated with an eating disorder. The one or more molecules associated with eating disorders may include, but are not limited to, molecules of the hypothalamic-pituitary-adrenal (HPA) axis, including vasopressin, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, epinephrine, or norepinephrine; and serotonin, dopamine, neuropeptide Y, leptin, or ghrelin.

[0259] In some cases, the compositions and methods are used to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumors), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that can be treated by the compositions and methods herein include apraxia, agraphia, alcoholism, dyslexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, ataxia telangiectasia, attention deficit hyperactivity disorder, auditory processing disorder, autism spectrum disorder, bipolar disorder, Bell's palsy, brachial plexus injury, brain tumors, and neurodegenerative disorders. Lesion, brain injury, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, burning pain, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, craniopathy, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, coffin syndrome Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital facial paraplegia, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cyclothymic disorder, cytomegalic inclusion body disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, developmental Sexual coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, speech disorder, autonomic neuropathy, dyscalculia, dysgraphia, dyskinesia, reading disorder, dystonia, empty sella syndrome, encephalitis, cerebral herniation, trigeminal nerve region hemangiomatosis, fecal incontinence, enuresis, epilepsy, intellectual disability in women, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry disease, Fahr's syndrome, syncope, familial convulsive paralysis, febrile seizures,Fisher syndrome, Friedreich's ataxia, fibromyalgia, Foville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy febrile seizures plus, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, heterotopic grey matter, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasms, hereditary spastic paraplegia, hereditary polyneuropathic ataxia, herpes zoster oticus, herpes zoster, Hirayama syndrome group, Hirschsprung's disease, Holmes-Addie syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hyperadrenocorticism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile Refsum's disease, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, isodicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kinsbone syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral medulla (Warren's syndrome) Berg syndrome, learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, cerebral leukodystrophy, leukoencephalopathy with disappearance of white matter, dementia with Lewy bodies, lissencephaly, locked-in syndrome, intervertebral disc disease, lumbar spinal stenosis, Lyme disease - neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, macropsia, mal de débarquement, macrocephalic leukoencephalopathy with subcortical cysts, megalencephaly, Merkelson-Rosenthal syndrome, Meniere's disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, micropsia, migraine, Miller-Fitch syndrome Sher syndrome, Ministroke (Transient Ischemic Attack), Sound Aversion Syndrome, Mitochondrial Myopathy, Moebius syndrome, Monolimbic muscular atrophy, Motor Skills Disorder, Moyamoya Disease, Mucopolysaccharidosis, Multiinfarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Muscular Dystrophy, Myalgic Encephalomyelitis, Myasthenia Gravis, Myelinolytic Diffuse Sclerosis, Infantile Myoclonic Encephalopathy, Myoclonus, Myopathy, Myotubular Myopathy, Myotonia Congenita, Narcolepsy, Neuro-Behçet's Disease, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Signs of AIDS,Neurological sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neuropathy, Nyman-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, latent spinal malunion, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, recurrent vision, paresthesia, Parkinson's disease, congenital paramyotonia, paraneoplastic disorders, seizures, Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, pervasive developmental disorder, photic sneeze reflex, phytanic acid storage disease, Pick's disease, compressed nerve, pituitary tumor, P MG, polyneuropathy, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, post-herpetic neuralgia (PHN), orthostatic hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemifacial atrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrant hemianopsia, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen's encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg's syndrome, chorea (Saint Vitus dance), Sandhoff disease, Schilder's disease, schizencephaly, sensory processing disorder, septo-optic dysplasia, shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal tumor, spinal muscular atrophy, spinal-bulbar muscular atrophy, spinocerebellar ataxia, split brain, Steele-Richardson-Oller syndrome Zewski syndrome, stiff-person syndrome, stroke, Sturge-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial hemosiderosis, Sydenham's chorea, fainting, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, tardive dysphrenia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered spinal cord syndrome, Thomsen's disease,Thoracic outlet syndrome, painful tics, Todd's paralysis, Tourette's syndrome, toxic encephalopathy, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, von Hippel-Lindau disease (VHL), Biliusk encephalomyelitis (VE), Wallenberg syndrome, West syndrome, cervical spasticity, William's syndrome, Wilson's disease, or Zellweger syndrome.

[0260] In some cases, the compositions and methods disclosed herein can be used to treat brain cancer or brain tumors.Non-limiting examples of brain cancer or tumors that may be suitable for treatment with the vectors and compositions described herein include gliomas, such as anaplastic astrocytoma (grade III glioma), astrocytoma (grade II glioma), brain stem glioma, ependymoma, ganglioglioma, ganglioneuroma, glioblastoma (grade IV glioma), glioma, juvenile pilocytic astrocytoma (JPA), low grade astrocytoma (LGA), medullablastoma, mixed glioma, oligodendroglioma, optic nerve glioma, pilocytic astrocytoma (grade I glioma), and primitive neuroectodermal tumor (PNE) tumor. T); skull base tumors, e.g., acoustic neuroma (vestibular schwannoma), acromegaly, adenoma, chondrosarcoma, chordoma, craniopharyngioma, epidermoid, glomus jugular tumor, infratentorial meningioma, meningioma, pituitary adenoma, pituitary tumor, Rathke's cyst; metastatic cancers, e.g., brain metastases, metastatic brain tumors; other brain tumors, e.g., brain cyst, choroid plexus papilloma, CNS lymphoma, colloid cyst, cystic mass, dermoid tumor, germinoma, lymphoma, nasal carcinoma, nasopharyngeal tumor, pineal tumor, pineoblastoma, pineocytoma, supratentorial meningioma, and vascular tumor; spinal tumors, e.g., astrocytoma, ependymoma, meningioma, and schwannoma.

[0261] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating pain in a subject. "Pain" refers to discomfort and / or strange sensations in the subject's body. Pain sensations can range from mild and occasional to severe and constant. Pain can be classified as acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain, or psychogenic pain. In some cases, pain is caused or associated with disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, Parkinson's disease, pain associated with epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and pain associated with central nervous system trauma, strain / sprain, and burn; nociceptive pain, including myocardial infarction, acute pancreatitis, post-operative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0262] The compositions and methods herein can be used to improve the level of pain in a subject.In some cases, the level of pain in a subject is improved by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or about 100% (including all ranges and subranges therebetween).The level of pain in a subject can be evaluated by various methods.In some cases, the level of pain is evaluated by self-report (i.e., the human subject expresses a verbal report of the level of pain he / she is experiencing).In some cases, the level of pain is evaluated by behavioral indicators of pain, such as facial expressions, limb movements, vocalization, anxiety and guarding. These types of assessments can be useful, for example, when a subject is unable to self-report (e.g., young children, unconscious subjects, non-human subjects). Pain levels can be assessed after treatment with a composition of the present disclosure, compared to the level of pain the subject experienced prior to treatment with the composition.

[0263] In various embodiments, a method for controlling, managing, preventing, or treating pain in a subject comprises administering to the subject an effective amount of an engineered receptor contemplated herein. Without wishing to be bound by any particular theory, the present disclosure contemplates using the vectors disclosed herein to modulate neuronal activity to reduce pain in a subject.

[0264] In various embodiments, a vector encoding an engineered receptor that activates or depolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells, e.g., inhibitory interneurons, that are to reduce pain sensation. In the presence of a ligand, the neuronal cells expressing the engineered receptor are activated, reducing their sensitivity to pain and enhancing the analgesic effect of stimulating these neuronal cells.

[0265] In various embodiments, a vector encoding an engineered receptor that inactivates or hyperpolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells that are to have increased sensitivity to nociception or pain, such as nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc. In the presence of the ligand, the neuronal cells expressing the engineered receptor are inactivated, decreasing sensitivity to pain and enhancing the analgesic effect.

[0266] Targeted expression of engineered receptors to subpopulations of nociceptors can be achieved by one or more of: choice of vector (e.g., AAV1, AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10(Y733F), and AAV-ShH10); choice of promoter; and delivery means.

[0267] In certain embodiments, the compositions and methods contemplated herein are effective in reducing pain.Illustrative examples of pain suitable for treatment by the vectors, compositions and methods contemplated herein include, but are not limited to, acute pain, chronic pain, neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-associated neuropathy, nerve injury, rheumatoid arthritis pain, osteoarthritis pain, burns, back pain, eye pain, visceral pain, cancer pain (e.g., bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, post-herpetic neuralgia, post-operative pain, post-stroke pain, and menstrual pain.

[0268] Pain can be classified as acute or chronic. "Acute pain" refers to pain that begins suddenly and is usually sharp in quality. Acute pain can be mild and last for a moment, or severe and last for weeks or months. In many cases, acute pain does not last longer than three months, and it disappears when the underlying cause of the pain is treated or cured. However, unrelieved acute pain can result in chronic pain. "Chronic pain" refers to ongoing or recurrent pain that lasts beyond the normal course of an acute illness or injury, or that lasts longer than three to six months and adversely affects the well-being of an individual. In certain embodiments, the term "chronic pain" refers to pain that continues when it should not. Chronic pain can be nociceptive pain or neuropathic pain.

[0269] In some embodiments, the pain is predicted or expected to occur in association with or as a result of an injury, infection, or medical intervention. In some embodiments, the infection causes nerve damage. In some embodiments, the medical intervention is surgery, such as surgery to the central core of the body. In some embodiments, the medical intervention is surgery to remove part or all of one or more tissues, tumors, or organs of the body. In some embodiments, the medical intervention is an amputation. In certain embodiments, the compositions and methods contemplated herein are effective in reducing acute pain. In certain embodiments, the compositions and methods contemplated herein are effective in reducing chronic pain.

[0270] Clinical pain exists when discomfort and abnormal sensitivity are characteristic of the patient's symptoms. Individuals may be present with a variety of pain symptoms. Such symptoms include: 1) spontaneous pain, which may be dull, burning, or stabbing; 2) exaggerated pain response to noxious stimuli (hyperalgesia); and 3) pain caused by normally innocuous stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Patients suffering from various forms of acute and chronic pain may have similar symptoms, but the underlying mechanisms may be different and therefore may require different treatment strategies. Thus, pain can also be divided into several different subtypes according to different pathophysiology, such as nociceptive pain, inflammatory pain, and neuropathic pain.

[0271] In certain embodiments, the compositions and methods contemplated herein are effective in reducing nociceptive pain. In certain embodiments, the compositions and methods contemplated herein are effective in reducing inflammatory pain. In certain embodiments, the compositions and methods contemplated herein are effective in reducing neuropathic pain.

[0272] Nociceptive pain is induced by tissue injury or by strong stimuli that may cause injury. Moderate to severe acute nociceptive pain is the main feature of pain from central nervous system trauma, strain / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumor-related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer treatment (e.g. post-chemotherapy syndrome, chronic postoperative pain syndrome or post-radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiation therapy. Back pain may be due to herniated or ruptured intervertebral disc or abnormality of lumbar facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament. Back pain may resolve naturally, but in some patients where it lasts for more than 12 weeks, it can become a chronic condition and be particularly debilitating.

[0273] Neuropathic pain can be defined as the pain that is initiated or caused by primary lesion or dysfunction in the nervous system.The etiology of neuropathic pain includes, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, and the pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0274] Neuropathic pain can be associated with pain disorders, a term that refers to diseases, disorders or conditions associated with or caused by pain. Examples of pain disorders include arthritis, allodynia, typical trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia, causalgia, sciatica disorders, osteoarthritis, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis or pain associated with cancer.

[0275] The inflammatory process is a complex series of biochemical and cellular events that are activated in response to tissue injury or the presence of foreign substances, resulting in swelling and pain. Joint pain is a common form of inflammatory pain.

[0276] Other types of pain amenable to treatment with the vectors, compositions, and methods contemplated herein include, but are not limited to, pain resulting from musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-arthritis, dystrophinopathy, glycogenolysis, polymyositis, and pyomyositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredema, and skeletal muscle ischemia; headaches, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache, mixed headache, and headaches associated with vascular disorders; and orofacial pain, including toothache, earache, burning mouth syndrome, and temporomandibular myofascial pain.

[0277] The effective amount of the compositions and methods contemplated herein for reducing the amount of pain experienced by human subjects can be determined using various pain scales.Patient self-report can be used to evaluate whether pain is reduced; see, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231.Alternatively, observational pain scales can be used.LANSS pain scale can be used to evaluate whether pain is reduced; see, for example, Bennett (2001) Pain 92:147.Visual analog pain scale can be used; see, for example, Schmader (2002) Clin. J. Pain 18:350.Lycr pain scale can be used; for example, 0 is no pain, 5 is moderate pain, and 10 is the worst possible pain.Self-report pain scales for children include, for example, facial pain scale; Wong-Baker facial pain rating scale; and color analog scale. Self-report pain scales for adults include, for example, visual analog scales; verbal numerical rating scales; verbal language scales; and the Brief Pain Questionnaire.Examples of pain measurement scales include the Alder Hey Triage pain score (Stewart et al. (2004) Arch. Dis. Child. 89:625); behavioral pain scales (Payen et al. (2001) Critical Care Medicine 29:2258); the Brief Pain Questionnaire (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23: 129); the Non-Verbal Pain Indicator Checklist (Feldt (2000) Pain Manag. Nurs. 1: 13); the Critical Care Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); the COMFORT scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); the Dallas Pain Questionnaire (Ozguler et al. (2002) Spine 27:1783); the Dolorimeter Pain Index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); the Facial Pain Scale-Revised (Hicks et al. (2001) Pain 93:173); the FLACC (Face Legs Activity Cry Consolability) scale; the McGill Pain Questionnaire (Melzack (1975) Pain 1 :277); the descriptive differential scale (Gracely and Kwilosz (1988) Pain 35:279); the numeric 11-point box (Jensen et al. (1989) Clin. J. Pain 5: 153); the numeric rating scale (Hartrick et al. (2003) Pain Pract. 3:310); the Wong-Baker FACES pain rating scale; and the visual analog scale (Huskisson (1982) J. Rheumatol. 9:768).

[0278] In certain embodiments, a method of alleviating pain in a subject is provided, comprising introducing an engineered receptor into a neuronal cell and controlling the activity of the cell by providing an effective amount of a ligand that activates the engineered receptor, thereby alleviating pain in the subject.The method provides significant analgesia for pain without off-target effects such as general central nervous system depression.In certain embodiments, the method provides a reduction in neuropathic pain in a subject of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including all ranges and subranges therebetween) compared to untreated subjects. In some embodiments, the method includes measuring pain in the subject before and after administration of the binding agent, and the pain in the subject is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including all ranges and subranges therebetween). In such examples, the measurement may be performed 4 hours or more after administration of the binding agent, for example, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, or 4 days or more after administration of the binding agent.

[0279] In certain embodiments, the vector contemplated herein is administered or introduced into one or more neuronal cells. The neuronal cells may be the same type of neuronal cells or a mixed population of different types of neuronal cells. In one embodiment, the neuronal cells are nociceptive neurons or peripheral sensory neurons. Examples of sensory neurons include, but are not limited to, dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In one embodiment, the neuronal cells are inhibitory interneurons involved in neuronal pain circuits.

[0280] In some cases, the vector encoding the engineered receptor is administered to a subject in need thereof. Non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cisternal administration, and intraneuronal administration. In some embodiments, administration may include injection of a liquid formulation of the vector and / or nucleic acid. In some cases, administration may include injection of a liquid formulation of the vector. In other cases, administration may include oral delivery of a solid formulation of the vector. In some cases, the oral formulation may be administered with food. In certain embodiments, the vector is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneuronally, intraganglionally, intraspinally, or intracerebroventricularly to a subject to introduce the vector into one or more neuronal cells. In various embodiments, the vector is rAAV.

[0281] In one embodiment, AAV is administered to sensory neurons or nociceptors, such as DRG neurons, TGG neurons, etc., by intrathecal (IT) or intraganglionic (IG) administration. The IT route delivers AAV to cerebrospinal fluid (CSF). This administration route may be suitable for treating, for example, chronic pain or other peripheral nervous system (PNS) or central nervous system (CNS) indications. In animals, IT administration has been achieved by inserting an IT catheter via the cisterna magna and advancing it caudally to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a routine clinical procedure that shows an excellent safety profile.

[0282] In certain cases, the vector can be administered to a subject by intraganglionic administration. Intraganglionic administration may include direct injection into one or more ganglia. The IG route can deliver AAV directly to the DRG or TGG parenchyma. In animals, IG administration to the DRG is performed by open neurosurgery, which is undesirable in humans because it requires complex and invasive procedures. In humans, minimally invasive, CT imaging-guided techniques can be used to safely target the DRG. AAV can be delivered into the DRG parenchyma using a custom-made needle assembly for convection-enhanced drug delivery (CED). In a non-limiting example, the vector of the present disclosure can be delivered to one or more dorsal root ganglia and / or trigeminal ganglia for the treatment of chronic pain. In another non-limiting example, the vector of the present disclosure can be delivered to the nodose ganglion (vagus nerve) for the treatment of epilepsy.

[0283] In yet another particular case, the vector can be administered to the subject by intracranial administration (i.e., directly to the brain). In a non-limiting example of intracranial administration, the vector of the present disclosure can be delivered to the cortex of the brain, for example, to treat epilepsy seizure focus, to the paraventricular hypothalamus, for example, to treat satiety disorder, or to the central amygdala, for example, to treat satiety disorder. In yet another particular case, the vector can be administered to the subject by intraneural injection (i.e., directly to the nerve). The nerve can be selected based on the indication to be treated, for example, injection into the sciatic nerve to treat chronic pain, or injection into the vagus nerve to treat epilepsy or satiety disorder. In yet another particular case, the vector can be administered to the subject by subcutaneous injection into sensory nerve terminals, for example, to treat chronic pain.

[0284] A vector dose can be expressed as the number of vector genome units delivered to a subject. "Vector genome units" as used herein refers to the number of individual vector genomes administered in a dose. The size of an individual vector genome will generally depend on the type of viral vector used. The vector genomes of the present disclosure can be from about 1.0 kilobase, 1.5 kilobase, 2.0 kilobase, 2.5 kilobase, 3.0 kilobase, 3.5 kilobase, 4.0 kilobase, 4.5 kilobase, 5.0 kilobase, 5.5 kilobase, 6.0 kilobase, 6.5 kilobase, 7.0 kilobase, 7.5 kilobase, 8.0 kilobase, 8.5 kilobase, 9.0 kilobase, 9.5 kilobase, 10.0 kilobase, to more than 10.0 kilobase. Thus, a single vector genome can contain up to 10,000 or more base pairs of nucleotides. In some cases, a vector dose can be about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×109 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×10 13 、8×10 13 、9×10 13 、1×10 14 、2×10 14 、3×10 14 、4×10 14 、5×10 14 、6×10 14 、7×10 14 、8×10 14 、9×10 14 、1×10 15 、2×10 15 、3×10 15 、4×10 15 、5×10 15 、6×10 15 、7×10 15, 8×10 15 , 9×10 15 , 1×10 16 , 2×10 16 , 3×10 16 , 4×10 16 , 5×10 16 , 6×10 16 , 7×10 16 , 8×10 16 , 9×10 16 , 1×10 17 , 2×10 17 , 3×10 17 , 4×10 17 , 5×10 17 , 6×10 17 , 7×10 17 , 8×10 17 , 9×10 17 , 1×10 18 , 2×10 18 , 3×10 18 , 4×10 18 , 5×10 18 , 6×10 18 , 7×10 18 , 8×10 18 , 9×10 18 , 1×10 19 , 2×10 19 , 3×10 19 , 4×10 19 , 5×10 19 , 6×10 19 , 7×10 19 , 8×10 19 , 9×10 19 , 1×10 20 , 2×10 20 , 3×10 20 , 4×10 20 , 5×10 20 , 6×10 20 , 7×10 20 , 8×10 20 , 9×10 20 or may be on a higher vector genome basis.

[0285] In certain embodiments, the vectors contemplated herein are at least about 1×10 9 Genomic particles / mL, at least approximately 1 x 10 10Genomic particles / mL, at least approximately 5 x 10 10 Genomic particles / mL, at least approximately 1 x 10 11 Genomic particles / mL, at least approximately 5 x 10 11 Genomic particles / mL, at least approximately 1 x 10 12 Genomic particles / mL, at least approximately 5 x 10 12 Genomic particles / mL, at least approximately 6 x 10 12 Genomic particles / mL, at least approximately 7 x 10 12 Genomic particles / mL, at least approximately 8 x 10 12 Genomic particles / mL, at least approximately 9 x 10 12 Genomic particles / mL, at least approximately 10 x 10 12 Genomic particles / mL, at least approximately 15 x 10 12 Genomic particles / mL, at least approximately 20 x 10 12 Genomic particles / mL, at least approximately 25 x 10 12 Genomic particles / mL, at least approximately 50 x 10 12 Genomic particles / mL, or at least about 100 x 10 12 The titer of genome particles / mL is administered to subjects.When used in reference to virus titer, the term "genome particles (gp)" or "genome equivalent" or "genome copies" (gc) refers to the number of virions that contain recombinant AAV DNA genome, regardless of infectivity or function.The number of genome particles in a particular vector preparation can be measured by methods well understood in the art, for example, by quantitative PCR of genome DNA, or as described, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039;Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0286] The vector of the present disclosure may be administered in a volume of fluid. In some cases, the vector may be administered in a volume of about 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, 6.0mL, 7.0mL, 8.0mL, 9.0mL, 10.0mL, 11.0mL, 12.0mL, 13.0mL, 14.0mL, 15.0mL, 16.0mL, 17.0mL, 18.0mL, 19.0mL, 20.0mL, or greater than 20.0mL. In some cases, the vector dose may be expressed as the concentration or titer of the vector administered to the subject. In this case, the vector dose may be expressed as the number of vector genome units per volume (i.e., genome units / volume).

[0287] In certain embodiments, the vectors contemplated herein are at least about 5×10 9 Infectious units / mL, at least approximately 6 × 10 9 Infectious units / mL, at least approximately 7 × 10 9 Infectious units / mL, at least approximately 8 × 10 9 Infectious units / mL, at least approximately 9 × 10 9 Infectious units / mL, at least approximately 1 × 10 10 Infectious units / mL, at least approximately 1.5 × 10 10 Infectious units / mL, at least approximately 2 × 10 10 Infectious units / mL, at least approximately 2.5 × 10 10 Infectious units / mL, at least approximately 5 × 10 10 Infectious units / mL, at least approximately 1 × 10 11 Infectious units / mL, at least approximately 2.5 × 10 11 Infectious units / mL, at least approximately 5 × 10 11 Infectious units / mL, at least approximately 1 × 10 12 Infectious units / mL, at least approximately 2.5 × 10 12 Infectious units / mL, at least approximately 5 × 10 12 Infectious units / mL, at least approximately 1 × 10 13 Infectious units / mL, at least approximately 5 × 10 13 Infectious units / mL, at least approximately 1 × 1014 It is administered to a subject in a titer of infectious units / mL. When used in reference to virus titer, the term "infectious unit (iu)", "infectious particle" or "replication unit" refers to the number of infectious and replication-competent recombinant AAV vector particles, as measured by infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0288] In certain embodiments, the vectors contemplated herein are at least about 5×10 10 Transducing units / mL, at least approximately 1 x 10 11 Transducing units / mL, at least approximately 2.5 x 10 11 Transducing units / mL, at least approximately 5 x 10 11 Transducing units / mL, at least approximately 1 x 10 12 Transducing units / mL, at least approximately 2.5 x 10 12 Transducing units / mL, at least approximately 5 x 10 12 Transducing units / mL, at least approximately 1 x 10 13 Transducing units / mL, at least approximately 5 x 10 13 Transducing units / mL, at least approximately 1 x 10 14 It is administered to subjects in a titer of transducing unit / mL.When used with reference to viral titer, the term "transducing unit (tu)" refers to the number of infectious recombinant AAV vector particles that produce functional transgene product when measured in functional assay such as those described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0289] Vector doses are generally determined by the route of administration. In a particular example, intraganglionic injections can be administered in a volume of about 0.1 mL to about 1.0 mL, with a dose of about 1×10 9 ~Approx. 1×10 13In another particular embodiment, the intrathecal injection may contain about 1×10 vector genome in a volume of about 1.0 mL to about 12.0 mL. 10 ~Approx. 1×10 15 In yet another particular embodiment, the intracranial injection may comprise about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In another particular embodiment, the intraneural injection may contain about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In another specific example, the intraspinal injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In yet another particular embodiment, the cisternal infusion may contain about 5×10 vector genomes in a volume of about 0.5 mL to about 5.0 mL. 9 ~Approx. 5×10 13 In yet another particular embodiment, the subcutaneous injection may contain about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 It may include a vector genome.

[0290] In some cases, the vector is delivered to the subject by infusion. The vector dose delivered to the subject by infusion can be measured as the vector infusion rate. Non-limiting examples of vector infusion rates include 1-10 μL / min for intraganglionic, intraspinal, intracranial or intraneural administration; and 10-1000 μL / min for intrathecal or cisternal administration. In some cases, the vector is delivered to the subject by MRI-guided convection-enhanced drug delivery (CED). This technique allows for increased viral diffusion and transduction distributed throughout a large volume of the brain and reduces vector reflux along the needle track.

[0291] In various embodiments, methods are provided that include administering to one or more neuronal cells that are to have increased nociception or sensitivity to pain a vector that encodes an engineered receptor that inactivates or hyperpolarizes the neuronal cells, and administering to a subject a ligand that specifically binds to neuronal cells expressing the engineered receptor, thereby inactivating the cells, decreasing sensitivity to pain, and enhancing the analgesic effect.

[0292] In various embodiments, methods are provided that include administering to one or more neuronal cells that are to be reduced in nociception or sensitivity to pain a vector that encodes an engineered receptor that activates or polarizes the neuronal cells, and administering to a subject a ligand that specifically binds to the neuronal cells expressing the engineered receptor, thereby activating the cells, reducing sensitivity to pain, and enhancing the analgesic effect.

[0293] The formulation of the ligand can be administered to the subject by various routes. Non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, transdermal administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, and intraneural administration. In some cases, administration may include injection of a liquid formulation of the ligand. In other cases, administration may include oral delivery of a solid formulation of the ligand. In certain cases, the ligand is administered by oral administration (e.g., pill, tablet, capsule, etc.). In some cases, the oral composition can be administered with food. In other certain cases, the ligand is administered by intrathecal injection (i.e., into the subarachnoid space of the spinal cord) for delivery to the cerebrospinal fluid (CSF) of the subject. In other certain cases, the ligand is administered locally (e.g., skin patch, cream, lotion, ointment, etc.).

[0294] The dosage of the ligand administered to a subject is not subject to absolute limitations, but depends on the nature of the composition and its active ingredients and its unwanted side effects (e.g., immune response to antibodies), the subject being treated, and the type of condition being treated and the mode of administration. Generally, the dosage is a therapeutically effective amount, e.g., an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce or attenuate the level of pain experienced by the subject. In certain embodiments, the dosage can also be a prophylactic or effective amount. The therapeutically effective amount of the ligand can depend on the route of administration, the indication being treated, and / or the ligand selected for use.

[0295] In one embodiment, the ligand is first administered to the subject before the administration of the vector. A therapeutically effective amount of the ligand may be administered to the subject at some time after the delivery of the vector. Generally, after the delivery of the vector, there is a period of time that is necessary for one or more cells of the subject to produce the protein (i.e., the engineered receptor) encoded by the vector. During this period, the administration of the ligand to the subject may not be beneficial to the subject. In this situation, it may be preferable to administer the ligand after a certain amount of the engineered receptor is produced by one or more cells of the subject.

[0296] In one embodiment, the ligand is first administered to the subject at about the same time that the vector is administered to the subject.

[0297] In one embodiment, the ligand is first administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 hours, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 years after administration of the vector to the subject. In some cases, the therapeutically effective amount of the ligand may be administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 days after delivery of the vector. In certain examples, the therapeutically effective amount of the ligand is administered to the subject at least one week after delivery of the vector. In further examples, the therapeutically effective amount of the ligand is administered to the subject daily for at least three consecutive days.

[0298] The therapeutically effective amount or dose of the ligand of the present disclosure can be expressed as mg or μg of ligand per kg of subject body weight. In some examples, the therapeutically effective amount of the ligand is about 0.001 μg / kg, about 0.005 μg / kg, about 0.01 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 50 μg / kg, about 60 μg / kg, about 70 μg / kg, about 80 μg / kg, about 90 μg / kg, about 100 μg / kg, about 150 μg / kg, about 200 μg / kg, about 300 μg / kg, about 400 μg / kg, about 50 ... g / kg, approximately 60μg / kg, approximately 70μg / kg, approximately 80μg / kg, approximately 90μg / kg, approximately 100μg / kg, approximately 120μg / kg, approximately 140μg / kg, approximately 160μg / kg, approximately 180μg / kg, approximately 200μg / k g, approximately 220μg / kg, approximately 240μg / kg, approximately 260μg / kg, approximately 280μg / kg, approximately 300μg / kg, approximately 320μg / kg, approximately 340μg / kg, approximately 360μg / kg, approximately 380μg / kg, approximately 400μg / kg, approximately 420μg / kg, approximately 440μg / kg, approximately 460μg / kg, approximately 480μg / kg, approximately 500μg / kg, approximately 520μg / kg, approximately 540μg / kg, approximately 560μg / kg, approximately 580μg / kg, approximately 600μ g / kg, approximately 620μg / kg, approximately 640μg / kg, approximately 660μg / kg, approximately 680μg / kg, approximately 700μg / kg, approximately 720μg / kg, approximately 740μg / kg, approximately 760μg / kg, approximately 780μg / kg, approximately 800 The dose may be about 820 μg / kg, about 840 μg / kg, about 860 μg / kg, about 880 μg / kg, about 900 μg / kg, about 920 μg / kg, about 940 μg / kg, about 960 μg / kg, about 980 μg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, or more than 10 mg / kg.

[0299] In certain embodiments, the dose of the ligand administered to the subject is at least about 0.001 micrograms per kg (μg / kg), at least about 0.005 μg / kg, at least about 0.01 μg / kg, at least about 0.05 μg / kg, at least about 0.1 μg / kg, at least about 0.5 μg / kg, 0.001 milligrams per kg (mg / kg), at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, or at least about 10 mg / kg or more.

[0300] In certain embodiments, the dose of ligand administered to the subject is at least about 0.001 μg / kg to at least about 10 mg / kg, at least about 0.01 μg / kg to at least about 10 mg / kg, at least about 0.1 μg / kg to at least about 10 mg / kg, at least about 1 μg / kg to at least about 10 mg / kg, at least about 0.01 mg / kg to at least about 10 mg / kg, at least about 0.1 mg / kg to at least about 10 mg / kg, or at least about 1 mg / kg to at least about 10 mg / kg, or any intervening ranges thereof.

[0301] In some embodiments, a therapeutically effective amount of a ligand can be expressed as a molar concentration (i.e., M or mol / L). In some cases, a therapeutically effective amount of a ligand can be about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 , 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or more.

[0302] The therapeutically effective amount of the ligand can be administered once or more than once daily. In some cases, the therapeutically effective amount of the ligand is administered as needed (e.g., when pain relief is required). The ligand may be administered continuously (e.g., every day, without a break in the duration of the treatment regimen). In some cases, the treatment regimen may be less than one week, one week, two weeks, three weeks, one month, or longer than one month. In some cases, the therapeutically effective amount of the ligand is administered for one day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or for more than at least ten consecutive days. In certain cases, the therapeutically effective amount of the ligand is administered for three consecutive days. In some cases, a therapeutically effective amount of the ligand can be administered once per week, twice per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per week, nine times per week, ten times per week, eleven times per week, twelve times per week, thirteen times per week, fourteen times per week, fifteen times per week, sixteen times per week, seventeen times per week, eighteen times per week, nineteen times per week, twenty times per week, twenty-five times per day, thirty-five times per day, thirty-five times per day, forty times per day, or more than forty times per day. In some cases, a therapeutically effective amount of the ligand can be administered once per day, twice per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, ten times per day, or more than ten times per day. In some cases, a therapeutically effective amount of the ligand is administered at least every hour, at least every 2 hours, at least every 3 hours, at least every 4 hours, at least every 5 hours, at least every 6 hours, at least every 7 hours, at least every 8 hours, at least every 9 hours, at least every 10 hours, at least every 11 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.Doses of the ligand can be administered to the subject continuously, or 1, 2, 3, 4, or 5 times a day; 1, 2, 3, 4, 5, 6, or 7 times a week, 1, 2, 3, or 4 times a month, once every 2, 3, 4, 5, or 6 months, or once a year, or even at longer intervals. The duration of treatment can last for 1 day, 1, 2, or 3 weeks, 1, 2, 3, 4, 5, 7, 8, 9, 10, or 11 months, 1, 2, 3, 4, 5 or more years, or longer.

[0303] The subject treated by the methods and compositions disclosed herein may be a human or a non-human animal. The term "treat" and grammatical equivalents used herein generally refer to the use of a composition or method to reduce, eliminate, or prevent the symptoms of a disease, including achieving therapeutic benefit and / or preventive benefit. Therapeutic benefit means slowing down, stopping, reversing, or eradicating or ameliorating the progression of the symptoms of the disorder or condition being treated. The preventive benefit of treatment includes reducing the risk of a condition, delaying the progression of a condition, or reducing the likelihood of the occurrence of a condition.

[0304] Non-limiting examples of non-human animals include non-human primates, livestock, household pets, and laboratory animals. For example, the non-human animal may be an ape (e.g., chimpanzee, baboon, gorilla, or orangutan), an Old World monkey (e.g., rhesus monkey), a New World monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a llama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal. The compositions and methods described herein are suitable for treating veterinary animals. Veterinary animals may include, but are not limited to, dogs, cats, horses, cows, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some aspects, contacting a tissue or cell population with the composition includes administering the composition to the cell population or subject. In some embodiments, the administration is performed in vitro, for example, by adding the composition to a cell culture system. In some embodiments, administration is performed in vivo, for example, by administration by a specific route. When more than one composition is administered, the compositions may be administered by the same route at the same time (e.g., on the same day), or by the same route at different times. Alternatively, the compositions may be administered by different routes at the same time (e.g., on the same day), or by different routes at different times.

[0305] The number of times that the composition is administered to the subject in need of it depends on the medical professional's discretion, the disorder, the severity of the disorder, and the subject's response to the formulation.In some embodiments, the composition is administered at least once.In further embodiments, the administration is performed more than once, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in a given period.The dosage and / or frequency of each administration can be adjusted as necessary based on the patient's condition and physiological response.

[0306] In some embodiments, the composition can be administered for a period of time sufficient to achieve a desired physiological effect or improvement in the subject's condition. If the subject's condition is not improved, at the physician's discretion, the composition can be administered chronically, i.e., for an extended period of time, e.g., for the subject's lifetime, to improve or otherwise control or limit the symptoms of the subject's disease or condition. If the subject's condition is improved, at the physician's discretion, the composition can be administered continuously, or the dose of the administered drug can be temporarily reduced or temporarily suppressed for a certain period of time (i.e., a "drug holiday"). The length of the drug holiday can vary from 2 days to 1 year, e.g., by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. Dose reductions during drug holidays can be from 10% to 100%, for example, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% (including all ranges and subranges therebetween).

[0307] When a composition is administered more than once, each administration may be performed by the same party and / or in the same geographic location, or each administration may be performed by a different party and / or in a different geographic location.

[0308] For human and veterinary treatment, the amount of a particular drug administered may depend on a variety of factors, including, for example, the disorder being treated and the severity of the disorder; the activity of the particular drug used; the age, weight, general health, sex and diet of the patient; the number of doses, the route of administration and the rate of excretion of the particular drug used; the duration of treatment; drugs used in combination with or simultaneously with the particular drug used; the judgment of the prescribing physician or veterinarian; and similar factors known in the medical and veterinary fields.Similarly, the effective concentration of a given composition may depend on a variety of factors, including the age, sex, weight, genetic condition and general health of the patient or subject.

[0309] All articles, publications, and patents cited herein are incorporated by reference as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in conjunction with which the publications are cited. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be, taken as an admission or any form of suggestion that they constitute legitimate prior art or form part of the common general knowledge in any country in the world.

[0310] Unless the context indicates otherwise, it is specifically contemplated that the various features described herein can be used in any combination.

[0311] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0312] It should be understood that the foregoing description and the following examples are intended to illustrate, rather than limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. EXAMPLES

[0313] Example 1 Construction of engineered receptors containing ion pore domains derived from human GlyRα1, GlyRα2, GlyRα3 or GABArho receptors To generate LGICs that conduct anion currents following exposure to non-natural small molecule agonists of the human α7-nAChR, chimeric ligand-gated ion channel (LGIC) receptors were constructed containing a ligand binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from human GlyRα1, GlyRα2, Gly or GABArho receptors according to Table 12.

[0314] Example 2 Characterization of engineered receptors using high-throughput fluorescence-based plate screening The engineered LGIC receptors of Example 1 were analyzed for their potency against their natural ligand acetylcholine (Ach), as well as non-natural ligands such as AZD-0328 (adisinsight.springer.com / drugs / 800018503), TC-6987 (drugbank.ca / drugs / DB14854), ABT-126 (medchemexpress.com / Nelonicline.html), APN-1125 (clinicaltrials.gov / ct2 / show / NCT02724917), TC-5619 (en.wikipedia.org / wiki / Bradanicline), and fascinicline / RG3487 (researchgate.net / figure / Molecular-structure-of-RG3487_fig1_47499934).

[0315] The response profile of these engineered receptors to ligands was assessed using an anion reporter assay. In this assay, cells expressing a YFP reporter, whose fluorescence is quenched in the presence of anions, are transfected with DNA encoding the engineered receptor of interest. Upon exposure to ligand, the channel is activated with influx anions, resulting in a dose-dependent quenching of YFP that can be detected in a plate reader.

[0316] Lenti-X 293T cells (LX293T, Clontech) were maintained in DMEM containing 10% FBS and 1% penicillin / streptomycin (Invitrogen). For plate reader assays, LX293T cells were infected with lentivirus to generate cells stably expressing a mutant YFP (H148Q / I152L) reporter, which shows enhanced sensitivity to anions. Two days prior to the assay, cells were split at a density of 20,000 cells / well in poly-d-lysine-coated 96-well tissue culture plates (Thermo Scientific). The next day, cells were transiently transfected with 0.1 μg of DNA per well using the standard Fugene 6 protocol (Promega). On the day of the assay, cells were washed twice in 1x extracellular solution (1xECS: 140mM NaCl, 5mM KCl, 1mM MgCl2, 2mM CaCl2, 10mM HEPES, 10mM glucose, pH 7.2, 300mOsm). After the final wash, 100μL of 1xECS was added to the wells and the plate was incubated at 37°C for 30 minutes. While the plate was incubating, drugs were diluted to 2x concentration in 1xECS-NaI (same components as 1xECS except that 140mM NaCl was replaced by 140mM NaI). Plates were then read on a Flexstation3 (Molecular Devices). Each well of the plate, eight wells at a time, is read for 2 minutes using a Flexstation3 (Molecular Devices) as follows: 1) baseline YFP fluorescence is read for 17 seconds, 2) 100 μL of ligand is added, and 3) the change in YFP fluorescence is then measured every 1.3 seconds for the remainder of the time.

[0317] FIG. 1 provides a heat map of percent quenching of YFP fluorescence after stimulation with various doses of either acetylcholine or the non-natural ligands indicated for cells expressing CODA71 (containing the GlyRα1 ion pore domain) or CODA1237 (containing the GlyRα2 ion pore domain). Non-natural ligands tested include AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and fascinicline / RG3487. A positive fluorescent signal indicated by blue-shaded cells indicates that the engineered receptor is activated by that concentration of the non-natural ligand. The results show that both groups of cells show similar responsiveness to multiple ligands. Cells expressing CODA1237 are more responsive to ABT-126, RG3487, and AZD-0328 than cells expressing CODA71.

[0318] Figure 2 provides a heat map of percent quenching of YFP fluorescence following stimulation with various doses of either acetylcholine or TC-5619 for cells expressing chimeric LGIC receptors based on CODA1237 that contain various amino acid mutations in the ligand-binding domain derived from the α7-nAChR, as summarized in Table 12. For most of these mutants, the corresponding cell lines show a significant shift to the right in the dose required to elicit a response to acetylcholine (i.e., low responsiveness to acetylcholine), while showing minimal to no significant shift in response to TC-5619. CODA75 is a non-responsive chimeric receptor used here as a negative control.

[0319] Figure 3 provides a heat map of percent quenching of YFP fluorescence following stimulation with the indicated doses of acetylcholine in cells expressing the indicated chimeric LGIC receptors. We tested four chimeric LGIC receptors (CODA1342, CODA1343, CODA1344 and CODA1345, summarized in Table 12) that contain the ion pore domain of GlyRα3 and compared their responsiveness to acetylcholine to that of CODA71 and CODA64. CODA75 is a non-responsive chimeric receptor used here as a negative control. The results show that, using CODA71 as a reference point, cells expressing CODA1342 and CODA1345 have similar responsiveness to acetylcholine, cells expressing CODA1344 have a lower responsiveness to acetylcholine, and cells expressing CODA1343 have a higher responsiveness to acetylcholine.

[0320] Table 13 below provides EC50 measurements of different ligands for CODA1316 using either the fluorescent plate screening method or the manual patch clamp method (described in Example 4 below). CODA1316 was able to bind to TC-5619 with an EC50 in the low nanomolar concentrations. Table 13: EC50 of engineered receptors [Table 13]

[0321] Example 3 Localization of engineered receptors The effectiveness of the engineered receptors disclosed herein in localizing to the surface of cells was assessed using fluorescently labeled α-bungarotoxin, which specifically binds to the engineered receptor, followed by flow cytometry to assess surface localization of the engineered receptor.

[0322] Alpha-bungarotoxin conjugated to biotin, Alexa Fluor 647 were all purchased from Thermo / Fisher (Waltham MA). Briefly, HEK-293T or HEK293T cells transduced with Ric3, Nacho, and Gcamp6s were seeded at 200,000 cells per well and transfected the next day with Fugene 6 at a 1:3 ratio of DNA to Fugene. Ric3 and Nacho are chaperones for the human α7 nicotinic acetylcholine receptor, and GCamp6s is a calcium reporter. Cells were analyzed the day after transfection using flow cytometry. For flow cytometry analysis, transfected HEK293T cells were manually picked, washed, and incubated in α-bungarotoxin (1:1000) in FACS buffer (2% BSA, 1× PBS without Ca+ and Mg+, and 1× penicillin-streptomycin) for 1 hour. Cells were then washed in FACS buffer and then analyzed on a Sony SH800 FACS sorter (San Jose, CA). Subsequent analysis was performed using FlowJo (San Jose, CA).

[0323] Figure 4A shows the percentage of α-bungarotoxin staining positive HEK293T cells transiently transfected with CODA71, CODA829, CODA1237, CODA1238, CODA1298, CODA1239, CODA1342, CODA1343, CODA1344, or CODA1345. The design of the LGIC constructs is summarized in Table 12. Figure 4B shows the mean fluorescence intensity of α-bungarotoxin staining positive cells from Figure 4A.

[0324] Figure 4C shows the percentage of α-bungarotoxin staining positive cells transiently transfected with CODA71, CODA102, CODA829, CODA1237, CODA1238, CODA1298, or CODA1239 using HEK293T cells transduced with Ric3, Nacho, and GCAMP6s. The design of the LGIC constructs is summarized in Table 12. Figure 4D shows the mean fluorescence intensity of α-bungarotoxin staining positive cells from Figure 4C.

[0325] The results in Figures 4A-4D show that for cells transfected with chimeric LGICs containing GlyRα1 IPD, the T225I mutation can improve the surface expression level of LGICs in α-bungarotoxin staining-positive cells according to a comparison between CODA71 and CODA829. For cells transfected with chimeric LGICs containing GlyRα2 IPD, the T225I mutation improves the surface expression level of LGICs in α-bungarotoxin staining-positive cells according to a comparison between CODA1237 and CODA1298. Furthermore, chimeric LGIC receptors containing the ion pore domain of GlyRα3 isoform K (CODA1343 and CODA1345) have a higher percentage of surface-expressing cells and higher surface expression than the corresponding LGIC receptors containing the ion pore domain of GlyRα3 isoform L (CODA1342 and CODA1344).

[0326] Figure 5A shows the percentage of α-bungarotoxin staining positive HEK293T cells transiently transfected with CODA71, CODA409, CODA1237, CODA1297, CODA1298, CODA1299, CODA1300, CODA1301, CODA1302, CODA1303, CODA1304, CODA1305, CODA1306, CODA1308, CODA1309, or CODA1310. The design of the LGIC constructs is summarized in Table 12. Figure 5B shows the mean fluorescence intensity of α-bungarotoxin staining positive cells from Figure 5A.

[0327] The results show that CODA1237 has higher surface expression than CODA71, and the T202I mutation further increases the surface expression of the engineered chimeric receptor. Mutations in the ligand-binding domain also affect the surface expression levels of the engineered chimeric receptor.

[0328] Example 4 Peak current measurements of engineered receptors using IonFlux and manual patch clamp Peak current and dose response measurements using IonFlux:

[0329] HEK 293 cells were routinely passaged in DMEM with 10% fetal bovine serum (ThermoFisher Scientific). Cells were seeded in 15 cm dishes 24 h prior to transfection with Fugene 6 (Promega, E2691) and plasmid DNA (20 μg DNA total). 24 h post-transfection, cells were lifted using Accutase (Sigma, A6964) and then resuspended in serum-free medium (CHO-S-SFM II, ThermoFisher Scientific 12052 with 25 mM HEPES, ThermoFisher Scientific, 15630080) and allowed to rest for 30–90 min before loading onto an IonFlux (Fluxion Biosciences) for experiments. Recordings were made in extracellular solution (ECS) containing: 140 mM sodium chloride (S7653), 4 mM potassium chloride (P9333), 1 mM magnesium chloride (M2670), 2 mM calcium chloride (C3881), 10 mM HEPES (54457), and 10 mM D-(+)-glucose (G7528) (pH=7.3 with sodium hydroxide, osmolality=305-315 mOsm). The intracellular solution (ICS) contains: 140 mM cesium chloride (289329), 5 mM potassium fluoride (60238), 2 mM calcium chloride (P9333), 2 mM magnesium chloride (M2670), 10 mM HEPES (54457), and 10 mM EGTA (3777) (pH = 7.2 with cesium hydroxide, osmolality = 290-295 mOsm).

[0330] Drug-induced currents were recorded in ensemble mode (20 cells patched per recording channel). Cells were held at -60 mV and transmembrane currents were recorded at a sampling rate of 1 kHz. Drugs tested included acetylcholine chloride (A6625) and other synthetic compounds. Drug doses were applied for 5 seconds with a 2 minute wash between dose applications unless otherwise stated. The holding current was subtracted from the peak to find the current amplitude. Doses were applied in half-log increments, increasing the concentration until the peak current amplitude plateaued. For each ensemble channel, the amplitude was normalized to the maximum current. The normalized current amplitude was averaged across channels and the data was fitted to a dose-response curve using a nonlinear regression model (Y=bottom+(top-bottom) / (1+EC50 / X)^Hill coefficient). The maximum peak currents (absolute amplitude) from all ensemble channels were averaged for each receptor and normalized to the average maximum peak current from CODA71HA transfected and run in the same batch.

[0331] Peak current and dose response measurements using manual patch clamp:

[0332] Cell culture: HEK 293 cells were routinely passaged in DMEM with 10% fetal bovine serum (ThermoFisher Scientific). Cells were seeded in 6-well dishes 24 h prior to transfection with Fugene 6 (Promega, E2691) and plasmid DNA (1 μg DNA in total). 24 h after transfection, cells were re-seeded on 10 mm PDL and used for patch clamp experiments 24–48 h after coating coverslips with collagen. For dorsal root ganglion neuron experiments, adult rat lumbar ganglia were harvested, dissociated according to standard protocols, cultured on glass coverslips coated with poly-L-lysine and mouse laminin, transduced with ires-GFP-containing lentivirus or AAV6 vectors at DIV1, and processed for immunocytochemistry at DIV7. For hippocampal neuron experiments, hippocampi were harvested from embryonic day 18 rat pups or postnatal day 0 mouse pups and dissociated according to standard protocols. Cells were cultured on poly-L-lysine-coated glass coverslips, transduced with ires-GFP-containing lentiviral or AAV6 vectors at DIV3, and processed for immunocytochemistry at DIV14 (surface expression experiments) or stained with calcein violet and imaged at DIV16 (neuron survival assays). 5-fluoro-deoxyuridine was added to hippocampal cultures at DIV3 (rat E18) or DIV7 (mouse P0) to inhibit glial growth. For surface expression experiments using lentiviral expression, cultures were transduced with human synapsin promoter-driven lentiviral vectors at MOI=20 (DRG) or MOI=5 (hippocampus). For lentiviral survival assays, cultures were transduced with ubiquitin promoter-driven lentiviral vectors at an MOI of 2 (DRG) or MOI of 1 (hippocampus). For all experiments using AAV6 expression, cultures were transduced with human synapsin promoter-driven AAV6 vectors at an MOI of 2e5 (rat DRG and E18 hippocampus) or MOI of 3e8 (mouse p0 hippocampus).

[0333] Whole-cell patch clamp recording protocol: Cells seeded on coverslips were visualized at 10x and 40x on an inverted fluorescent microscope (Olympus). Recordings were made at room temperature using 3-6 MOhm glass patch electrodes (Sutter, BF150-86-10) with an Axopatch 200B amplifier and Digidata 1550B (Molecular Devices). Chemicals were obtained from Sigma, except for TC-5619, which was manufactured by Attenua / Targacept. Unless otherwise stated, recordings were made in extracellular solution (ECS) containing: 140 mM sodium chloride (S7653), 4 mM potassium chloride (P9333), 1 mM magnesium chloride (M2670), 2 mM calcium chloride (C3881), 10 mM HEPES (54457), and 10 mM D-(+)-glucose (G7528) (pH=7.3 with sodium hydroxide, osmolality=305-315 mOsm). An 8-line reservoir (AutoMate Scientific) combined with an 8-channel zero-dead-volume-perfusion pencil (AutoMate Scientific) was used for perfusion. Data were acquired using pClamp software (Molecular Devices). Traces were analyzed in Clampfit (Molecular Devices) or using custom-written Python code. Plotting and statistics were performed in GraphPad Prism.

[0334] Dose-response experiments: Drug-evoked currents were recorded in voltage clamp mode. For HEK cell experiments, recordings were performed in normal ECS with a cesium-based internal solution containing: 140 mM cesium chloride (289329), 5 mM potassium fluoride (60238), 2 mM calcium chloride (P9333), 2 mM magnesium chloride (M2670), 10 mM HEPES (54457), and 10 mM EGTA (3777) (pH = 7.2 with cesium hydroxide, osmolality = 290-295 mOsm). For DRG neuron experiments, cells were patched with normal ECS and then switched to NMDG-based ECS containing the following to prevent endogenous cation curren...

Claims

1. 1. An engineered receptor that is a chimeric ligand-gated ion channel (LGIC) receptor, (a) a ligand-binding domain derived from a first wild-type Cys-loop LGIC receptor; and (b) The ion pore domain from a second wild-type Cys-loop LGIC receptor. Engineered receptors, including

2. 2. The engineered receptor of claim 1, wherein the first wild-type Cys-loop LGIC receptor comprises a receptor of the nicotinic acetylcholine receptor family.

3. 3. The engineered receptor of claim 1 or 2, wherein the first wild-type Cys-loop LGIC receptor is a human α7 nicotinic acetylcholine receptor (α7-nAChR, SEQ ID NO:4).

4. 4. The engineered receptor of claim 1, wherein the second wild-type Cys loop LGIC receptor is a chloride permeable Cys loop ligand-gated ion channel receptor.

5. 5. The engineered receptor of claim 4, wherein the second wild-type Cys-loop LGIC receptor is a glycine receptor or a GABA-A receptor.

6. 6. The engineered receptor of claim 5, wherein the second wild-type Cys-loop LGIC receptor comprises glycine receptor α1, glycine receptor α2, glycine receptor α3, GABA-A receptor ρ1, GABA-A receptor ρ2, or GABA-A receptor ρ3.

7. 7. The engineered receptor of claim 1, wherein the second wild-type Cys-loop LGIC receptor is not a glycine receptor α1.

8. 8. The engineered receptor of claim 1 , wherein part or all of the Cys loop domain of the ligand binding domain is derived from the second wild-type Cys loop LGIC receptor.

9. 9. The engineered receptor of any one of claims 1 to 8, wherein part or all of the β1-2 loop domain of the ligand binding domain is derived from the second wild-type Cys-loop LGIC receptor.

10. 10. The engineered receptor of any one of claims 1 to 9, comprising a pre-M1 linker derived from the first wild-type Cys-loop LGIC receptor or the second wild-type Cys-loop LGIC receptor.

11. 10. The engineered receptor of any one of claims 1 to 9, comprising a pre-M1 linker comprising an N-terminal segment derived from the first wild-type Cys-loop LGIC receptor and a C-terminal segment derived from the second wild-type Cys-loop LGIC receptor.

12. 12. The engineered receptor of claim 10 or 11, wherein the pre-M1 linker comprises or consists of a sequence having at least 70% identity to any one of SEQ ID NOs: 72-91.

13. 13. The engineered receptor of any one of claims 1 to 12, wherein some or all of the M2-M3 linker of the ion pore domain is derived from the first wild-type Cys-loop LGIC receptor.

14. 14. The engineered receptor of any one of claims 1 to 13, wherein the M2-M3 linker of the ion pore domain comprises one or more mutations.

15. 15. The engineered receptor of any one of claims 1 to 14, wherein the M2-M3 linker of the ion pore domain comprises an amino acid sequence having at least 70% identity according to amino acids 283 to 295 of SEQ ID NO:

4.

16. 16. The engineered receptor of any one of claims 1 to 15, which forms a homomeric ion channel when expressed on the cell surface.

17. 17. The engineered receptor of claim 16, wherein greater than 50% of the engineered receptors expressed on the cell surface form homomeric ion channels.

18. 18. The engineered receptor of any one of claims 1 to 17, wherein the second wild-type Cys-loop LGIC receptor is the human glycine receptor α1 subunit (GlyRα1).

19. 20. The engineered receptor of claim 18, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 248-457 of SEQ ID NO:

2.

20. 20. The engineered receptor of claim 18 or 19, wherein the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 166-180 of SEQ ID NO:

2.

21. 21. The engineered receptor of any one of claims 18 to 20, wherein the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 80 to 85 of SEQ ID NO:

2.

22. 22. The engineered receptor of any one of claims 18-21, wherein the ion pore domain does not contain any amino acids between the amino acid positions corresponding to K353 and E362 of SEQ ID NO:

2.

23. 22. The engineered receptor of any one of claims 18 to 21, wherein the ion pore domain comprises an amino acid sequence having less than 50% sequence identity to SEQ ID NO:96 between amino acid positions corresponding to K353 and E362 of SEQ ID NO:

2.

24. 24. The engineered receptor of any one of claims 18 to 23, wherein the ion pore domain comprises one or more mutations in a region corresponding to the nuclear localization signal (NLS) / ER retention signal (ERRS) sequence of the human GlyRα1.

25. 25. The engineered receptor of any one of claims 18 to 24, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 16 or 33.

26. 18. The engineered receptor of any one of claims 1 to 17, wherein the second wild-type Cys-loop LGIC receptor is the human glycine receptor α2 subunit (GlyRα2).

27. 27. The engineered receptor of claim 26, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 254-452 of SEQ ID NO:

59.

28. 28. The engineered receptor of claim 26 or 27, wherein the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 172-186 of SEQ ID NO:

59.

29. 29. The engineered receptor of any one of claims 26 to 28, wherein the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 86-91 of SEQ ID NO:

59.

30. 30. The engineered receptor of any one of claims 26 to 29, wherein the ion pore domain comprises one or more mutations in a region corresponding to the NLS / ERRS sequence of human GlyRα2.

31. 31. The engineered receptor of any one of claims 26 to 30, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 62 or 65.

32. 18. The engineered receptor of any one of claims 1 to 17, wherein the second wild-type Cys-loop LGIC receptor is the human glycine receptor α3 subunit (GlyRα3).

33. 33. The engineered receptor of claim 32, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 253-464 of SEQ ID NO:61 or amino acids 253-449 of SEQ ID NO:

69.

34. 34. The engineered receptor of claim 32 or 33, wherein the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 171-185 of SEQ ID NO: 61 or 69.

35. 35. The engineered receptor of any one of claims 32 to 34, wherein the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 85-90 of SEQ ID NO: 61 or 69.

36. 36. The engineered receptor of any one of claims 32-35, wherein the ion pore domain does not contain an amino acid residue between the amino acid positions corresponding to K357 and D358 of SEQ ID NO:

69.

37. 36. The engineered receptor of any one of claims 32-35, wherein the ion pore domain comprises an amino acid sequence having less than 50% sequence identity to SEQ ID NO:95 between amino acid positions corresponding to K357 and D358 of SEQ ID NO:

69.

38. 38. The engineered receptor of any one of claims 32-37, wherein the ion pore domain comprises one or more mutations in a region corresponding to the NLS / ERRS sequence of human GlyRα3.

39. 39. The engineered receptor of any one of claims 32 to 38, comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 63, 66, 70 and 71.

40. 18. The engineered receptor of any one of claims 1 to 17, wherein the second wild-type Cys-loop LGIC receptor is the human GABA-A receptor ρ1 subunit (GABA-A ρ1).

41. 41. The engineered receptor of claim 40, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity according to amino acids 281-479 of SEQ ID NO:

10.

42. 42. The engineered receptor of claim 40 or 41, wherein the Cys loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 198-212 of SEQ ID NO:

10.

43. 43. The engineered receptor of any one of claims 40 to 42, wherein the β1-2 loop domain of the ligand binding domain comprises an amino acid sequence having at least 80% identity according to amino acids 112 to 117 of SEQ ID NO:

10.

44. 44. The engineered receptor of any one of claims 40 to 43, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 64 or 67.

45. 45. The engineered receptor of any one of claims 1 to 44, wherein the pre-M1 linker of the ligand binding domain comprises one or more mutations.

46. 46. ​​The engineered receptor of claim 45, wherein the ligand binding domain is derived from the human α7-nAChR and the one or more mutations in the pre-M1 linker are at one or more positions corresponding to T225, M226, and / or T230 of the human α7-nAChR.

47. 47. The engineered receptor of claim 46, wherein the ligand binding domain is derived from the human α7-nAChR and the one or more mutations include a mutation corresponding to T225I in the human α7-nAChR.

48. 48. The engineered receptor of any one of claims 45 to 47, wherein said one or more mutations increase surface expression of said engineered receptor.

49. 49. The engineered receptor of claim 48, wherein said surface expression is measured by an alpha-bungarotoxin (a-BTX) assay.

50. 50. The engineered receptor of any one of claims 3 to 49, wherein the ligand binding domain comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to amino acids 23-220 of SEQ ID NO:

4.

51. 51. The engineered receptor of any one of claims 3 to 50, wherein the ligand binding domain comprises an amino acid substitution at one or more residues including W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, Y140, L141, Y151, S170, W171, S172, Y173, S188, Y190, Y210, C212, C213, E215, Y217, or any combination thereof, of human α7-nAChR.

52. 52. The engineered receptor of any one of claims 3 to 51, wherein the ligand binding domain comprises two amino acid substitutions at pairs of residues including R101 and L131, Y115 and Y210, or R101 and Y210 of the human α7-nAChR.

53. 52. The engineered receptor of any one of claims 3 to 51, wherein the ligand binding domain comprises amino acid substitutions corresponding to L131N, W77F, and S172D of SEQ ID NO:

4.

54. 52. The engineered receptor of any one of claims 3 to 51, wherein the ligand binding domain comprises amino acid substitutions corresponding to Q139W and S172D of SEQ ID NO:

4.

55. 52. The engineered receptor of any one of claims 3 to 51, wherein the ligand binding domain comprises one or more amino acid substitutions listed in Table 12.

56. 56. The engineered receptor of any one of claims 3 to 55, wherein the ligand binding domain comprises amino acid substitutions corresponding to R101W, Y115E, and Y210W of the human α7-nAChR.

57. 57. The engineered receptor of any one of claims 1 to 56, comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:65 and comprising amino acid substitutions corresponding to R101W, Y115E, Y210W, and T225I of SEQ ID NO:

65.

58. 58. The engineered receptor of any one of claims 1 to 57, wherein the potency of the engineered receptor for a natural ligand of the first wild-type Cys-loop LGIC receptor is lower than the potency of the first wild-type Cys-loop LGIC receptor for the natural ligand.

59. 59. The engineered receptor of claim 58, wherein the potency of the engineered receptor against the natural ligand is at least 2-fold lower than the potency of the first wild-type Cys-loop LGIC receptor against the natural ligand.

60. 60. The engineered receptor of any one of claims 1 to 59, wherein the potency of the engineered receptor for a non-natural ligand is approximately the same as the potency of the first wild-type Cys-loop LGIC receptor for the non-natural ligand.

61. 60. The engineered receptor of any one of claims 1 to 59, wherein the potency of the engineered receptor for the non-natural ligand is greater than the potency of the first wild-type Cys-loop LGIC receptor for the non-natural ligand.

62. 62. The engineered receptor of claim 61, wherein the potency of the engineered receptor for the non-natural ligand is at least two-fold higher than the potency of the first wild-type Cys-loop LGIC receptor for the non-natural ligand.

63. 63. The engineered receptor of any one of claims 58 to 62, wherein determining the potency comprises determining the EC50.

64. 64. The engineered receptor of any one of claims 1 to 63, wherein the efficacy of the engineered receptor in the presence of a non-natural ligand is greater than the efficacy of the first wild-type Cys-loop LGIC receptor in the presence of the non-natural ligand.

65. 65. The engineered receptor of claim 64, wherein the efficacy of the engineered receptor in the presence of a non-natural ligand is at least two-fold greater than the efficacy of the first wild-type Cys-loop LGIC receptor in the presence of the non-natural ligand.

66. 66. The engineered receptor of claim 64 or 65, wherein determining efficacy comprises determining the amount of current passed through the engineered receptor in vitro in the presence of the non-natural ligand.

67. 67. The engineered receptor of any one of claims 60-66, wherein the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and facinicline / RG3487.

68. 68. The engineered receptor of claim 67, wherein the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and APN-1125.

69. 69. The engineered receptor of claim 68, wherein the non-natural ligand is TC-5619.

70. 70. A polynucleotide comprising a nucleic acid encoding the engineered receptor of any one of claims 1 to 69.

71. 71. The polynucleotide of Claim 70, comprising a promoter operably linked to said nucleic acid encoding said engineered receptor.

72. 72. The polynucleotide of claim 71, wherein the promoter is a regulatable promoter.

73. 73. The polynucleotide of claim 72, wherein the regulatable promoter is active in excitable cells.

74. 74. The polynucleotide of claim 73, wherein the excitable cell is a neuron or a muscle cell.

75. 75. The polynucleotide of claim 74, wherein the excitable cell is a neuron.

76. 76. A vector comprising a polynucleotide according to any one of claims 70 to 75.

77. 77. The vector of claim 76 which is a plasmid or viral vector.

78. 78. The vector of claim 77, which is a viral vector selected from the group consisting of an adenoviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, and a herpes simplex 1 virus vector (HSV-1).

79. 79. The vector of claim 78, wherein the viral vector is an AAV vector, wherein the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof.

80. 80. A composition comprising an engineered receptor according to any one of claims 1 to 69, a polynucleotide according to any one of claims 70 to 75, or a vector according to any one of claims 76 to 79.

81. 80. A pharmaceutical composition comprising an engineered receptor according to any one of claims 1 to 69, a polynucleotide according to any one of claims 70 to 75, or a vector according to any one of claims 76 to 79; and a pharma- ceutically acceptable carrier.

82. 82. A method of producing an engineered receptor in a neuron, comprising contacting the neuron with a polynucleotide described in any one of claims 70-75, a vector described in any one of claims 76-79, a composition described in claim 80, or a pharmaceutical composition described in claim 81.

83. 83. The method of claim 82 or the polynucleotide of claim 75, wherein the neuron is a neuron of the peripheral nervous system.

84. 84. The method of claim 82 or 83, or the polynucleotide of claim 75, wherein the neuron is a neuron of the central nervous system.

85. 85. The method of any one of claims 82 to 84 or the polynucleotide of claim 75, wherein the neuron is a nociceptive neuron.

86. 86. The method of any one of claims 82 to 85 or the polynucleotide of claim 75, wherein the neuron is a non-nociceptive neuron.

87. 87. The method of any one of claims 82 to 86 or the polynucleotide of claim 75, wherein the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron.

88. 88. The method of any one of claims 82 to 87 or the polynucleotide of claim 75, wherein the neuron is an A-delta afferent fiber, a C fiber or an A-beta afferent fiber.

89. 89. The method of claim 88 or the polynucleotide of claim 75, wherein the neuron is an Aβ afferent fiber.

90. 90. The method of claim 89 or the polynucleotide of claim 75, wherein the Aβ afferent fiber is a damaged Aβ afferent fiber.

91. 90. The method of claim 89 or the polynucleotide of claim 75, wherein the Aβ afferent fibers are uninjured Aβ afferent fibers.

92. 92. The method of any one of claims 82 to 91 or the polynucleotide of claim 75, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5.

93. 93. The method of any one of claims 82 to 92 or the polynucleotide of claim 75, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.

1.

94. 94. The method of any one of claims 82 to 93, wherein the contacting step is performed in vitro, ex vivo, or in vivo.

95. 95. The method of claim 94, wherein said contacting step is performed in vivo in a subject.

96. 96. The method of claim 95, wherein the contacting step comprises administering the polynucleotide, the vector, the composition, or the pharmaceutical composition to the subject.

97. 95. The method of claim 94, wherein said contacting step is carried out in vitro or ex vivo.

98. 98. The method of claim 97, wherein the contacting step comprises lipofection, nanoparticle delivery, biolistics, electroporation, sonication, or microinjection.

99. 99. The method of any one of claims 82 to 98, wherein the engineered receptor is capable of localizing to the cell surface of the neuron.

100. 11. A method of inhibiting neuronal activity comprising the steps of: (a) contacting the neuron with an engineered receptor of any one of claims 1-69, a polynucleotide of any one of claims 70-75, a vector of any one of claims 76-79, a composition of claim 80, or a pharmaceutical composition of claim 81; and (b) contacting the neuron with a non-natural ligand of the engineered receptor.

101. 101. The method of claim 100, wherein the neuron is a neuron of the peripheral nervous system.

102. 101. The method of claim 100, wherein the neuron is a neuron of the central nervous system.

103. 103. The method of any one of claims 100 to 102, wherein the neuron is a nociceptive neuron.

104. 103. A method according to any of claims 100 to 102, wherein the neuron is a non-nociceptive neuron.

105. 105. The method of any one of claims 100 to 104, wherein the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron.

106. 106. The method of any one of claims 100 to 105, wherein the neurons are A-delta afferent fibers, C-fibers, or A-beta afferent fibers.

107. 107. The method of claim 106, wherein the neuron is an Aβ afferent fiber.

108. 108. The method of claim 107, wherein the Aβ afferent fibers are damaged Aβ afferent fibers.

109. 108. The method of claim 107, wherein the Aβ afferent fibers are uninjured Aβ afferent fibers.

110. 110. The method of any one of claims 100 to 109, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5.

111. 111. The method of any one of claims 100 to 110, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.

1.

112. 112. The method of any one of claims 100 to 111, wherein the contacting step (a) is carried out in vitro, ex vivo, or in vivo.

113. 113. The method of any one of claims 100 to 112, wherein the contacting step (b) is performed in vitro, ex vivo, or in vivo.

114. 114. The method of any one of claims 100 to 113, wherein the contacting steps (a) and / or (b) are performed in vivo in a subject.

115. The method of Claim 114, wherein the contacting step (a) comprises administering the engineered receptor, the polynucleotide, the vector, or the pharmaceutical composition to the subject; and / or the contacting step (b) comprises administering the non-natural ligand to the subject.

116. 116. The method of any one of claims 100 to 115, wherein the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, biolistics, electroporation, sonication, or microinjection.

117. 117. A method according to any one of claims 100 to 116, wherein the engineered receptor is capable of localising to the cell surface of the neuron.

118. 1. A method of treating and / or delaying the onset of a neurological disorder in a subject in need thereof, comprising: (a) administering to said subject a therapeutically effective amount of an engineered receptor of any one of claims 1-69, a polynucleotide of any one of claims 70-75, a vector of any one of claims 76-79, a composition of claim 80, or a pharmaceutical composition of claim 81; and (b) administering to said subject a non-natural ligand of said engineered receptor. A method comprising:

119. 119. The method of claim 118, wherein the subject is administered the non-natural ligand after step (a).

120. The method of claim 118, wherein the subject is administered the non-natural ligand simultaneously with step (a).

121. 121. The method of any one of claims 118 to 120, wherein the neurological disorder is a seizure disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spastic disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation.

122. 122. The method of claim 121, wherein the neurological disorder is allodynia.

123. 123. The method of any one of claims 118 to 122, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487.

124. 124. The method of any one of claims 118-123, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously.

125. The method of claim 124, wherein the non-natural ligand is administered orally.

126. 126. The method of any one of claims 118 to 125, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinal, or cisterna magna.

127. 127. The method of any one of claims 118 to 126, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally.

128. The method of any one of claims 118 to 127, wherein the subject is suffering from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

129. The method of any one of claims 118 to 127, wherein the subject is suffering from neuropathic pain and the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject.

130. 130. The method of any one of claims 118 to 129, wherein the subject is a human.

131. 131. The method of any one of claims 118 to 130, wherein the therapeutically effective amount reduces the severity of signs and / or symptoms of the neurological disorder.

132. 132. The method of any one of claims 118 to 131, wherein the therapeutically effective amount delays the onset of signs and / or symptoms of the neurological disorder.

133. 133. The method of any one of claims 118 to 132, wherein the therapeutically effective amount eliminates signs and / or symptoms of the neurological disorder.

134. 134. The method of any one of claims 131 to 133, wherein the symptom of the neurological disorder is nerve damage, nerve atrophy, and / or seizures.

135. 135. The method of claim 134, wherein the nerve injury is a peripheral nerve injury.

136. 136. The method of any one of claims 131 to 135, wherein the symptom of the neurological disorder is pain.

137. 1. A method of treating and / or delaying the onset of pain in a subject in need thereof, comprising: (a) administering to said subject a therapeutically effective amount of an engineered receptor of any one of claims 1-69, a polynucleotide of any one of claims 70-75, a vector of any one of claims 76-79, a composition of claim 80, or a pharmaceutical composition of claim 81; and (b) administering to said subject a non-natural ligand of said engineered receptor. A method comprising:

138. 138. The method of claim 137, wherein the subject is administered the non-natural ligand after step (a).

139. 138. The method of claim 137, wherein the subject is administered the non-natural ligand simultaneously with step (a).

140. 140. The method of any one of claims 137 to 139, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487.

141. 141. The method of any one of claims 137 to 140, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously.

142. The method of claim 141, wherein the non-natural ligand is administered orally.

143. 143. The method of any one of claims 137 to 142, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinal, or cisterna magna.

144. 144. The method of any one of claims 137 to 143, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally.

145. The method of any one of claims 137 to 144, wherein the subject is suffering from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

146. The method of any one of claims 137 to 145, wherein the subject is suffering from neuropathic pain and the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject.

147. 147. The method of any one of claims 137 to 146, wherein the subject is a human.

148. 148. The method of any one of claims 137 to 147, wherein the pain is neuropathic pain.

149. 149. The method of any one of claims 137-148, wherein the pain is associated with, caused by, or results from chemotherapy.

150. 150. The method of any one of claims 137 to 149, wherein the pain is associated with, caused by, or results from trauma.

151. 151. The method of any one of claims 137 to 150, wherein the subject is suffering from allodynia.

152. 152. The method of any one of claims 137 to 151, wherein the pain occurs following a medical procedure.

153. 153. The method of any one of claims 137 to 152, wherein the pain is associated with, caused by, or results from childbirth or a Caesarean section.

154. 154. The method of any one of claims 137-153, wherein the pain is associated with, caused by, or results from a migraine headache.

155. 155. The method of any one of claims 137 to 154, wherein the therapeutically effective amount temporarily reduces pain in the subject, permanently reduces pain in the subject, prevents the onset of pain in the subject, and / or eliminates pain in the subject.

156. 156. The method of any one of claims 137 to 155, wherein steps (a) and (b) are performed prior to the appearance of pain in the subject.

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