Ligand gated ion channels and methods of use thereof

Abstract

Compositions and methods are provided for modulating cell activity using engineered receptors, engineered receptors encoded by polynucleotides, and gene therapy vectors comprising polynucleotides encoding engineered receptors. These compositions and methods are particularly useful for modulating the activity of neurons, for example for treating disease or for studying the neuronal circuit.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of US Provisional Application No. 63 / 311,735, filed on February 18, 2022, and US Provisional Application No. 63 / 348,560, filed on June 3, 2022, all of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] 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 its entirety. Field of the Invention

[0005] 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 Art

[0006] Refractory neurological diseases are often associated with abnormally functioning neurons. Attempts to develop therapies to treat these conditions have been hampered by the lack of tractable target proteins associated with the disease.

[0007] The most commonly used treatments for chronic pain are opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive failure, hallucinations and other systemic side effects. Despite the widespread use of drugs, their effectiveness in pain relief has a surprisingly low success rate. More invasive options for treating pain include nerve blocks and electrical stimulation. The most invasive and least preferred method for managing pain is to completely remove the nerve or part of it that causes the pain through surgery.

[0008] There is a long-felt and unmet need for safe, efficient and cost-effective treatments for neurological disorders. The present disclosure provides safe, efficient and cost-effective treatments for neurological disorders, including pain management. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present disclosure may be best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application document contains at least one drawing in color. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fee. It should be emphasized that, in accordance with common practice, the various features of the drawings are not drawn to scale. Instead, the dimensions of the various features are arbitrarily expanded or reduced for clarity. The following figures are included in the drawings:

[0010] Figure 1 Shown is a heat map of the percentage quenching of YFP fluorescence of cells transfected with CODA71 or CODA1237 after stimulation by various doses of acetylcholine or indicated non-natural ligands. The ligand dose is written at the top of each chart. The numbers in the boxes indicate the absolute amount of quenching observed. Dark blue indicates strong quenching of the YFP reporter. Light blue indicates moderate quenching. Orange indicates weaker or minimal quenching. Negative values ​​represent non-responders with negative quenching due to stimulation artifacts.

[0011] Figure 2 Heatmap showing the percent quenching of YFP fluorescence in cells transfected with chimeric CODA1237-based LGIC receptors containing various amino acid mutations following stimulation by increasing doses of acetylcholine or TC-5619. CODA75 is a non-responsive chimera that served as a negative control. Color coding and labeling follows that of Figure 1 Same rules.

[0012] Figure 3 Heatmap showing the percent quenching of YFP fluorescence in cells transfected with CODA 64, CODA71, or chimeric receptors containing the ion pore domain of GlyRα3 (CODA1342, CODA1343, CODA1344, CODA1345) after stimulation by different doses of acetylcholine. CODA75 is a non-responsive chimera that served as a negative control. Color coding and labeling follows that of Figure 1 Same rules.

[0013] Figure 4A The percentage of HEK293T cells transiently transfected with the indicated receptors that stained positive for α-bungarotoxin is shown. Figure 4B Shows the Figure 4A The mean fluorescence intensity (MFI) of α-bungarotoxin-positive cells. Figure 4C Shown are the percentages of cells positive for α-bungarotoxin staining in HEK293T cells transiently transfected with the indicated receptors in transduced with Ric3, Nacho, and GCAMPI6s. Figure 4D Shows the Figure 4C The mean fluorescence intensity of α-bungarotoxin-positive cells was shown in Figure 2. The data represent n=2-6 replicate samples.

[0014] Figure 5A The percentage of HEK293T cells transiently transfected with the indicated receptors that stained positive for α-bungarotoxin is shown. Figure 5B Shows the Figure 5A The mean fluorescence intensity of α-bungarotoxin-positive cells was shown in Figure 2. The data represent n=2-6 replicate samples. Figure 5CShown are the dose responses of TC-5619 and acetylcholine for CODA71 in HEK cells. Figure 5D Shown are the dose responses of TC-5619 and acetylcholine for CODA1055 in HEK cells. Figure 5E Shown is the dose response of CODA1316 to TC-5619 in HEK cells. Fig. 5F Shown are the peak currents of the indicated receptors in HEK cells upon activation by TC-5619.

[0015] Figure 6 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-Aρ1 (SEQ ID NO: 10), human GABA-Aρ2 (SEQ ID NO: 12), and human GABA-Aρ3 (SEQ ID NO: 14) protein sequences is shown. The β1-2 loop, Cys loop, pre-M1 linker, and M2-M3 linker regions are labeled. Summary of the invention

[0016] In one aspect, the present disclosure provides an engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor and comprises (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 nicotinic acetylcholine receptor family receptor. 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 includes 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 a glycine receptor α1. In some embodiments, part 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, part 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, which is 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, which comprises 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, part 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 to amino acids 283-295 of SEQ ID NO: 4. In some embodiments, the engineered receptor forms a homomeric ion channel when expressed on the cell surface. In some embodiments, more than 50% of the engineered receptors expressed on the cell surface form homomeric ion channels.

[0017] 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 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 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 to amino acids 80-85 of SEQ ID NO:2. In some embodiments, the ion pore domain does not comprise amino acids between the 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 SEQID NO:96 between the amino acid positions corresponding to K353 and E362 of SEQ ID NO:2. In some embodiments, the cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor comprising an amino acid sequence according to 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.

[0018] 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 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 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 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.

[0019] In some embodiments, the second wild-type Cys loop LGIC receptor is a human glycine receptor α3 subunit (GlyRα3). In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity 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 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 to amino acids 85-90 of SEQ ID NO:61 or 69. In some embodiments, 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 ion pore domain comprises an amino acid sequence having less than 50% sequence identity with SEQ ID NO: 95 between the amino acid positions corresponding to K357 and D358 of SEQ ID NO: 69. In some embodiments, the cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor comprising an ion pore domain comprising an IPD derived from human GlyRα3 isoform L (SEQ ID NO: 61) and comprising 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 with any one of SEQ ID NO: 63, 66, 70, and 71.

[0020] In some embodiments, the second wild-type Cys loop LGIC receptor is a human GABA-A receptor ρ1 subunit (GABA-Aρ1). In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity 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 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 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.

[0021] 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 one or more mutations in the pre-M1 linker are at one or more positions corresponding to T225, M226 and / or T230 of human α7-nAChR. In some embodiments, the ligand binding domain is derived from human α7-nAChR, and one or more mutations comprise a mutation corresponding to T225I of human α7-nAChR. In some embodiments, one or more mutations increase the surface expression of engineered receptors. In some embodiments, surface expression is measured by α-bungarotoxin (a-BTX) assay.

[0022] In some embodiments, the ligand binding domain comprises a sequence that is 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% identical to amino acids 23-220 of SEQ ID NO:4.

[0023] In some embodiments, the ligand binding domain comprises an amino acid substitution at one or more residues of human α7-nAChR, the one or more residues comprising 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. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of residues of human α7-nAChR, the pair of residues comprising R101 and L131, Y115 and Y210, or R101 and Y210. 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.

[0024] In some embodiments, the potency of the engineered receptor for 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 for the natural ligand. In some embodiments, the potency of the engineered receptor for the natural ligand is at least 2 times lower than the potency of the first wild-type Cys loop LGIC receptor for the natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is about the same as the potency of the first wild-type Cys loop LGIC receptor for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is higher than the potency of the first wild-type Cys loop LGIC receptor for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least 2 times higher than the potency of the first wild-type Cys loop LGIC receptor for the non-natural ligand. In some embodiments, determining the potency includes determining EC50. In some embodiments, the potency of the engineered receptor in the presence of a non-natural ligand is higher than the potency of the first wild-type Cys loop LGIC receptor in the presence of a non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of a non-natural ligand is at least 2 times higher than the efficacy of the first wild-type Cys loop LGIC receptor in the presence of a non-natural ligand. In some embodiments, wherein determining the efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of a 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.

[0025] In one aspect, the disclosure provides polynucleotides comprising nucleic acids encoding engineered receptors of the disclosure. In some embodiments, the polynucleotides comprise a promoter operably connected to the nucleic acids encoding the engineered receptors. In some embodiments, the promoter is a regulatable promoter. In some embodiments, the regulatable promoter is active in excitable cells. In some embodiments, the excitable cells are neurons or muscle cells. In some embodiments, the excitable cells are neurons.

[0026] In one aspect, the present disclosure provides a vector comprising a 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: an adenoviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, and a herpes simplex-1 viral vector (HSV-1). In some embodiments, the viral vector is an AVV vector, and wherein the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof.

[0027] 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.

[0028] 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 pharmaceutically acceptable carrier.

[0029] In one aspect, the present disclosure provides a method for 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 impaired Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an unimpaired Aβ afferent fiber. In some embodiments, the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5. In some embodiments, the neuron does not express TrpV1, prostatic acid phosphatase, or 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 a subject. In some embodiments, the contacting step comprises administering a polynucleotide, a vector, a composition, or a 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, particle bombardment, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor is capable of localizing to the cell surface of a neuron.

[0030] In one aspect, the present disclosure provides a method for inhibiting neuronal activity, comprising (a) contacting a 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 a neuron with a non-natural ligand of an 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 a damaged Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an undamaged Aβ afferent fiber. In some embodiments, the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5. In some embodiments, 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) includes administering an engineered receptor, a polynucleotide, a carrier or a pharmaceutical composition to the subject; and / or the contacting step (b) includes administering a non-natural ligand to the subject. In some embodiments, the contacting steps (a) and / or (b) include lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication or microinjection. In some embodiments, the engineered receptor is capable of being localized to the cell surface of neurons.

[0031] On the one hand, the present disclosure provides a method for treating a neurological disorder in a subject in need thereof and / or delaying its onset, comprising: (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 non-natural ligand is administered to the subject after step (a). In some embodiments, the non-natural ligand is administered to the subject in parallel with step (a). In some embodiments, the neurological disorder is epilepsy, movement disorders, eating disorders, spinal cord injury, neurogenic bladder, abnormal pain, spastic disorders, 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 nervous system 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, carrier, composition or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionically, intraneuronally, intracranially, intraspinally or to the cisterna magna. In some embodiments, the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered by transforaminal injection or intrathecal administration. In some embodiments, the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain, and wherein 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 the signs and / or symptoms of a nervous system disorder. In some embodiments, the therapeutically effective amount delays the onset of the signs and / or symptoms of a nervous system disorder. In some embodiments, the therapeutically effective amount eliminates the signs and / or symptoms of a nervous system disorder. In some embodiments, the signs of a nervous system disorder are nerve damage, nerve atrophy and / or epileptic seizures. In some embodiments, the nerve damage is peripheral nerve damage. In some embodiments, the symptom of a nervous system disorder is pain.

[0032] On the one hand, the present disclosure provides a method for treating pain in a subject in need thereof and / or delaying its onset, comprising: (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 non-natural ligand is administered to the subject after step (a). In some embodiments, the non-natural ligand is administered to the subject in parallel 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, carrier, composition or pharmaceutical composition is administered subcutaneously, orally, intrathecally, superficially, intravenously, intraganglionically, intraneuronally, intracranially, intraspinally or to the cisterna magna. In some embodiments, the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered by transforaminal injection or intrathecal administration. In some embodiments, the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain, and wherein the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. In some embodiments, the subject is a person. In some embodiments, pain is neuropathic pain. In some embodiments, pain is associated with chemotherapy, caused by chemotherapy or caused by chemotherapy. In some embodiments, pain is associated with trauma, caused by trauma or caused by trauma. In some embodiments, the subject suffers from allodynia. In some embodiments, pain appears after medical procedures. In some embodiments, the pain is associated with, caused by, or results from childbirth or cesarean section. In some embodiments, the pain is associated with, caused by, or results from migraine. In some embodiments, the therapeutically effective amount temporarily reduces pain in a subject, permanently reduces pain in a subject, prevents the onset of pain in a subject, and / or eliminates pain in a subject. In some embodiments, steps (a) and (b) are performed before pain manifests in the subject. DETAILED DESCRIPTION

[0033] Overview

[0034] Refractory neurological diseases are usually associated with abnormally active neurons. Attempts to develop therapies for these conditions are hampered by the lack of tractable target proteins associated with the disease. For example, unrelieved chronic pain is a serious health problem in the US and around the world. A report from the Institute of Medicine estimates that 116 million Americans suffer from pain that lasts for weeks to years, which results in annual costs of more than $560 million. There are not enough long-term therapies for chronic pain patients, which leads to huge costs for both society and individuals. Pain usually leads to disability, and even when not disabled, it has a great effect on quality of life. Pain treatment often fails even in the following situations: care provision is optimal, such as careful, well-trained doctors; easy access to opioids; use of adjuvant analgesics; availability of patient-controlled analgesia; and the evidence-based use of procedures such as nerve blocks and IT pumps.

[0035] The most commonly used treatment for chronic pain is the use of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and can cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive failure, hallucinations and other systemic side effects. Despite the widespread use of drugs, their effectiveness in pain relief has a surprisingly low success rate. Large randomized studies using various drugs found that only one in every two or three patients achieved at least 50% pain relief (Finnerup et al., 2005). Follow-up studies using the most developed pharmacological treatments found the same results, indicating that there was no improvement in the efficacy of pain drugs (Finnerup et al., Pain, 150 (3): 573-81, 2010).

[0036] More invasive options for treating pain include nerve blocks and electrical stimulation. A nerve block is an injection of a local anesthetic, usually in the spinal cord, to interrupt pain signals to the brain, with effects that last only weeks to months. In most cases, nerve blocks are not a recommended treatment option (Mailis and Taenzer, Pain Res Manag. 17(3):150–158, 2012). Electrical stimulation involves delivering an electric current to block pain signals. While the effects can last longer than a nerve block, the electrical leads themselves can have complications: dislocation, infection, breakage, or battery dying. One review found that 40% of patients treated for neuropathy with electrical stimulation experienced one or more device problems (Wolter, 2014).

[0037] The most invasive and least preferred method of managing pain is to completely remove the nerve or part of it that is causing the pain through surgery. This option is recommended only when the patient has exhausted the previous and other less invasive treatments and found them to be ineffective. Radiofrequency nerve ablation uses heat to destroy the problematic nerve and provides longer-lasting pain relief than nerve blocks. However, one study found no difference between the 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 painful nerves suffer from similar disadvantages and have long-term serious side effects, including sensory or motor deficits, or cause pain elsewhere.

[0038] Provided are compositions and methods for modulating cell activity using engineered ligand-gated ion channel (LGIC) receptors, polynucleotides encoding engineered LGIC receptors, and gene therapy vectors comprising polynucleotides encoding engineered LGIC receptors. These compositions and methods are particularly useful for modulating neuronal activity, such as for treating disease or for studying neuronal circuits. In addition, reagents, devices, and kits thereof for practicing the subject methods are also provided.

[0039] In particular, the present disclosure provides engineered receptors that bind to known drugs, ligands and / or binding agents and send signals in response to known drugs, ligands and / or binding agents. In some embodiments, the engineered receptors described herein show increased affinity for agonists or agonist binding agents. In some embodiments, the engineered receptors described herein show affinity for antagonists or regulator binding agents and respond to antagonists and / or regulators as if they were agonist agents. The present disclosure further provides methods for treating neurological disorders in subjects in need thereof. The present disclosure increases the number of clinical indications for which known drugs can be used by utilizing engineered receptors that respond to known drugs in a manner different from that of wild-type endogenous receptors.

[0040] Before describing the methods and compositions of the present invention, it will be understood that the present disclosure is not limited to the specific methods or compositions described, and therefore may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0041] When providing a numerical range, it is understood that, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed, to one tenth of the lower limit unit. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded from the range, and each range in which any one limit, neither limit, or both limits are included in the smaller range is also included in the present disclosure, subject to the influence of any specifically excluded limits in the specified range. Where the range includes one or both of the limits, the range excluding any one or both of these included limits is also included in the present disclosure.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials associated with the cited publications. It should be understood that if there is a conflict, the disclosure will supersede the disclosure of any incorporated publication.

[0043] As will be apparent to those skilled in the art reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any of the described methods can be performed in the order of events recited or in any other order that is logically possible.

[0044] Publications throughout this disclosure are provided only as they were disclosed prior to the filing date of this application. Nothing in this disclosure should be construed as an admission that the disclosure is not entitled to publish earlier than that disclosed by virtue of prior disclosure. Further, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0045] definition

[0046] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art, and so forth.

[0047] As used in this specification, the term "and / or" means "and" or "or" in this disclosure unless otherwise stated.

[0048] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" means the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In addition, the recitation of numerical ranges throughout this specification specifically includes all integers and decimal points therebetween.

[0049] Throughout this specification, unless the context requires otherwise, the phrase "consisting essentially of" means limiting the scope of the described composition, method, or kit to specified materials or steps that do not materially affect the basic and novel features of the disclosure. For example, a ligand binding domain that "consists essentially of" a disclosed sequence has the amino acid sequence of the disclosed sequence, plus or minus about 5 amino acid residues at the sequence boundaries, such as about 5 residues, 4 residues, 3 residues, 2 residues, or about 1 residue less than the described binding amino acid residues, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the described binding amino acid residues.

[0050] Throughout this specification, unless the context requires otherwise, the phrase "consisting of" means excluding from the composition, method or kit any element, step or ingredient not specified in the claim. For example, a ligand binding domain "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0051] As used in the present application, the terms "about" and "approximately" are used as equivalents. With or without about / approximately, any numeral used in the present application is intended to cover any normal fluctuations understood by those of ordinary skill in the relevant art. In certain embodiments, the terms "about" or "approximately" refer to a numerical range falling 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 reference value (greater than or less than), unless otherwise stated or apparent from context (unless such numerals exceed 100% of possible values).

[0052] As used throughout the disclosure, the term "isolated" means that the material is substantially or essentially free from normally accompanying components as found in its native state. In some embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0053] The terms "subject", "individual" and "patient" are used interchangeably and refer to vertebrates, such as mammals. The mammal can be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee) or human. Tissues, cells or derivatives thereof of the subject (whether obtained in vivo or cultured in vitro) are also contemplated. The human subject can be an adult, teenager, child (2 to 14 years old), infant (1 to 24 months) or newborn (up to 1 month). In some embodiments, the adult is an elderly person of about 65 years old or older or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who intends to become pregnant.

[0054] The term "sample" refers to a certain volume and / or mass of biological material analyzed. In some embodiments, the sample includes a tissue sample, a cell sample, a fluid sample, etc. In some embodiments, the sample is taken from a subject (e.g., a human subject) or provided by it. In some embodiments, the sample includes a portion of tissue taken from any internal organ, cancerous, precancerous or non-cancerous tumor, brain, skin, hair (including roots), eyes, muscles, bone marrow, cartilage, white adipose tissue and / or brown adipose tissue. In some embodiments, the fluid sample includes oral swabs, blood, cord blood, saliva, semen, urine, ascites, pleural fluid, spinal fluid, lung lavage fluid, tears, sweat, etc. It will be understood by those of ordinary skill in the art that in some embodiments, "sample" is a "raw sample" because it is obtained directly from a source (e.g., a subject). In some embodiments, "sample" is the result of processing the raw sample, such as removing certain potential contaminating components, separating certain components and / or purifying certain components of interest. In some embodiments, the sample is a cell or cell population (e.g., a neuronal cell). The cell sample can be directly derived from a subject (e.g., a primary sample) or can be a cell line. The cell line can include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0055] As used throughout this disclosure, "treating" or "treatment" refers to delivering a composition (e.g., an engineered receptor and / or binding agent) to a subject and / or cell population to affect a physiological outcome. In specific embodiments, treatment results in an improvement (e.g., reduction, alleviation, or remedy) in one or more symptoms of a disease. The improvement may be an observable or measurable improvement, or may be an improvement in the subject's overall sense of well-being. Treatment of a disease may refer to reducing the severity of disease symptoms. In some embodiments, treatment may refer to reducing the severity of disease symptoms to a level comparable to the severity 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., continuous or chronic) reduction in disease symptoms. In some embodiments, treatment may refer to the alleviation of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of subjects at risk of developing a particular disease to prevent disease development. Preventing disease development may refer to preventing disease symptoms completely, delaying the onset of disease, alleviating the severity of symptoms in a subsequently developed disease, or reducing the likelihood of disease development.

[0056] As used throughout this disclosure, "management" or "control" refers to the use of the compositions or methods contemplated by this disclosure to improve the quality of life of an individual suffering from a particular disease. In certain embodiments, the compositions and methods of this disclosure provide analgesia to a subject suffering from pain.

[0057] A "therapeutically effective amount" is the amount of a composition required to achieve the desired therapeutic outcome. A therapeutically effective amount can vary depending on factors such as, but not limited to, the disease state and age, sex, and weight of the subject. Generally, a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes an amount of a composition that is effective for treating a subject.

[0058] "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 can 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. An increase also means an increase 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) higher than a reference or control level.

[0059] "Reduced," "decreased," "attenuated," or their synonyms refer to a decrease in a value of at least 5% compared to a reference or control level (e.g., decreased binding affinity, decreased physiological response, decreased therapeutic reduction, decreased pain in a subject, etc.). For example, a decrease can 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 means 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.

[0060] The terms "reference" or "control" levels are used interchangeably throughout the present disclosure and refer to the value of a particular physiological and / or therapeutic effect in a subject or sample that has not been treated with a composition of the present disclosure, or a subject or sample that has been treated with a vehicle control. In some embodiments, a reference level refers to the value of a particular physiological and / or therapeutic effect measured in a subject or sample prior to administration of a composition of the present disclosure (e.g., a baseline level).

[0061] Throughout this specification, "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 changes the function of the receptor - activating or inhibiting 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 an ion channel.

[0062] "Receptor-ligand binding" and "ligand binding" are used interchangeably throughout this disclosure to refer to the physical interaction between a receptor (e.g., LGIC) and a ligand. As used throughout this disclosure, the term "ligand" can 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 (PIP 2 )), amino acids (e.g., glycine), or nucleotides (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., synthetic or non-naturally occurring ligand (e.g., binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by a variety of methods known in the art (e.g., detecting association with a radiolabeled ligand).

[0063] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site on 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., a receptor and a ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K d Affinity can be measured by common methods known in the art, including the methods described in this disclosure.

[0064] The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims to refer to the binding that occurs between paired species of molecules (e.g., receptors and ligands). The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims to refer to the binding that occurs between paired species of molecules (e.g., receptors and ligands). In various embodiments, the specific binding between one or more species is direct. In one embodiment, the affinity of the 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.

[0065] "Signaling" refers to the biochemical or physiological response generated as a result of the binding of a ligand to a receptor (eg, as a result of the binding of a binding agent to an engineered receptor of the disclosure).

[0066] The term "wild type" or "original" is a term of art understood by those skilled in the art and refers to the typical form of an organism, strain, gene, protein or feature as it occurs in nature, as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of the protein that occurs in nature.

[0067] Throughout the specification and claims, the terms "non-naturally occurring," "variant," and "mutant" are used interchangeably to refer to mutants of a native or wild-type composition, such as a variant polypeptide having less than 100% sequence identity with a native or wild-type sequence.

[0068] The amino acid modification can be an amino acid substitution, an amino acid deletion and / or an amino acid insertion. The amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. Conservative substitutions (also referred to as conservative mutations, conservative substitutions or conservative variations) are amino acid substitutions in proteins that change a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used throughout this disclosure, "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another; or substitution of one polar residue for another, such as substitution of arginine for lysine, substitution of glutamic acid for aspartic acid, or substitution of glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the following changes: 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 proline; 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.

[0069] The terms "parent" or "starting material" are used interchangeably throughout the specification and claims to refer to an initial composition, or a protein that is mutated, modified or derivatized to create an engineered composition with new properties. In some embodiments, the parent protein is a chimeric protein.

[0070] The term "engineered" is used throughout the specification and claims to refer to a non-naturally occurring composition, or a protein that has properties that differ from the parent composition, or the protein from which it is derived.

[0071] In general, "sequence identity" or "sequence homology" refers to the correspondence between nucleotides and nucleotides or amino acids and amino acids of two polynucleotides or polypeptide sequences, respectively. Typically, the technique for determining sequence identity includes 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 (polynucleotides or amino acids) can be compared by determining the "percent identity" of the two or more sequences (polynucleotides or amino acids). The percent identity of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of close matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. The identity percentage can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health. BLAST program is based on the alignment method of Karlin and Altschul, Proc.Natl.Acad.Sci.USA 87:2264-2268 (1990), and as Altschul, et al., J.Mol.Biol.215:403-410 (1990); Karlin and Altschul, Proc.Natl.Acad.Sci.USA 90:5873-5877 (1993); One Foundation Altschul et al., Nucleic Acids Res.25:3389-3402 (1997) described. In short, BLAST program defines identity as the number of identical aligned symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The program can be used to determine the percentage identity of the entire length of the compared protein. Default parameters are provided to optimize searches using short query sequences, such as using blastp program. The program also allows the use of a SEG filter program to mask segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 80% to 100% and intermediate integer values. Typically, the percent identity between a disclosed sequence and a claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0072] As used throughout this disclosure, "substantially identical" refers to sequences having 85% or more, such as 90% or more, such as 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, wherein the activity of the composition is not altered by sequence modifications that result in differences in sequence identity.

[0073] 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 a transcription start site, 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 most 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 connection 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, wherein the expression control sequence directs the transcription of a nucleic acid corresponding to the second sequence.

[0074] As used throughout this disclosure, the term "viral vector," "viral vector," or "gene delivery vector" refers to a viral particle that acts as a vehicle for nucleic acid delivery and comprises a nucleic acid (e.g., an AAV expression cassette) packaged within a virion. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0075] As used throughout this disclosure, "neuronal activity," "neuronal activity," "neuronal discharges," and variants and synonyms thereof refer to electrical activity resulting from stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using an automated or manual patch clamp technique. In some embodiments, determining the activity of a neuron includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the activity level of a neuron depends on, or is affected by, an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential.

[0076] 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 a structural, biochemical, and / or electrical abnormality of the brain, spinal cord, nerves, or any component of the nervous system.

[0077] As used throughout this disclosure, "signs" of disease refer to physical or mental characteristics of a condition that are considered indicative of disease. In some embodiments, a sign is an objective indication of disease. In some embodiments, a sign is objectively evaluated, examined, observed, or measured by someone other than the patient, such as a physician.

[0078] As used throughout this disclosure, "symptoms" of a disease refer to physical or mental characteristics of a condition that are considered indicative of the disease, particularly characteristics that are noticeable to the patient. In some embodiments, the symptom is subjectively assessed by the patient. For example, in some embodiments, the symptom is pain.

[0079] 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 or closing of an ion channel. In some embodiments, determining potency includes determining the half-maximal effective concentration (EC50) of a protein (such as LGIC) for a ligand under specific conditions. EC50 refers to the concentration of ligand that causes a response halfway between baseline and maximum after a specific exposure time.

[0080] 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 through a LGIC under specific conditions, such as in the presence of a specific concentration of a ligand. In some embodiments, determining efficacy includes determining the amount of current through a receptor, and / or the base strength of a receptor.

[0081] 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 can include determining and taking into account one or more factors, such as potency, efficacy, and subcellular localization of the protein.

[0082] As used throughout this disclosure with respect to amino acid positions, the term "corresponds to" or "corresponds to" refers to an amino acid in a first polypeptide sequence that is aligned with a given amino acid in a reference polypeptide sequence when the first polypeptide is aligned with the reference polypeptide sequence. The alignment is performed by one skilled in the art using software designed for this purpose (e.g., Clustal Omega version 1.2.4 with default parameters for this version). As an example, amino acid position D449 in SEQ ID NO:69 corresponds to D464 of SEQ ID NO:61.

[0083] As used throughout this disclosure, "amino acid mutation" refers to any difference in an amino acid sequence relative to the corresponding parent sequence, such as an amino acid substitution, deletion and / or insertion.

[0084] Engineered receptors

[0085] The present disclosure relates to engineered receptors, engineered receptor mutants and methods of using them. As used herein, the term "receptor" refers to any protein located on the surface of a cell and that can mediate signal transduction to and / or from a cell. The term "engineered receptor" is used herein to refer to a receptor that has been experimentally altered to physically and / or functionally differ from a corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. "Wild-type receptor" is used herein to refer to a receptor having a polypeptide sequence identical to that of a protein found in nature. Wild-type receptors include receptors naturally present in humans and orthologs naturally present in other eukaryotic organisms (e.g., protists, fungi, plants or animals, such as yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates). In some embodiments, the parent receptor is a non-natural receptor; that is, it is a receptor that does not exist in nature, for example, a receptor engineered from a wild-type receptor. For example, a parent receptor can be an engineered receptor comprising one or more subunits from a wild-type receptor and one or more subunits from a second wild-type receptor. Thus, the resulting protein comprises subunits from two or more wild-type receptors. Thus, in some embodiments, the parent receptor is a chimeric receptor. Engineered receptors of the present disclosure include, for example, parent receptor mutants and switch receptors.

[0086] The engineered receptors disclosed herein include at least one amino acid mutation relative to the corresponding parent receptor, for example, one or more mutations in one or more domains of a 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 lower sequence identity with the corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the engineered receptor has 85% or higher sequence identity with the corresponding parent receptor, for example, 90% or higher or 95% or higher with the corresponding parent receptor, for example, about 96%, about 97%, about 98% or about 99% identity, including all values ​​and subranges therebetween. In some embodiments, the engineered receptor (for example, parent receptor mutant) is generated by error-prone PCR.

[0087] In some embodiments, the ligand binding domain (LBD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation relative to the corresponding ligand binding domain of the parent receptor, such as 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 has a sequence identity of about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or less with the ligand binding domain of the corresponding parent receptor, including all values ​​and sub-ranges therebetween. In some embodiments, the ligand binding domain of the engineered receptor has a sequence identity of 85% or more with the ligand binding domain of the corresponding parent receptor, such as 90% or more or 95% or more with the ligand binding domain of the corresponding parent receptor, such as about 96%, about 97%, about 98% or about 99%, including all values ​​and sub-ranges therebetween. In some embodiments, the ligand binding domain of the engineered receptor is generated by error-prone PCR.

[0088] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation relative to the corresponding ion pore domain of the parent receptor, such as 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 with the ion pore domain of the corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the ion pore domain of the engineered receptor has 85% or more sequence identity with the ion pore domain of the corresponding parent receptor, such as 90% or more or 95% or more, such as about 96%, about 97%, about 98% or about 99% identity with the ion pore domain of the corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the ion pore domain of the engineered receptor is generated by error-prone PCR.

[0089] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation relative to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, substantially reduce, or eliminate the function of the engineered receptor relative to the parent receptor, for example by reducing the binding of an endogenous ligand to the engineered receptor relative to the binding of an endogenous ligand to the parent receptor or by reducing the activity of one or more signaling pathways downstream of the engineered receptor that are normally activated in response to binding of a binding agent to the corresponding parent receptor.

[0090] In some embodiments, amino acid mutations are functional gain amino acid mutations relative to the corresponding parent receptor. "Function gain" amino acid mutations refer to, for example, by changing or enhancing the affinity of the engineered receptor to the binding agent relative to the endogenous ligand and the parent receptor, or by changing or enhancing the activity of the signal transduction pathway activated in response to the binding of the binding agent to the engineered receptor relative to the endogenous ligand and the corresponding parent receptor, one or more mutations of the function of the engineered receptor modified relative to the parent receptor. In some embodiments, functional gain mutations cause the affinity of the engineered receptor to the binding agent to increase. In specific embodiments, functional gain mutations cause the affinity of the engineered receptor to the agonist binding agent to increase. In some embodiments, functional gain mutations cause the antagonist binding agent to act as an agonist binding agent when combined with the engineered receptor (for example, causing an agonist signal transduction pathway rather than an antagonist signal transduction pathway to be activated). In some embodiments, functional gain mutations cause the regulator binding agent to act as an agonist binding agent when combined with the engineered receptor. In some embodiments, the presently disclosed subject engineered receptors, or ligand binding domains and / or ion pore domains thereof, include one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations relative to the corresponding parent receptor.

[0091] 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 subject engineered receptor (or its ligand binding domain and / or ion pore domain) comprising a loss-of-function mutation and / or a gain-of-function mutation has a sequence identity of about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% (including all ranges and subranges therebetween) or less with the corresponding parent receptor, such as a wild-type receptor or a non-natural receptor (or its corresponding ligand binding domain and / or ion pore domain). In some embodiments, the subject engineered receptor (or its ligand binding domain and / or ion pore domain) shares 85% or more sequence identity with the corresponding parent receptor (or its corresponding ligand binding domain and / or ion pore domain), such as 85%, 90% or 95% or more sequence identity, and in some cases 96%, 97%, 98% or more sequence identity, such as, 99% or 99.5% or more sequence identity, including all 4 and subranges therebetween.

[0092] In some aspects, the engineered receptors of the present disclosure comprise a combination of one or more amino acid sequences, such as a subunit from a wild-type receptor having one or more amino acid sequences, such as a receptor produced from a subunit from a second wild-type receptor. In other words, the engineered receptors include amino acid sequences that are heterologous to each other, where "heterologous" means not occurring together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, chimeric receptors are used as parent receptors from which the engineered receptors disclosed herein are generated.

[0093] In some embodiments, the parent receptor mutant exhibits increased affinity for an agonist binding agent.In some embodiments, a ligand or binding agent that acts as an antagonist or modulator when bound to a wild-type receptor acts as an agonist when bound to a parent receptor mutant.

[0094] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGIC refers to a large group of transmembrane proteins that allow ions to pass through when activated by a specific ligand (e.g., a chemical agent or binding agent). LGICs are composed of at least two domains: a ligand binding domain and a transmembrane ion pore domain. Binding of a ligand 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 one or more specific ions; in fact, when the channel is inactive or closed, no ions can pass through the channel, but when a ligand is bound, up to 10 ions can pass through the channel per second. 7 In some embodiments, the LGIC responds to extracellular ligands (e.g., neurotransmitters) and promotes ion influx into the cytosol. In some embodiments, the LGIC responds to intracellular ligands (e.g., nucleotides such as ATP and mitochondria such as PIP 2 Importantly, LGIC activation leads to ion transport across the cell membrane (e.g., Ca 2+ 、Na + , K + , Cl - etc.) without causing the transport of the ligand itself.

[0095] LGIC receptors contain multiple subunits and can be homomeric or heteromeric receptors. Homomeric receptors contain subunits that are all of the same type. Heteromeric receptors contain subunits in which at least one subunit is different from at least one other subunit included in the receptor. For example, the glycine receptor contains 5 subunits, of which there are two types: the α subunit, of which there are four isoforms (α 1 -α 4 ); and β subunit, of which there is a single known isoform. An exemplary homomeric GlyR is a 1-GlyR subunits of GlyR. Similarly, homomeric GABA A The receptors may contain β 3 -GABA A subunits, and nAchR receptors can contain α 7 -nAchR subunits. Exemplary heteromeric GlyRs may include one or more α-subunits and one or more β-subunits (e.g., α 1 Table 1 shows the subunits of exemplary LGIC receptors.

[0096] Table 1: LGIC receptors and subunits

[0097]

[0098]

[0099]

[0100] Illustrative examples of LGIC families suitable for use in particular embodiments include, but are not limited to, Cys loop receptors, such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), λ-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).

[0101] Specific non-limiting examples of LGICs suitable for use in 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; GRI A4; GRID1; GRID2; GRIK1; GRIK2; GRIK3; GRIK4; GRIK5; GRIN1; GRIN2A; GRIN2B; GRIN2C; GRIN2D; GRIN3A; GRIN3B; ITPR1; ITPR2; ITPR3; CHRNA1; CHRNA2; CHRNA3; CHRNA4; CHR NA5; CHRNA6; CHRNA7; CHRNA9; CHRNA10; CHRNB1; CHRNB2; CHRNB3; CHRNB4; CHRNG; CHRND; CHRNE; P2RX1; P2RX2; P2RX3; P2RX4; P2RX5; P2RX6; P2RX7; RYR1; RYR2; RYR3; and ZACN.

[0102] TRPV1, TRPM8, and P2X 2 They are members of a larger family of LGICs that share structural features and gating principles. For example, TRPV4, like TRPV1, is also triggered by heat rather than capsaicin; and P2X 3 is triggered by ATP, but is more potent than P2X 2 Desensitization is faster. Therefore, TRPV1, TRPM8 and P2X 2 are non-limiting examples of LGICs suitable for use in particular embodiments.

[0103] In one embodiment, the engineered receptor is a TRPV1 or TRPM8 receptor or a mutant protein thereof. TRPV1 and TRPM8 are vanilloid receptors and menthol receptors expressed by nociceptive neurons of the peripheral nervous system. It is believed that both channels are used as non-selective sodium and calcium permeable homotetramers. In addition, these two channels and their main agonists, respectively, 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 receptor sites for light-labile blocking groups. The association of light-labile blocking groups with such receptors will produce ligand-gated ion channels in which light acts as an indirect trigger by releasing active ligands.

[0104] In one embodiment, the engineered receptor is a P2X 2 Receptor or its mutant protein. P2X 2 It is an ATP-gated non-selective cation channel distinguished by its slower desensitization rate. 2 can serve as a selectively addressable source of depolarizing currents and presents a platform for generating engineered channel-ligand combinations that completely lack natural agonists.

[0105] Non-limiting examples of sequences of wild-type LGIC receptors that can be used to generate the engineered receptors of the present disclosure include the following. In the sequence, the signal peptide is italicized, the ligand binding domain is bold, and the ion pore domain is underlined:

[0106] In some embodiments, the wild-type LGIC receptor is the human α1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 2), which is encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1 (SEQ ID NO: 1)):

[0107]

[0108] (SEQ ID NO:2).

[0109] In some embodiments, the wild-type LGIC receptor is the human α2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO:59), which is encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1, SEQ ID NO:58):

[0110]

[0111] (SEQ ID NO:59).

[0112] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO:61), which is encoded by the GLRA3 gene (GenBank Accession No. NM_006529.2, SEQ ID NO:60):

[0113]

[0114] (SEQ ID NO:61)

[0115] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO:69), which is encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.2, SEQ ID NO:68):

[0116]

[0117] (SEQ ID NO:69)

[0118] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor α7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO:4), which is encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5 (SEQ ID NO:3)):

[0119]

[0120] In some embodiments, the wild-type LGIC receptor is human 5-hydroxytryptamine receptor 3A (5HT3A, GenBank Accession No. NP_998786.2, SEQ ID NO:6), which is encoded by the HTR3A gene (GenBank Accession No. NM_213621.3, SEQ ID NO:5):

[0121]

[0122] In some embodiments, the wild-type LGIC receptor is human 5-hydroxytryptamine receptor 3B (5HT3B GenBank Accession No. NP_006019.1, SEQ ID NO:57), which is encoded by the HTR3B gene (GenBank Accession No. NM_006028.4, SEQ ID NO:56):

[0123]

[0124] In some embodiments, the wild-type LGIC receptor is human gamma-aminobutyric acid receptor A (GABA-A), subunit beta-3 (GABA-A β3) (GenBank Accession No. NP_000805.1, SEQ ID NO:8), which is encoded by the GABRB3 gene (GenBank Accession No. NM_000814.5, SEQ ID NO:7):

[0125]

[0126] (SEQ ID NO:8).

[0127] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho1 (ρ1) (GABA-Aρ1) (GenBank Accession No. NP_002033.2, SEQ ID NO: 10), which is encoded by the GABRR1 gene (GenBank Accession No. NM_002042.4, SEQ ID NO: 9):

[0128]

[0129] (SEQ ID NO: 10).

[0130] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho2 (ρ2) (GABA-Aρ2) (GenBank Accession No. NP_002034.3, SEQ ID NO: 12), which is encoded by the GABRR2 gene (GenBank Accession No. NM_002043.4, SEQ ID NO: 11):

[0131]

[0132] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho3 (ρ3) (GABA-Aρ3) (GenBank Accession No. NP_001099050.1, SEQ ID NO: 14), which is encoded by the GABRR3 gene (GenBank Accession No. NM_001105580.2, SEQ ID NO: 13):

[0133]

[0134] In some aspects, the subject engineered receptor is a chimeric receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence and an ion pore conduction domain sequence derived from at least a first LGIC, or more simply, an "ion pore domain sequence" derived from at least a second LGIC. 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 in at least one amino acid position compared to the corresponding region of the original amino acid sequence. In some embodiments, the amino acid sequence derived from the original amino acid sequence differs from the corresponding region of the original amino acid sequence by at most 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% (including all ranges and subranges therebetween) sequence identity with the corresponding region of the original amino acid sequence.

[0135] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequences and ion pore domain sequences 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 with 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 with the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is bold and the ion pore domain is underlined.

[0136] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO: 2). 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 about amino acids 29-235 of GlyRα1, such as 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, 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.

[0137] In some embodiments, the ligand binding domain of the chimeric receptor includes 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 about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), such as 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 human α7-nAChR.

[0138] 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 with about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), such as amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, 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 human α7-nAChR.

[0139] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the sequence of the ligand binding domain 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 about amino acids 23-247 of 5HT3A (SEQ ID NO: 6), e.g., amino acids 23-240, amino acids 30-245, amino acids 23-247, amino acids 23-250, and in some cases 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 approximately amino acids 21-239 of 5HT3B (SEQ ID NO: 57), e.g., amino acids 21-232, amino acids 21-235, amino acids 21-240, amino acids 21-245, and in some cases 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 human 5-hydroxytryptamine receptor 3.

[0140] 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-Aβ3. 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 about amino acids 26-245 of GABA-Aβ3 (SEQ ID NO: 8), e.g., amino acids 26-240, amino acids 26-245, amino acids 26-248, amino acids 26-250, and in some cases 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.

[0141] In some embodiments, the ion pore domain fused to the ligand binding domain conducts anions, for example, it comprises the ion pore domain sequence of a human glycine receptor or a human serotonin receptor. In other embodiments, the ion conducting pore domain fused to the ligand binding domain conducts cations, for example, it comprises the ion pore domain sequence of a human acetylcholine receptor or a human γ-aminobutyric acid receptor A.

[0142] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of a 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 with about amino acids 245-457 of GlyRα1 (SEQ ID NO: 2), such as 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.

[0143] In some embodiments, the ion pore domain of the chimeric receptor comprises 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 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 with 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.

[0144] 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 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 that 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 to 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 that is identical to an 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.

[0145] In some embodiments, the ion pore domain of the chimeric receptor comprises 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 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 that 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 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 that is 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.

[0146] In some embodiments, the ion pore domain is derived from the 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 with about amino acids 230-502, such as amino acids 227-502, amino acids 230-502, amino acids 231-502, amino acids 232-502 or amino acids 235-502. 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.

[0147] In some embodiments, the ion pore domain is derived from the 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 about 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 about 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 230-441 of SEQ ID NO:57, consists essentially of amino acids 235-441 of SEQ ID NO:57, consists essentially of amino acids 240-441 of SEQ ID NO:57, consists essentially of amino acids 245-441 of SEQ ID NO:57, or consists essentially of amino acids 250-441.

[0148] In some embodiments, the ion pore domain is derived from the ion pore domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-Aβ3. 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 with about amino acids 246-473 of GABA-Aβ3 (SEQ ID NO: 8), such as 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 240-473 of SEQ ID NO:8, amino acids 245-473 of SEQ ID NO:8, amino acids 247-473 of SEQ ID NO:8, amino acids 250-473 of SEQ ID NO:8, or amino acids 253-473 of SEQ ID NO:8.

[0149] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ1 (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-Aρ1 (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 that 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 with the ion pore domain sequence of human GABA-Aρ1 (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 that is identical to the ion pore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain sequence of human GABA-Aρ1 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-Aρ1 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-Aρ1 comprises, consists essentially of, or consists of amino acids 290-479 of SEQ ID NO: 10.

[0150] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ2 (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-Aρ2 (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 that 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 with the ion pore domain sequence of human GABA-Aρ2 (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 that is identical to the ion pore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain sequence of human GABA-A p2 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 p2 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 p2 comprises, consists essentially of, or consists of amino acids 271-466 of SEQ ID NO: 12.

[0151] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ3 (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-Aρ3 (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 that 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 with the ion pore domain sequence of human GABA-Aρ3 (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 that is identical to the ion pore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain sequence of human GABA-A p3 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 p3 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 p3 comprises, consists essentially of, or consists of amino acids 277-467 of SEQ ID NO: 14.

[0152] 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-terminal end of the receptor's β10 region and at its carboxyl (C) terminus by the N-terminal end of the transmembrane region 1 (M1) of the receptor. The pre-M1 linker of the 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 the LGIC receptor connects the ligand binding domain and the ion pore domain. In some embodiments, the pre-M1 linker of the human α7-nAchR corresponds to amino acids 224-233 of SEQ ID NO:4. In some embodiments, the pre-M1 linker of the 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.

[0153] In some embodiments, the pre-M1 linker of the chimeric LGIC receptor comprises or consists of a pre-M1 linker sequence of one of the wild-type LGIC receptors 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 those of the wild-type LGIC receptor.

[0154] 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.

[0155] 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.

[0156] Table 10: List of pre-M1 connectors

[0157]

[0158] 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-Aρ1 receptor, a GABA-Aρ2 receptor, and a GABA-Aρ3 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.

[0159] Table 11: List of pre-M1 connectors

[0160] name sequence SEQ ID NO: α7-nAchRpre-M1 linker VTMRRRTLYY 72 GABA-Aρ1, ρ2pre-M1 junction FTLRRHIFFF 80 GABA-Aρ3pre-M1 junction FVLRRHVFFF 81

[0161] name sequence SEQ ID NO: Pre-M1 connector #7 VTMRRRTLYF 82 Pre-M1 connector #8 VTMRRRTLFF 83 Pre-M1 connector #9 VTMRRRTFFF 84 Pre-M1 connector #10 VTMRRRIFFF 85 Pre-M1 connector #11 VTMRRHIFFF 86 Pre-M1 connector #12 VTLRRHIFFF 87 Pre-M1 connector #13 VTMRRRVFFF 88 Pre-M1 connector #14 VTMRRHVFFF 89 Pre-M1 connector #15 VTLRRHVFFF 90 Pre-M1 connector #16 VVLRRHVFFF 91

[0162] In some embodiments, the ion pore domain of the 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 by the C-terminal end of the transmembrane domain 2 (M2) of the receptor at its amino (N) terminal and by the N-terminal end of the transmembrane domain 3 (M3) of the receptor at its carboxyl (C) terminal. The M2-M3 linker of the 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, 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 the subject ligand-gated ion channel comprises a ligand binding domain from an AChR and an ion pore domain from a GlyR, its ion pore domain sequence may comprise an M2-M3 linker sequence derived from an 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 M2-M3 linker sequence removed from the ion pore domain corresponds to approximately 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 approximately 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 approximately amino acids 307-318 of GlyRα3 (SEQ ID NO: 61 or 69). In some embodiments, the ion pore domain is derived from GABA-Aρ1 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 approximately amino acids 335-346 of GABA-Aρ1 (SEQID NO: 10). In some embodiments, the ion pore domain is derived from GABA-Aρ2 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 approximately amino acids 315-326 of GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain is derived from GABA-Aρ3 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 321-332 of GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the inserted M2-M3 linker is derived from about amino acids 283-295 of α7-nAChR (SEQ ID NO: 4), such as 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 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 to 16 amino acids, 10 to 15 amino acids, 11 to 14 amino acids, or 12 to 13 amino acids in length, including all ranges and subranges therebetween.

[0163] 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 within the ligand binding domain of the Cys loop LGIC that forms a ring structure flanked by cysteines at the N-terminus and C-terminus. Without wishing to be bound by theory, it is believed that upon binding of the ligand to the ligand binding domain, the Cys loop structurally moves to be adjacent to the M2-M3 loop, and this movement mediates the biophysical transition of ligand binding in the extracellular domain to signal transduction in the ion pore domain (as reviewed in Miller and Smart, Trends in Pharmacological Sci 2009: 31(4)). Replacing the endogenous Cys loop sequence with a heterologous Cys loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases at least 5 times or 6 times, and at least 7 times, 8 times, 9 times or 10 times at certain doses. The Cys loop domain of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the 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 the subject chimeric ligand-gated ion channel comprises a ligand binding domain from an AChR and an ion pore domain from a GlyR, the subject ligand-gated ion channel can comprise a ligand binding domain sequence from an AChR (except for the sequence of the Cys loop domain, which is instead derived from a GlyR). In some embodiments, the ligand binding domain is derived from α7-nAChR and the Cys loop sequence is from GlyRα1, GlyRα2 or GlyRα3. In some such embodiments, the Cys loop sequence removed from the ligand binding domain corresponds to about amino acids 150-164 of α7-nAChR (SEQ ID NO: 4), such as amino acids 150-157 of α7-nAChR. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 166-180 of GlyRα1 (SEQ ID NO: 2), such as 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 inserted Cys loop sequence is derived from approximately amino acids 172-186 of GlyRα2 (SEQ ID NO: 59), such as 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 about amino acids 171-185 of GlyRα3 (SEQ ID NO: 61 or 69), such as 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 about amino acids 198-212 of GABA-ρ1 (SEQ ID NO: 10), such as amino acids 198-204 of GABA-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-ρ1. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 178-192 of GABA-ρ2 (SEQ ID NO: 12), such as amino acids 178-184 of GABA-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-ρ2. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 184-198 of GABA-Aρ3 (SEQ ID NO: 14), such as amino acids 184-190 of GABA-Aρ3, 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-Aρ3.

[0164] 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 within the ligand binding domain of the Cys loop LGIC, which is flanked by the C-terminus of the β1 sheet at its N-terminus and by the N-terminus of the β2 sheet at its C-terminus. Without wishing to be bound by theory, it is believed that the β1-2 loop contributes to the biophysical transition of mediating ligand binding in the extracellular domain to the ion pore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). It is believed that, after ligand binding, the β1-2 loop, together with the Cys loop, comes to the immediate vicinity of the M2-M3 loop to mediate the biophysical transition of signal transduction in the ion pore domain where the ligand in the extracellular domain resides (as reviewed by Miller and Smart above). Replacing the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases at least 5 times or 6 times, and at least 7 times, 8 times, 9 times or 10 times at certain doses. The β1-2 loop of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the 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 subject chimeric ligand-gated ion channel comprises a ligand binding domain derived from an AChR and an ion pore domain derived from a GlyR, the sequence of the β1-2 loop domain of the ligand binding domain can 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 about amino acids 67-70 of α7-nAChR (SEQ ID NO: 4), such as amino acids 67-70, amino acids 66-71, or amino acids 64-72 of α7-nAChR. In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to about amino acids 66-71 of α7-nAChR (SEQ ID NO: 4). In some embodiments, the ion pore domain is derived from GlyRα1, and the inserted β1-2 loop corresponds to about amino acids 80-85 of GlyRα1 (SEQ ID NO: 2), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα2 and the inserted β1-2 loop corresponds to approximately amino acids 86-91 of GlyRα2 (SEQ ID NO:59) with up to 3, up to 2, up to 1, or no amino acid mutations.In some embodiments, the ion pore domain is derived from GlyRα3, and the inserted β1-2 loop corresponds to about amino acids 85-90 of GlyRα3 (SEQ ID NO: 61 or 69), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Aρ1, and the inserted β1-2 loop corresponds to about amino acids 112-117 of GABA-Aρ1 (SEQ ID NO: 10), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Aρ2, and the inserted β1-2 loop corresponds to about amino acids 92-97 of GABA-Aρ2 (SEQ ID NO: 12), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Ap3 and the inserted β1-2 loop corresponds to approximately amino acids 98-103 of GABA-Ap3 (SEQ ID NO: 14) with up to 3, up to 2, up to 1, or no amino acid mutations.

[0165] Non-limiting examples of chimeric LGIC receptor sequences of the present disclosure include sequences disclosed herein as SEQ ID NO: 15 - SEQ ID NO: 52. In some embodiments, the chimeric LGIC receptor or polynucleotide encoding the same has 85% or more sequence identity to the sequences provided herein as SEQ ID NO: 15 - SEQ ID NO: 52, such as 90% or more, 93% or more, or 95% or more, i.e., about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences provided in SEQ ID NO: 15 - SEQ ID NO: 52. In the sequences, the signal peptide is italicized, the ligand binding domain is bold, and the ion pore domain is underlined.

[0166] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 linked) comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0167]

[0168] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (R228 linked) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0169]

[0170] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (V224 junction) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0171]

[0172] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (Y233 linked) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0173]

[0174] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 linked) comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GlyRα1 ion pore domain (underlined) comprising an α7-nAChRM2-M3 linker (lowercase):

[0175] (a)

[0176] (b)

[0177]

[0178] (c)

[0179] (d)

[0180]

[0181] (e)

[0182] (f)

[0183]

[0184] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1 Cys loop sequence (lowercase); fused to a 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 the following SEQ ID NO: 33:

[0185]

[0186] (a)

[0187]

[0188] 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 GlyRα1β1-2 loop sequence (lowercase); fused to the human GlyRα1 ion pore domain (underlined):

[0189] (a)

[0190] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1β1-2 loop sequence (lowercase) and a Cys loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined):

[0191] (a)

[0192]

[0193] (b)

[0194] (c)

[0195]

[0196] (d)

[0197] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1β1-2 loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined) comprising a human α7-nAChRM2-M3 linker (lowercase):

[0198] (a)

[0199]

[0200] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1 Cys loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined) comprising a human α7-nAChR M2-M3 linker (lowercase):

[0201] (a)

[0202] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (R241 linked), comprising the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold), fused to the human GlyRα1 ion pore domain (underlined):

[0203] (a)

[0204]

[0205] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (V236 junction) comprising the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0206] (a)

[0207] In some embodiments, the chimeric LGIC receptor is a GABRB3 / GLRA1 chimera (Y245 linked) comprising the human GABA-Aβ3 signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0208] (a)

[0209]

[0210] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA2 (R229 linked) 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 the following SEQ ID NO:62:

[0211] (a)

[0212] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 linked) 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 the following SEQ ID NO:63:

[0213] (a)

[0214]

[0215] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 linked) 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 the following SEQ ID NO:70:

[0216] (a)

[0217]

[0218] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GABRR1 (R229 linked) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GABA-Aρ1 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 the following SEQ ID NO:64:

[0219] (a)

[0220]

[0221] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA2 (R229 linked) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα2Cys loop sequence (lowercase); 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 the following SEQ ID NO: 65:

[0222] (a)

[0223]

[0224] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 linked) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα3Cys loop sequence (lowercase); 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 with the following SEQ ID NO: 66:

[0225] (a)

[0226] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA3 (R229 linked) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα3Cys loop sequence (lowercase); 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 with the following SEQ ID NO: 71:

[0227] (a)

[0228]

[0229] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GABRR1 (R229 linked) chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GABA-Aρ1 Cys loop sequence (lowercase); fused to a human GABA-Aρ1 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 the following SEQ ID NO: 67:

[0230] (a)

[0231]

[0232] Cell surface expression

[0233] In some embodiments, the engineered receptor comprises one or more amino acid mutations compared to the corresponding region of the wild-type receptor, which changes the expression level of the engineered receptor on the cell surface. In some embodiments, such 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 the one or more mutations. In some embodiments, the one or more amino acid mutations that change the surface expression of the engineered receptor do not negatively affect the effectiveness, efficacy and / or responsiveness of the engineered receptor.

[0234] In some embodiments, the one or more amino acid mutations are at 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 1 amino acid mutation.

[0235] 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 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 human α7-nAChR (SEQ ID NO: 4).

[0236] 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 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 α7-nAChR (SEQ ID NO: 4), wherein 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). 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.

[0237] 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 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 α7-nAChR (SEQ ID NO: 4), wherein 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). 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.

[0238] 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 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 α7-nAChR (SEQ ID NO: 4), wherein 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). 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.

[0239] 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 of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the T225I mutation of human α7-nAChR (SEQ ID NO: 4), wherein 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).

[0240] 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 M226I of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the M226I mutation of human α7-nAChR (SEQ ID NO: 4), wherein 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).

[0241] 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 T230P of SEQ ID NO: 4. In some embodiments, the ligand binding domain of the engineered receptor comprises a mutation corresponding to the T230P mutation of human α7-nAChR (SEQ ID NO: 4), wherein 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).

[0242] In some embodiments, the cell surface expression of an engineered receptor comprising such 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 a corresponding engineered receptor without one or more mutations. In some embodiments, the cell surface expression of an engineered receptor comprising such 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 a corresponding engineered receptor without one or more mutations.

[0243] 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-Aρ1 (SEQ ID NO: 10), human GABA-Aρ2 (SEQ ID NO: 12), or human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, such engineered receptors have higher surface expression than corresponding engineered receptors 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-Aρ1 (SEQ ID NO: 10).

[0244] In some embodiments, the cell surface expression of the engineered receptor can be measured using an α-bungarotoxin (a-BTX) binding assay. For example, fluorescently labeled α-bungarotoxin can be used to evaluate relative cell surface expression based on the fluorescence intensity of α-bungarotoxin-stained positive cells.

[0245] Without being bound by any particular theory, the increase in cell surface expression of the engineered receptor may be due to one or more of the following factors:

[0246] (a) Increased trafficking of engineered receptors to the cell surface;

[0247] (b) reduced trafficking of engineered receptors to non-cell surface destinations;

[0248] (c) longer retention of engineered receptors on the cell surface;

[0249] (d) reduced internalization of the engineered receptor from the cell surface;

[0250] (e) reduced degradation of the engineered receptor; and / or

[0251] (f) Increased overall expression of engineered receptors.

[0252] In some embodiments, the increase of the cell surface expression of the engineered receptor makes the stress of the endoplasmic reticulum (ER) lower. In some embodiments, the increase of the cell surface expression of the engineered receptor makes the stress of the cell degradation mechanism lower. In some embodiments, the increase of the cell surface expression of the engineered receptor makes the stress of the cell degradation mechanism lower. In some embodiments, the increase of the cell surface expression of the engineered receptor increases the vigor (for example, long-term vigor) of the cells expressing the engineered receptor. In some embodiments, the increase of the cell surface expression of the engineered receptor increases the sensitivity of the cells expressing the engineered receptor to one or more ligands (for example, 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 sub-ranges therebetween) of the total engineered receptors are expressed on the cell surface.

[0253] In some embodiments, the engineered receptor forms a homomeric receptor on the cell surface. In some embodiments, the engineered receptor forms a pentamer channel containing homomeric subunits. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα1. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα2. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human GlyRα3. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human gaba-aρ1. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human gaba-aρ2. In some embodiments, the engineered receptor forming a homomeric channel comprises an ion pore domain derived from the ion pore domain of human gaba-aρ3. 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 surface of cells form homomeric ion channels.

[0254] Mutations in engineered receptors

[0255] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO: 2), and wherein the eight amino acids corresponding to amino acids 354-361 of SEQ ID NO: 2 are removed from the ion pore domain ("SPMLNLFQ", SEQ ID NO: 96). In some embodiments, the engineered receptor that does not comprise these eight amino acids exhibits higher cell surface expression, enhanced export 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 that does not comprise these eight amino acids exhibits higher potency, higher efficacy, and / or high 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 comprise 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 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 the amino acid positions corresponding to K353 and E362 of SEQ ID NO: 2, wherein the ion pore domain comprises an amino acid sequence having 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, wherein the Cys loop domain is optionally substituted with a sequence derived from the corresponding Cys loop domain of GlyRα1. In some embodiments, such engineered receptors have correspondingly higher cell surface expression than a corresponding engineered receptor comprising an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO: 2) and containing 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 a corresponding engineered receptor comprising an ion pore domain of an IPD derived from human GlyRα1 (SEQ ID NO: 2) and containing these eight amino acids. In some embodiments, the cell surface expression of such engineered receptors is increased by at least 50% compared to a corresponding engineered receptor comprising an ion pore domain of an IPD derived from human GlyRα1 (SEQ ID NO: 2) and containing these eight amino acids.

[0256] 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 compared to human GlyRα3 isoform K (SEQ ID NO: 69), human GlyRα3 isoform L (SEQ ID NO: 61) contains an additional 15 amino acid residues in the ion pore domain, which correspond to amino acids 358-372 of SEQ ID NO: 61 ("TEAFALEKFYRFSDM", SEQ ID NO: 95). In some embodiments, an engineered receptor comprising an IPD derived from the IPD of GlyRα3 isomer K (i.e., without the 15 amino acid fragment) exhibits higher cell surface expression, enhanced export to the cell surface, slower internalization from the cell surface, and / or slower degradation compared to a corresponding engineered receptor comprising an IPD derived from the IPD of GlyRα3 isomer L (i.e., comprising the 15 amino acid segment). In some embodiments, an engineered receptor that does not comprise the 15 amino acid segment exhibits higher potency, higher efficacy, and / or a high responsiveness segment compared to a corresponding engineered receptor comprising the 15 amino acid segment.

[0257] In some embodiments, the engineered receptor comprises an ion pore domain derived from an IPD of human GlyRα3 isoform K (SEQ ID NO: 69), wherein the ion pore domain does not comprise amino acid residues between 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 an 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 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 an amino acid sequence having 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 (including all ranges and subranges therebetween) with SEQ ID NO: 95 between the amino acid positions corresponding to K357 and D358 of SEQ ID NO: 69. In some embodiments, such engineered receptors comprise a ligand binding domain derived from human α7-nAChR, wherein the Cys loop domain is optionally substituted with a sequence derived from the corresponding Cys loop domain of GlyRα3. In some embodiments, such engineered receptors have correspondingly higher cell surface expression than a corresponding engineered receptor comprising an ion pore domain derived from the IPD of human GlyRα3 isoform L (SEQ ID NO: 61) and containing these eight amino acids. In some embodiments, the cell surface expression of such engineered receptor 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 comprising an ion pore domain of an IPD derived from human GlyRα3 isoform L (SEQ ID NO: 61) and containing these eight amino acids. In some embodiments, the cell surface expression of such engineered receptor is increased by at least 50% compared to a corresponding engineered receptor comprising an ion pore domain comprising an IPD derived from human GlyRα3 isoform L (SEQ ID NO:61) comprising 15 amino acid residues corresponding to amino acids 358-372 of SEQ ID NO:61.

[0258] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα1 (SEQ ID NO: 2), and wherein 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 6 amino acids corresponding to amino acids 346-351 of SEQ ID NO: 2 ("RRKRRH", SEQ ID NO: 97), is composed of or is located within it. In some embodiments, the mutation includes 1, 2, 3, 4, 5 or more than 5 non-lysine, non-arginine substitutions. In some embodiments, the mutation includes the deletion of 1, 2, 3, 4, 5 or more than 5 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 the amino acid sequence of SEQ ID NO: 98 ("RRRQKR") or an amino acid sequence having at most 1 or at most 2 mutations according to SEQ ID NO: 98. In some embodiments, the NLS / ERRS sequence of the engineered receptor corresponding to amino acids 346-350 ("RRKRR") of SEQ ID NO: 2 is replaced by the first five amino acids ("RRRQK") of SEQ ID NO: 98. In some embodiments, the engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher cell surface expression, enhanced export to the cell surface, slower internalization from the cell surface, and / or slower degradation compared to the corresponding engineered receptor containing the complete NLS / ERRS sequence. In some embodiments, the engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher potency, higher efficacy, and / or higher responsiveness compared to the corresponding engineered receptor containing the complete NLS / ERRS sequence.

[0259] In some embodiments, the engineered receptor comprises an ion pore domain derived from the IPD of human GlyRα2 (SEQ ID NO: 59), and wherein 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 6 amino acids ("RRRQKR", SEQ ID NO: 97) corresponding to amino acids 352-357 of SEQ ID NO: 98, consisting of, or located within. In some embodiments, the mutation comprises 1, 2, 3, 4, 5 or more than 5 non-lysine, non-arginine substitutions. In some embodiments, the mutation comprises 1, 2, 3, 4, 5 or more than 5 amino acid deletions within the NLS / ERRS sequence. In some embodiments, the engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher cell surface expression, enhanced export to the cell surface, slower internalization from the cell surface, and / or slower degradation compared to the corresponding engineered receptor containing the complete NLS / ERRS sequence. In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit increased potency, increased efficacy, and / or increased responsiveness compared to corresponding engineered receptors containing the intact NLS / ERRS sequence.

[0260] 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 wherein 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 6 amino acids corresponding to amino acids 351-356 of SEQ ID NO: 61 or 69 ("RRKRKN", SEQ ID NO: 99), consists of, or is located within. In some embodiments, the mutation comprises 1, 2, 3, 4, 5 or more than 5 non-lysine, non-arginine substitutions. In some embodiments, the mutation comprises a deletion of 1, 2, 3, 4, 5 or more than 5 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 the amino acid sequence of SEQ ID NO: 98 ("RRRQKR") or an amino acid sequence having up to 1 or up to 2 mutations according to SEQ ID NO: 98. In some embodiments, the NLS / ERRS sequence of the 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, the engineered receptor having one or more mutations in the NLS / ERRS sequence exhibits higher cell surface expression, enhanced export to the cell surface, slower internalization from the cell surface, and / or slower degradation compared to the corresponding engineered receptor containing the complete NLS / ERRS sequence. In some embodiments, engineered receptors having one or more mutations in the NLS / ERRS sequence exhibit increased potency, increased efficacy, and / or increased responsiveness compared to corresponding engineered receptors containing the intact NLS / ERRS sequence.

[0261] Amino acid mutation

[0262] As described above, in some aspects, the subject engineered receptor comprises at least one amino acid mutation that changes the potency of the ligand on the engineered receptor relative to the potency of the ligand on the unmutated parent receptor. In other words, one or more amino acid mutations, such as loss-of-function mutations or gain-of-function mutations, change the responsiveness of the engineered receptor to the ligand relative to the responsiveness of the unmutated parent receptor. In some such embodiments, one or more mutations are in the ligand binding domain of the engineered receptor. In some embodiments, such as when the ligand binding domain of the engineered receptor is a Cys loop receptor protein, the one or more amino acid mutations are substitutions at residues corresponding to the following residues of α7-nAChR (SEQ ID NO: 4), the residues 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, one or more substitutions are within the α7-nAChR sequence.

[0263] In some embodiments, one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine and non-natural ligands by, e.g., 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. In certain embodiments, one or more substitutions are substitutions corresponding 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 cases, one or more substitutions selectively reduce the potency of acetylcholine to the engineered receptor. In other words, one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine while substantially maintaining the responsiveness to non-natural ligands, or, compared with its reduction of the responsiveness of the engineered receptor to non-natural ligands, otherwise reduce the responsiveness of the engineered receptor to acetylcholine by 2 times or more, such as 3 times, 4 times, 5 times or more, in some cases 10 times, 20 times, 50 times, 100 times or more. Exemplary substitutions, i.e., substitutions corresponding to L131E, L131S, L131T, L131D or S172D of α7-nAChR. In other embodiments, one or more substitutions selectively reduce the effectiveness of non-natural ligands to engineered receptors. In other words, one or more substitutions reduce the responsiveness of the engineered receptor to non-natural ligands while substantially maintaining the responsiveness to acetylcholine, or, compared with its reduction of the responsiveness of the engineered receptor to acetylcholine, otherwise reduce the responsiveness of the engineered receptor to non-natural ligands by 2 times or more, such as 3 times, 5 times or more, in some cases 10 times, 20 times or 50 times or more. Exemplary substitutions include substitutions corresponding to W77M, Y115W, S172T or S172C of α7-nAChR. In certain embodiments, 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.

[0264] In other embodiments, one or more substitutions increase, for example, 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 the responsiveness of engineered receptors to acetylcholine and / or non-natural ligands. Exemplary substitutions include substitutions 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 cases, one or more substitutions increase the potency of acetylcholine and non-natural ligands, such as substitutions corresponding to L131N, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q, and S188T of α7-nAChR. In other cases, one or more substitutions selectively increase the potency of acetylcholine for engineered receptors. In other words, it increases the responsiveness of the engineered receptor to acetylcholine by 2-fold or more, such as 3-fold, 4-fold or 5-fold or more, in some cases 10-fold, 20-fold, 50-fold or 100-fold, compared to one or more substitutions that increase the responsiveness of the engineered receptor to the non-natural ligand, such as substitutions corresponding to L141W, S172T, S172C, S188P or S188W of α7-nAChR. In certain embodiments, 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. In yet other cases, 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 the non-natural ligand by 2-fold or more, e.g., 3-fold, 5-fold or more, in some cases 10-fold, 20-fold or 50-fold or more, than the one or more substitutions increase the responsiveness of the engineered receptor to acetylcholine.

[0265] In some embodiments, the amino acid residue mutated in the subject engineered receptor is not an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of wild-type a7 nAChR (SEQ ID NO: 4). In some embodiments, the amino acid residue mutated in the subject engineered receptor is an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of 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 is 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, when such a substitution is present in an engineered receptor, it is present in combination with one or more amino acid mutations described herein.

[0266] For example, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 4) have been found to mediate the binding of the natural ligand acetylcholine. Mutations at these residues will reduce the binding of acetylcholine and thus result in 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 the non-natural ligand AZD0328 to the receptor, and mutations of these residues can increase the affinity of AZD0328 and / or other ligands to the receptor and are therefore gain-of-function mutations. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain of a α7-nAChR (SEQ ID NO:4) or a chimeric receptor comprising the ligand binding domain of a 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 of the α7-nAChR (SEQ ID NO:4) or a chimeric receptor comprising the ligand binding domain of the α7-nAChR is a substitution at 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.

[0267] As another example, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 4) have been found to mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues will reduce the binding of acetylcholine and / or nicotine. R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR mediate the binding of the non-natural ligand ABT126, and mutations at one or more of these residues are expected to increase the affinity of ABT126 and / or other ligands for α7-nAChR. R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of α7-nAChR mediate the binding of the non-natural ligand TC6987, and mutations in one or more of these residues are expected to increase the affinity of TC6987 and / or other ligands for α7-nAChR. R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of α7-nAChR mediate the binding of the non-natural ligand Facinicline / RG3487, and mutations in one or more of these residues are expected to increase the affinity of Facinicline / RG3487 and / or other ligands for α7-nAChR. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues of the ligand binding domain of the α7-nAChR or a chimeric receptor comprising the ligand binding domain of the 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, mutation of one or more amino acid residues alters the binding of ABT126 to α7-nAChR, wherein the 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, mutations in one or more amino acid residues alter the binding of TC6987 to α7-nAChR, wherein 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 α7-nAChR. In certain such embodiments, the amino acids are selected from R101, T128, N129, Y210, and C213. In some embodiments, mutations in one or more amino acid residues alter the binding of Facinicline / RG3487 to α7-nAChR, wherein 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 α7-nAChR. In certain such embodiments, the amino acid is selected from Y210, R101, and N129.

[0268] As another example, residues W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3 (SEQ ID NO: 6) have been found to mediate the binding of serotonin, and mutations at one or more of these residues will 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 cilansetron, and mutations at one or more of these residues are expected to increase the affinity of cilansetron and / or other ligands for 5HT3. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain of a chimeric receptor comprising the ligand binding domain of 5HT3A, wherein 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 E2311. In some embodiments, mutations in one or more amino acid residues alter the binding of serotonin to 5HT3, wherein the one or more amino acids are 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 acids are selected from Y136, Y138, N147, K149, and L179. In some embodiments, mutations in one or more amino acid residues alter 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 acid is selected from D64, I66, Y89, N123, T176, W190, R191, F221, E224, and E231.

[0269] In some embodiments, one or more mutations that affect the ability of a ligand to modulate LGIC activity are in the ion pore domain of the LGIC. For example, residue T279 of the serotonin receptor 5HT3A mediates the manner in which ligands modulate channel activity, such that mutation of this residue to, for example, serine (T279S) switches the effect from antagonism (i.e., reducing the activity of the LGIC) to agonism (i.e., promoting the activity of the channel). In some embodiments, the subject ligand-gated ion channel 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 comprising the 5HT3A ion pore domain, wherein the substitution is in the amino acid corresponding to 279 of SEQ ID NO: 6. In certain embodiments, the substitution is a T279S substitution relative to SEQ ID NO: 6.

[0270] The present disclosure provides engineered receptors having two or more mutations (such as amino acid substitutions) compared to a parent receptor. 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 a parent receptor comprising the amino acid sequence of SEQ ID NO:33.

[0271] In some embodiments, 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.

[0272] The present invention provides an engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor and comprises: (a) a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprising a Cys ring domain 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 an amino acid residue selected from the group consisting of Y140, R101, L131, Y115, and Y210, wherein the amino acid residue corresponds to an amino acid residue of α7-nAChR. In some embodiments, the engineered receptor comprises an amino acid sequence of SEQ ID NO: 33, wherein the amino acid sequence further comprises one or more amino acid substitutions at an amino acid residue selected from the group consisting of Y140, R101, L131, Y115, and Y210, wherein the amino acid residue corresponds to an amino acid residue of α7-nAChR.

[0273] 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.

[0274] 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 an amino acid residue 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 an amino acid residue 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, 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.

[0275] In some embodiments, the potency of the engineered receptor to acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine. In some embodiments, the potency of the engineered receptor to acetylcholine is at least about 1.5 times lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine (e.g., about 2 times, 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).

[0276] In some embodiments, the effectiveness of engineered receptors to non-natural ligands is roughly the same as that of human α7 nicotinic acetylcholine receptors (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of engineered receptors to non-natural ligands is higher than that of human α7 nicotinic acetylcholine receptors (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of engineered receptors to non-natural ligands is at least about 1.5 times higher than that of human α7 nicotinic acetylcholine receptors (α7-nAChR) to non-natural ligands (e.g., low about 2 times, 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 sub-ranges and values ​​therebetween). In some embodiments, determining effectiveness includes determining EC50.

[0277] In some embodiments, the efficacy of the engineered receptor in the presence of non-natural ligands is higher than the efficacy of human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of non-natural ligands. In some embodiments, the efficacy of the engineered receptor in the presence of non-natural ligands is higher than the efficacy of human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of non-natural ligands by at least about 1.5 times (for example, low about 2 times, 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 sub-ranges and values ​​therebetween). In some embodiments, wherein determining the efficacy includes determining the amount of the current through the engineered receptor in vitro in the presence of the non-natural ligands.

[0278] In some aspects, the subject ligand-gated ion channel comprises one or more non-desensitizing mutations. When used for ligand-gated ion channels, "desensitization" refers to the gradual reduction of ion flux under the long-term presence of agonists. This causes the responsiveness of neurons to ligands to be gradually lost. The so-called non-desensitizing mutation means an amino acid mutation that prevents LGIC from becoming desensitized to the ligand, thereby preventing neurons from becoming less responsive or unresponsive to the ligand. By introducing LGIC carrying mutations into neurons and analyzing the current flux during long-term exposure to the ligand, non-desensitizing mutations can be easily identified. If the LGIC does not include a non-desensitizing mutation, the current will recover from the peak value to the steady state during the long-term exposure, and if the LGIC includes a non-desensitizing mutation, the current will maintain the peak flux during exposure to the ligand. Exemplary amino acid mutations that cause desensitization include the V322L mutation in human GlyRα1 (V294L post-processes the preprotein to remove the signal peptide) and the L321V mutation in the human GABA-A receptor GABRB3 (L296V post-processes the preprotein to remove the signal peptide). In some embodiments, the desensitizing mutation is a substitution of an amino acid residue at or near the C-terminus of LGIC with a desensitizing sequence (e.g., a sequence having 90% or greater identity to IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53) derived from the C-terminus of a protein encoded by GABAR1, e.g., substitution 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.

[0279] In some aspects, the subject ligand-gated ion channel comprises one or more conversion mutations. The so-called conversion mutation refers to a mutation that changes the permeability of the LGIC ion pore domain so that it becomes a mutation that allows the conduction of non-natural ions (i.e., ions that are not naturally allowed to pass). In some cases, the mutation converts the permeability from cations to anions, such as replacing amino acid residues 260-281 (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 54) in human α7-nAChR (CHRNA7) with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 55), or the corresponding amino acids in another LGIC that is permeable to cations. In some cases, the mutation converts the permeability from anion to cation, for example, amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with glutamic acid (E) (such as A293E substitution in GLRA1, which converts the LGIC from anion permeable to calcium permeable), or amino acid residue 278 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is deleted, amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with glutamic acid (E), and amino acid residue 293 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with valine (V) (such as P278Δ, A279E, T293V in GLRA1, which converts the LGIC from anion permeable to cation permeable).

[0280] In addition to those described herein, additional engineered receptors can be easily identified by in vitro screening and verification methods. In some embodiments, parent receptor mutant libraries are produced from a limited number of parent receptors. Parent receptors can be mutated using methods known in the art (including fallible PCR). In some embodiments, the parent receptor mutant library is then transfected into yeast or mammalian cells, and screened with high throughput to identify functional receptors (for example, to identify the parent receptor mutants that can be responsive to binding agents or ligands for signal transduction). In some embodiments, the functional parent receptor mutants identified in the primary screening are then expressed in mammalian cells, and the responsiveness to binding agents or ligands is screened, for example, by plate readers and / or electrophysiological assays as described herein. Then it is possible to select the binding affinity that increases to agonist binding agents in secondary screening or to enable the use of antagonists or regulator binding agents as agonist parent receptor mutants, and further in vitro and / or in vivo verification and characterization assays are performed. Such screening assays are known in the art, e.g., 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.

[0281] Exemplary LGIC receptors

[0282] Table 12 below provides exemplary LGIC receptors of the present disclosure. Chimeric LGIC receptors are described with their ligand binding domain (LBD) and ion pore domain (IPD) separated by a colon. "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.

[0283] For example, CODA1298 in Table 12 comprises a ligand binding domain derived from a human α7-nAChR receptor and an ion pore domain derived from GlyRα2, wherein the Cys loop of the ligand binding domain is substituted with the Cys loop of GlyRα2, and wherein the receptor comprises a T225I mutation at an amino acid position corresponding to T225 of the human α7-nAChR receptor (SEQ ID NO:4).

[0284] Table 12: Exemplary LGIC receptors

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] Binder

[0291] The terms "binding agent" or "agent" are used interchangeably herein and refer to an exogenous drug or compound with a known mechanism of action on mammalian cells (e.g., an agonist, antagonist, or modulator known to act as a receptor). Binding agents may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, binding agents include drugs or compounds that have been approved by the U.S. Food and Drug Administration (FDA) for clinical use in the treatment of specific diseases (e.g., neurological diseases). In some embodiments, binding agents include drugs or compounds that have not been approved by the FDA for clinical use, but have been tested in one or more clinical trials, are currently being tested in one or more clinical trials, and / or are expected to be tested in one or more clinical trials. In some embodiments, binding agents include drugs or compounds that are not approved by the FDA for clinical use but are routinely used in laboratory research. In some embodiments, the binding agent is an analog of one of the above agents. In a specific embodiment, 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.

[0292] 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 its R or S enantiomer:

[0293]

[0294] 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.

[0295] In some embodiments, the binding agent is an anxiolytic, anticonvulsant, antidepressant, antipsychotic, antiemetic, nootropic, antibiotic, antifungal, antiviral, or antiparasitic.

[0296] Table 2: Glycine receptor (GlyR) binders

[0297]

[0298]

[0299] Table 3: GABA-A receptor binding agents

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Table 4: 5-HT3 receptor binding agents

[0310]

[0311]

[0312]

[0313]

[0314] Table 5: Binders for nicotinic acetylcholine receptor (nAchR)

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] Table 6: Binders for ATP-gated P2X receptor cation channels (P2X)

[0321]

[0322]

[0323] Table 7: Binders for the inward rectifier potassium channel (Kir)

[0324]

[0325]

[0326] Table 8: Binders for voltage-dependent potassium channels (KCNQ / Kv7)

[0327]

[0328]

[0329] Table 9: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Binding Agents

[0330] Agonists Modulators / Binders Antagonists 8-Cyclopentyl-1,3-dipropylxanthine Bumetanide 8-Methoxypsoralen Crofelemer Apigenin Glibenclamide CTP-656 Ibuprofen Genistein IBMX Ivacaftor Lumacaftor

[0331] Polynucleotide

[0332] In various illustrative embodiments, the present disclosure contemplates, in part, polynucleotides, polynucleotides encoding engineered receptor polypeptides and fusion polypeptides comprising LGIC and its subunits and mutant proteins, viral vector polynucleotides, and compositions comprising the same.

[0333] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence" or "nucleic acid" are used interchangeably. The terms refer to a polymeric form of nucleotides (deoxynucleotides or ribonucleotides or their analogs) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined according to connection analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (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 can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the modification of the nucleotide structure can be performed before or after assembling the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The polynucleotide may be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a DNA / RNA hybrid. The polynucleotide may be single-stranded or double-stranded. The polynucleotide includes, but is not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozyme, 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 of 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 or more (including all ranges and subranges therebetween) nucleotides (ribonucleotides or deoxynucleotides or modified forms of either type of nucleotides) in length, as well as all intermediate lengths. It will be readily understood that in this context, "intermediate lengths" refers to any length between the cited values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.In specific embodiments, the 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, to a reference sequence described herein or known in the art, wherein, unless otherwise specified, the variants typically retain at least one biological activity of the reference sequence.

[0334] As used herein, the term "gene" may refer to a polynucleotide sequence including enhancers, promoters, introns, exons, etc. In a specific embodiment, the term "gene" refers to a polynucleotide sequence encoding a polypeptide, regardless of whether the polynucleotide sequence is the same as a genomic sequence encoding the polypeptide.

[0335] As used herein, "cis-acting sequence", "cis-acting regulatory sequence" or "cis-acting nucleotide sequence" or equivalents refer to polynucleotide sequences associated with the expression, e.g., transcription and / or translation, of a gene. In one embodiment, a cis-acting sequence regulates transcription because the cis-acting sequence is a binding site for a polypeptide that inhibits or reduces transcription or a polynucleotide sequence associated with a transcription factor binding site that contributes to transcriptional inhibition. Examples of cis-acting sequences that regulate the expression of a polynucleotide sequence and that can be operably linked to the polynucleotides of the present disclosure to regulate the expression of a subject engineered receptor 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.

[0336] As a non-limiting example, a promoter sequence is a DNA regulatory region that can bind to the RNA polymerase in the cell and initiate transcription of the downstream (3' direction) coding sequence. For the purpose of defining the present invention, the promoter sequence is defined by the transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background. A transcription start site and a protein binding domain responsible for binding RNA polymerase will be found within the promoter sequence. Eukaryotic promoters will often but not always contain "TATA" boxes and "CAT" boxes. Each promoter can be used to drive each vector of the present invention. For example, a promoter can be a constitutively active promoter, i.e., a promoter that is active in the absence of an externally applied agent, for example, CMV IE1 promoter, SV40 promoter, GAPDH promoter, actin promoter. A promoter can be an inducible promoter, i.e., a promoter whose activity is regulated when an agent is applied to a cell, for example, doxycycline, tet-on or tet-off promoter, estrogen receptor promoter, etc. The promoter may be a tissue-specific promoter, ie, a promoter that is active in certain types of cells.

[0337] In some embodiments, the promoter is active in excitable cells. "Excitable cells" means cells that are activated by changing membrane potential, such as neurons or muscle cells, such as dorsal root ganglion neurons, motor neurons, excitatory neurons, inhibitory neurons, or sensory neurons. Promoters active in excitable cells that would be useful in the polynucleotide compositions of the present invention would include neuronal promoters, such as synaptobrevin (SYN), TRPV1, Na v 1.7 Na v 1.8 Na v 1.9, CamKII, NSE and Advillin promoters; myocyte promoters, such as desmin (Des), α-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC) promoters; and ubiquitous promoters, such as CAG, CBA, E1Fa, Ubc, CMV and SV40 promoters.

[0338] As used herein, "regulatory elements for inducible expression" refers to a polynucleotide sequence 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, an enhancer, or a functional fragment thereof. Illustrative regulatory elements for inducible expression include, but are not limited to, tetracycline responsive promoters, ecdysone responsive promoters, cumate responsive promoters, glucocorticoid responsive promoters, estrogen responsive promoters, RU-486 responsive promoters, PPAR-γ promoters, and peroxide inducible promoters.

[0339] "Regulatory elements for transient expression" refers to polynucleotide sequences that can be used to briefly or temporarily express a polynucleotide sequence. In specific 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 about minutes, hours, or days. Illustrative regulatory elements for transient expression include, but are not limited to, nuclease target sites, recombinase recognition sites, and inhibitory RNA target sites. In addition, to some extent, in specific embodiments, regulatory elements for inducible expression can also help control the duration of polynucleotide expression.

[0340] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or to a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" encompass polynucleotides in which one or more nucleotides have been added or deleted or modified or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. In specific embodiments, the 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, to a reference sequence described herein or known in the art, wherein, unless otherwise specified, the variants typically retain at least one biological activity of the reference sequence.

[0341] In one embodiment, polynucleotides include nucleotide sequences that hybridize with target nucleic acid sequences under stringent conditions. Hybridization under "stringent conditions" describes a hybridization scheme in which nucleotide sequences at least 60% identical to each other maintain hybridization. Typically, stringent conditions are selected to be approximately 5 ° C lower than the thermal melting point (Tm) of a specific sequence under a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize with the target sequence when balanced. Since the target sequence is usually present in excess, 50% of the probes are occupied when balanced under Tm.

[0342] As used herein, the statement "sequence identity" or, for example, including "a sequence that is 50% identical to ..." refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within a comparison window, 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) appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size) and multiplying the result by 100 to produce a 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 typically 15 to 18 and often at least 25 monomer units in length, comprising nucleotides and amino acid residues. Because two polynucleotides can each include (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) a sequence that differs between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides within a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, typically about 50 to about 100, more typically about 100 to about 150, wherein a sequence is compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. The comparison window can include about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (the reference sequence does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementations of the algorithm (GAP, BESTFIT, FASTA, and TFASTA of the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., producing the highest percentage of homology over the comparison window) generated by any of the various methods selected.Reference may also be made to the BLAST family of programs as disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis may be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0343] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that occur on either side of it in its naturally occurring state, for example, a fragment that has been removed from sequences that are normally adjacent to a DNA fragment. In specific embodiments, "isolated polynucleotide" refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but have been made by the hand of man.

[0344] Terms describing the orientation of a polynucleotide include: 5' (usually the end of the polynucleotide with a free phosphate group) and 3' (usually the end of the polynucleotide with a free hydroxyl group (OH)). Polynucleotide sequences can be annotated with a 5'-3' orientation or a 3'-5' orientation. For DNA and mRNA, the 5'-3' strand is named the "sense", "positive" or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, rather than thymine (T) in DNA]. For DNA and mRNA, the 3'-5' complementary strand, which is the strand transcribed by RNA polymerase, is named the "template", "antisense", "negative" or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation or a 3' to 5' sequence written in a 5' to 3' orientation.

[0345] The term "flanking" refers to a polynucleotide sequence between an upstream polynucleotide sequence and / or a downstream polynucleotide sequence, i.e., 5' and / or 3', relative to the sequence. For example, a sequence "flanking" two other elements (e.g., ITRs) indicates that one element is located 5' of the sequence and the other element is located 3' of the sequence; however, there may be an intervening sequence between them.

[0346] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AG T CAT G 3' is 3'T CAG TAC 5'. The latter sequence is often written as the reverse complement 5'CAT GA CT 3' with the 5' end on the left and the 3' end on the right. A sequence that is equal to its reverse complement is called a palindrome. Complementarity can be "partial", in which only some of the bases of the nucleic acids are matched according to the base pairing rules. Alternatively, there can be "complete" or "full" complementarity between nucleic acids.

[0347] As used herein, the term "nucleic acid box" or "expression cassette" refers to a polynucleotide sequence within a larger polynucleotide such as a vector, which is sufficient to express one or more RNAs from the polynucleotide. The expressed RNA can be translated into a protein, can be used as a guide RNA or an inhibitory RNA to target other polynucleotide sequences for cleavage and / or degradation. In one embodiment, the nucleic acid box contains one or more polynucleotides of interest. In another embodiment, the nucleic acid box contains one or more expression control sequences operably linked to one or more polynucleotides of interest. The polynucleotide comprises one or more polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide used as a template for a transcriptional inhibitory polynucleotide, such as LGIC and its subunits and mutant proteins, as contemplated herein. In a specific embodiment, the polynucleotide of interest encodes a polypeptide or fusion polypeptide having one or more enzymatic activities (such as nuclease activity) and / or chromatin remodeling or epigenetic modification activity.

[0348] The vector may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. In a preferred embodiment of the present disclosure, the nucleic acid cassette includes one or more expression control sequences (e.g., a promoter or enhancer operable in a neuronal cell) operably linked to a polynucleotide encoding an engineered receptor, such as a LGIC or a subunit thereof or a mutant protein. The cassette may be removed from or inserted into other polynucleotide sequences, such as a plasmid or a viral vector, as a single unit.

[0349] In one embodiment, the polynucleotides contemplated herein include 1, 2, 3, 4, 5, 6, 7, 8, 9 or more nucleic acid cassettes, any number or combination of which may be in the same or opposite orientation.

[0350] In addition, it will be appreciated by those of ordinary skill in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences that can encode fragments of polypeptides or their variants, as contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon selection, are specifically contemplated by the present disclosure. In one embodiment, polynucleotides comprising specific allele sequences are provided. Alleles are endogenous polynucleotide sequences that are altered due to one or more mutations such as deletions, additions and / or substitutions of nucleotides.

[0351] Carrier

[0352] In some aspects of the present disclosure, nucleic acid molecules, i.e., polynucleotides encoding engineered receptors, are delivered to a subject. In some cases, nucleic acid molecules encoding engineered receptors are delivered to a subject via a vector. In various embodiments, a vector includes one or more polynucleotide sequences considered herein. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred polynucleotides are usually connected to a carrier nucleic acid molecule, such as inserted into a carrier nucleic acid molecule. The vector may contain a sequence that guides autonomous replication in a cell or may contain a sequence sufficient to allow integration into host cell DNA. The vector may deliver a target polynucleotide to an organism, a cell, or a cell component. In some cases, the vector is an expression vector. As used herein, "expression vector" refers to a vector, such as a plasmid, that can promote expression and replication of a polynucleotide incorporated therein. Typically, the nucleic acid sequence to be expressed is operably connected to a cis-acting regulatory sequence, such as a promoter and / or enhancer sequence and is controlled by transcriptional regulation of a 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.

[0353] In a specific embodiment, any carrier suitable for expression cassette or polynucleotides encoding engineered receptors can be introduced into neuronal cells.Illustrative examples of suitable carriers include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes and viral vectors.In some cases, carriers are circular nucleic acids, such as plasmids, BACs, PACs, YACs, cosmids, Fox plasmids, etc.In some cases, circular nucleic acid molecules can be used to deliver nucleic acid molecules encoding engineered receptors to a subject.For example, the plasmid DNA molecules encoding engineered receptors can be introduced into the cells of the subject, thus the DNA sequence encoding the engineered receptors is transcribed into mRNA and mRNA "message" is translated into a protein product.The circular nucleic acid vector will generally include a regulatory element for regulating the expression of the target protein.For example, the circular nucleic acid vector can include any number of promoters, enhancers, terminators, splicing signals, replication origins, start signals, etc.

[0354] In some cases, the vector may include a replicon. A replicon may be any nucleic acid molecule capable of self-replication. In some cases, a replicon is an RNA replicon derived from a virus. Various suitable viruses (e.g., RNA viruses) may be obtained, including but not limited to alphaviruses, picornaviruses, flaviviruses, coronaviruses, pestiviruses, rubella viruses, caliciviruses, and hepatitis viruses.

[0355] In some embodiments, the vector is a non-viral vector. "Non-viral vector" means any delivery vehicle that does not include 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, microcarrier DNA, ccDNA, synthetic RNA, exosomes, etc. Non-viral vectors can be delivered by any suitable method as will be fully understood in the art, including, for example, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. See, for example, Chen et al. Mol. Therapy, Methods and Clinical Development. 2016 Jan; Vol 3, issue 1; and Hardy, CE et al. Genes (Basel). 2017 Feb; 8 (2): 6.5.

[0356] In other embodiments, the vector is a viral vector. "Viral vector" means a delivery vehicle including a viral capsid or envelope around a polynucleotide encoding a 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 nucleic acid molecules of the present invention to a subject include those derived from adenovirus, retrovirus (e.g., lentivirus), adeno-associated virus (AAV) and herpes simplex-1 (HSV-1). Illustrative examples of suitable viral vectors include, but are not limited to, retroviral vectors (e.g., lentiviral vectors), vectors based on herpes virus and vectors based on parvovirus (e.g., vectors based on adeno-associated virus (AAV), AAV-adenovirus chimeric vectors and vectors based on adenovirus).

[0357] As used herein, the term "parvovirus" encompasses all parvoviruses, including autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include members of the genus Parvovirus, the genus Erythrovirus, the genus Densovirus, the genus Iteravirus, and the genus Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute viruses of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other autonomous 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).

[0358] 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, AAVrh10, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

[0359] In a preferred embodiment, the vector is an AAV vector. In a specific case, the viral vector is an AAV-5, AAV6 or AAV-9 vector.

[0360] The genome organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule with a length of less than about 5,000 nucleotides (nt). Reverse terminal repeats (ITRs) are flanked by unique coding nucleotide sequences of non-structural replication (Rep) proteins and structural (VP) proteins. VP proteins (VP1, VP2 and VP3) form capsids and contribute to the tropism of the virus. The terminal 145nt ITR is self-complementary and is organized so that the energetically stable intramolecular duplex formed by the formation of a T-shaped hairpin can be formed. These hairpin structures are used as the starting point for viral DNA replication, thereby being used as primers for cellular DNA polymerase complexes. After wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and plays a role in the replication of the viral genome.

[0361] In some cases, the outer protein "capsid" of the viral vector is present in nature, 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 properties that do not exist in nature, such as altered tropism, increased transduction efficiency, or immune evasion. An example of a rationally designed capsid is a 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 whose VP3 capsid proteins have 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 that have been engineered by directed evolution 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 comprising an amino acid mutation at one or more positions corresponding to T492, Y705, Y731, or any combination thereof of an AAV6 capsid protein, wherein the AAV capsid protein is 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 comprising one or more amino acid substitutions corresponding to T492V, Y705F, or Y731F of an AAV6 capsid protein, or any combination thereof, wherein the AAV capsid protein is 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.

[0362] 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 an amino acid mutation 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.

[0363] 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 an amino acid mutation 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.

[0364] 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 an amino acid mutation 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.

[0365] In some embodiments, a viral vector comprising an AAV capsid protein (or a mutant of the disclosure) facilitates targeting expression of an engineered receptor to a subpopulation of cells or neurons in a subject. In some embodiments, the neurons are nociceptors.

[0366] "Recombinant parvovirus or AAV vector" (or "rAAV vector") herein refers to a vector comprising one or more polynucleotides considered herein flanked by one or more AAV ITRs. Such polynucleotides are referred to as "heterologous" to ITRs because such combinations do not usually exist in nature. When present in insect host cells expressing AAVrep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors can replicate and be packaged into infectious viral particles. When the rAAV vector is incorporated into a larger nucleic acid construct (e.g., in a chromosome or in another vector such as a plasmid or baculovirus for cloning or transfection), the rAAV vector is often referred to as a pro-vector that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary auxiliary functions.

[0367] In certain embodiments, any AAV ITR can be used in an 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 include one or more AAV2 ITRs.

[0368] The payload capacity of a rAAV vector comprising two ITRs is about 4.4 kB. A self-complementary rAAV vector contains a third ITR and packages both strands of the recombinant portion of the vector, leaving only about 2.1 kB for the polynucleotides contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0369] The use of a dual rAAV vector strategy has achieved an expanded packaging capacity that is approximately twice the packaging capacity of rAAV (about 9kB). The dual vector strategy useful in generating the rAAV considered herein includes but is not limited to splicing (trans-splicing), homologous recombination (overlapping) or a combination of the two (hybridization). In the dual AAV trans-splicing strategy, the splicing donor (SD) signal is placed at the 3' end of the 5' half vector, and the splicing acceptor (SA) signal is placed at the 5' end of the 3' half vector. After co-infection of the same cell by a dual AAV vector and head-to-tail concatenation mediated by inverted terminal repeats (ITRs) of the two halves, trans-splicing results in the production of mature mRNA and full-size proteins (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 large transgene expression cassettes contained in the two halves of the dual AAV vector can contain homologous overlapping sequences (the dual AAV overlaps at the 3' end of the 5' half vector and at the 5' end of the 3' half vector) that will mediate the reconstruction of a single large genome by homologous recombination (Duan et al., 2001). This strategy depends on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on adding highly recombinogenic regions from exogenous genes (i.e., alkaline phosphatase; Ghosh et al., 2008; Ghosh et al., 2011) to the trans-splicing vector. To increase recombination between the dual AAVs, the added region is placed downstream of the SD signal in the 5' half vector and upstream of the SA signal in the 3' half vector.

[0370] "Hybrid AAV" or "hybrid rAAV" refers to a rAAV genome packaged with a capsid of a different AAV serotype (and preferably, a different serotype than the one or more AAV ITRs) and may otherwise be referred to as a pseudotyped rAAV. For example, the rAAV type 1, rAAV type 2, rAAV type 3, rAAV type 4, rAAV type 5, rAAV type 6, rAAV type 7, rAAV type 8, rAAV type 9, rAAV type 10, rAAV type 11, rAAV type 12, rAAV type 13, rAAV type 14, rAAV type 15, or rAAV type 16 genome can be in 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 10, AAV type 11, AAV type 12, AAV type 13, AAV type 14, AAV type 15, or AAV type 16. 16 capsid or a variant thereof, provided that the AAV capsid and genome (and preferably, the one or more AAV ITRs) are of different serotypes. In certain embodiments, the pseudotyped rAAV particles can be referred to as having an "x / y" type, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid, e.g., a 2 / 5 rAAV particle has ITRs from AAV2 and a capsid from AAV6.

[0371] "Host cells" include cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the present disclosure. Host cells may include cells that produce viruses and cells infected with viral vectors. In specific embodiments, host cells are infected in vivo with the 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.

[0372] High titer AAV preparations can be produced using techniques known in the art, for example, as described in U.S. Pat. Nos. 5,658,776, 6,566,118, 6,989,264, and 6,995,006; U.S. 2006 / 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 have also been reported (e.g., WO 00 / 28004), as well as various modifications or formulations of AAV vectors to reduce their immunogenicity when administered in vivo (see, e.g., WO 01 / 23001; WO 00 / 73316; WO 04 / 112727; WO 05 / 005610; WO 99 / 06562).

[0373] Pharmaceutical composition

[0374] Also provided is a pharmaceutical preparation, a pharmaceutical preparation comprising a carrier and a pharmaceutical preparation of a binding agent. The pharmaceutical preparation comprises a subject polynucleotide (RNA or DNA) encoding an engineered receptor, a carrier carrying a polynucleotide (RNA or DNA) encoding the subject engineered receptor, or a binding agent present in a pharmaceutically acceptable medium. "Pharmaceutically acceptable medium" can be a medium used in mammals such as humans, approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia (US Pharmacopeia) or other recognized pharmacopeias. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier for the compound of the present disclosure to be formulated with it for use in mammals. Such pharmaceutical vehicles can be liquids, such as water and oil, including those oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can be saline, gum arabic, gelatin, starch paste, talcum, keratin, glue silicon, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants and colorants can be used. When administered to mammals, the compounds and compositions of the present disclosure and pharmaceutically acceptable vehicles, excipients or diluents may be sterile. In some cases, when the compounds of the present disclosure are administered intravenously, aqueous media such as water, saline, and aqueous glucose and glycerol solutions may be employed as vehicles.

[0375] The pharmaceutical composition can take the form of capsules, tablets, pills, spheres, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories or their sustained release formulations or any other form suitable for administration to mammals. In some cases, the pharmaceutical composition is formulated according to conventional procedures for use as a pharmaceutical composition suitable for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulating them are described below: 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, incorporated herein by reference.

[0376] The choice of excipient will be determined in part by the particular carrier, as well as by the particular method used to administer the composition.Thus, there is a wide variety of formulations suitable for the pharmaceutical compositions of the present disclosure.

[0377] For example, the carrier can be formulated into a preparation for injection by dissolving, suspending or emulsifying the carrier 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; and if desired, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0378] As another example, the carrier 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) a capsule, sachet or tablet, each containing a predetermined amount of the active ingredient such as a solid or granules; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. The tablet form can contain one or more of the following: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, cross-linked sodium carboxymethyl cellulose, talc, magnesium stearate, stearic acid and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents and pharmacologically compatible excipients. Lozenge forms may contain the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, as well as pastilles containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia, emulsions, gels, and similar dosage forms containing in addition to the active ingredient such excipients as described herein.

[0379] As another example, the subject formulations of the present disclosure can be formulated into aerosol formulations for administration by inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. Aerosol formulations can also be formulated into medicaments for non-pressurized preparations, such as for use in a nebulizer or atomizer.

[0380] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulation may be presented in unit dose or multi-dose sealed containers such as ampoules and vials and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid excipients, such as water, for injection immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0381] The formulation suitable for topical application can be presented in creams, gels, pastes or foams, which, in addition to the active ingredient, also contain a suitable carrier. In some embodiments, the topical formulation contains one or more components selected from structurants, thickeners or gelling agents and emollients or lubricants. The structurants often used include long-chain alcohols such as stearyl alcohol and glycerol ethers or esters and oligo(ethylene oxide) ethers or their esters. Thickeners and gelling agents include polymers and esters thereof of, for example, acrylic acid or methacrylic acid, polyacrylamide and naturally occurring thickeners, such as agar, carrageenan, gelatin and guar gum. The example of an emollient includes triglycerides, fatty acid esters and amides, waxes (such as beeswax, spermaceti or carnauba wax), phospholipids (such as lecithin) and sterols and fatty acid esters thereof. The topical formulation may further include other components, such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (i.e., sunscreens), etc.

[0382] The compounds of the present disclosure can be formulated for topical application. The vehicle for topical application can be in one of various forms, such as lotion, cream, gel, ointment, stick, spray or paste. The vehicle can include various types of carriers, including but not limited to solution, aerosol, emulsion, gel and liposome. The carrier can be formulated into an emulsion such as having an oil-in-water or water-in-oil matrix. The suitable hydrophobic (oily) component used in the emulsion includes, for example, vegetable oil, animal fat and oil, synthetic hydrocarbon and its ester and alcohol (including polyester) and organopolysiloxane oil. Such emulsions also include emulsifiers and / or surfactants, such as nonionic surfactants, to disperse and suspend the discontinuous phase in the continuous phase.

[0383] 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 presented as pessaries, tampons, creams, gels, pastes, foams.

[0384] Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be provided, wherein each dosage unit (e.g., teaspoonful, tablespoonful, tablet or suppository) contains a predetermined amount of a composition comprising one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may include one or more inhibitors in a composition as a solution in sterile water, saline or another pharmaceutically acceptable carrier.

[0385] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present disclosure and a pharmaceutically acceptable diluent, carrier or vehicle, the predetermined amount of which is calculated to be sufficient to produce the desired effect. The specifications of the novel unit dosage forms of the present disclosure depend on the specific compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0386] Dosage levels may vary depending on the specific compound, the nature of the delivery vehicle, etc. The desired dosage for a given compound can be readily determined by a variety of means.

[0387] The dosage administered to animals, particularly humans, in the context of the present disclosure should be sufficient to achieve a prophylactic or therapeutic response in the animal within a reasonable time frame, for example, as described in more detail below. The dosage will depend on a variety of factors, including the strength of the specific compound employed, the condition of the animal and the weight of the animal, as well as the severity of the illness and the stage of the disease. The size of the dosage will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a specific compound.

[0388] In pharmaceutical dosage forms, the ASC inducer compound may be administered in the form of a free base, a pharmaceutically acceptable salt of the compound, or the compound may also be used alone or in suitable combination with other pharmaceutically active compounds and in combination therewith.

[0389] Clinical applications and treatment methods

[0390] The compositions and methods disclosed herein can be used to treat neurological diseases or conditions. In some aspects of the present disclosure, a method for treating a neurological disease or condition in a subject is provided, the method comprising introducing an engineered receptor into a neuronal cell and providing an effective amount of a ligand for activating the engineered receptor to control the activity of the cell, thereby alleviating the pain in the subject. In some aspects, the carrier or composition disclosed herein is used to manufacture a drug for treating a neurological disease or condition.

[0391] In some cases, the methods and compositions of the present disclosure are used to treat epilepsy. Compositions described herein can be used to prevent or control epileptic seizures. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonic, absence or atonic seizures. In some cases, the compositions and methods herein can prevent epileptic seizures 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 sub-ranges therebetween.

[0392] In some cases, the methods and compositions of the present disclosure are used to treat eating disorders. Eating disorders may be mental disorders limited by abnormal eating behaviors that have a negative impact on the physical or mental health of the subject. In some cases, eating disorders are anorexia nervosa. In other cases, eating disorders are bulimia nervosa. In some cases, eating disorders are pica, rumination, avoidance / restrictive food intake disorders, binge eating disorder (BED), other specified feeding and diet disorders (OSFED), compulsive overeating, diabetic eating disorders, health food anorexia, selective eating disorders, alcohol anorexia, hyperphagia during pregnancy (pregorexia) or gourmand syndrome (Gourmand syndrome). In some cases, the composition includes a G protein-coupled receptor that increases or decreases the production of one or more molecules related to the eating disorder. In some cases, the composition includes a ligand-gated ion channel that changes the production of one or more molecules related to the eating disorder. The one or more molecules associated with eating disorders may include, but are not limited to, molecules of the hypothalamus-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.

[0393] 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 tumor), 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: aspirin, agraphia, alcoholism, alexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger syndrome, ataxia, ataxia telangiectasia, attention deficit hyperactivity disorder, speech and auditory processing disorder, autism spectrum, bipolar disorder, Bell's palsy, brachial plexus injury, brain injury, brain trauma, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, cephalic vein disorder (Cephalic disorder), cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cerebral spinal stenosis, Charcot-Marie-Tooth disease, cerebellar tonsillar herniation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, Coffin-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital bilateral facial paralysis, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative injury disorder, Cushing's syndrome, cyclothymic inclusion disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson disease, DeMorsier's syndrome syndrome, Dejerine-Klumpkepalsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, developmental coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndromesyndrome), Duchenne muscular dystrophy, articulation disorders, autonomic disorders, calculation disorders, writing disorders, movement disorders, reading disorders, muscle tone disorders, empty sella syndrome, encephalitis, encephalocele, cerebral trigeminal angiomatosis, fecal incontinence, enuresis, epilepsy, intellectual disorders of female epilepsy, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry's disease, Fahr's syndrome, syncope, familial spastic palsy, febrile seizures, Fisher syndrome, Friedreich's ataxia, fibromyalgia, Fovego's syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher's disease, generalized epilepsy with febrile seizures plus, Gerstmann's syndrome syndrome), giant cell arteritis, giant cell inclusion disease, spherocytoid leukoencephalopathy, gray matter heterotopia, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, globulin pigment syndrome, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary ataxia of the polyneuritic type, herpes zoster oticus, herpes zoster, Hirayama syndrome, Hirschsprung's disease, Holmes-Adie syndrome, holoprosencephaly, Huntington's disease, hydrocephalus, hydrocephalus, hypercortisolism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile Refsum disease Refsum disease, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, homomorphic dicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kingsborne syndrome, Kleine-Levine syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndromesyndrome, Landau-Kleffner syndrome, lateral medullary (Wallenberg) syndrome, learning disorders, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, ablative leukoencephalopathy, dementia with Lewy bodies, lissencephaly, locked-in syndrome, intervertebral disk disease, lumbar spinal stenosis, Lyme disease-neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, macromegaly, Mal de debarquement, megalencephalic leukoencephalopathy with subcortical cysts, megalencephaly, Melkersson-Rosenthal syndrome, Menieres disease, meningitis, Menkes disease disease), metachromatic leukodystrophy, microcephaly, microsomia, migraine, Miller Fisher syndrome, mini-stroke (transient ischemic attack), misophonia, mitochondrial myopathy, Mobius syndrome, monomelic muscular atrophy, motor skill disorders, moyamoya disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinoclastic diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neuro-Behcet's disease disease), neurofibromatosis, neuroleptic malignant syndrome, neurologic manifestations of AIDS, neurologic sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal translocation disease, neuropathy, neurasthenia, Niemann-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, Ohtawara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, persistent afterimage, paresthesia, Parkinson's disease, paramyotonia congenita, paraneoplastic disease, paroxysmal seizures, Parry-Romberg syndrome syndrome), PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, generalized developmental disorder, optical sneeze reflex, phytanic acid storage disease, Pick's disease, nerve compression, pituitary tumor, PMG, polyneuropathy, poliomyelitis, polymicrogyria, polymyositis, trepanencephaly, postpoliomyelitis, postherpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemiatrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrantanopia, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen encephalitis encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retroviral myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg syndrome, Saint Vitus dance, Sandhoff disease, Schilder's disease, schizencephaly, sensory processing disorder, developmental failure, shaken baby syndrome, herpes zoster, hypotensive syndrome, Sjögren's syndrome syndrome), sleep apnea, narcolepsy, Snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinobulbar muscular atrophy, spinocerebellar ataxia, schizencephaly, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, stroke, Stitch-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, Sydenham's chorea, syncope, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, advanced psychosis, Tarlov cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered cord syndrome, myotonic cataract, thoracic outlet syndrome, trigeminal neuralgia Douloureux, Todd's palsy, Tourette's syndrome, toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trichotillomania, trigeminalneuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, Von Hippel-Lindau disease (VHL), Viliuisk's encephalomyelitis (VE), Wallenberg's syndrome, West's syndrome, whiplash, Williams syndrome, Wilson's disease, or Zellweger's syndrome.

[0394] In some cases, the compositions and methods disclosed herein can be used to treat brain cancer or brain tumors. Non-limiting examples of brain cancers or tumors that may be amenable to treatment with the vectors and compositions described herein include: gliomas, including anaplastic astrocytoma (grade III glioma), astrocytoma (grade II glioma), brain stem glioma, ependymoma, ganglioglioma, ganglioneuroma, malignant glioma (grade IV glioma), glioma, juvenile pilocytic astrocytoma (JPA), low grade astrocytoma (LGA), medullablastoma, mixed glioma, oligodendroglioma, optic nerve glioma, fibrous astrocytoma (grade I glioma), and primitive neuroectodermal (PNET); skull base tumors, including acoustic neuroma (vestibular schwannoma), acromegaly, adenoma, chondrosarcoma, chordoma, craniopharyngioma, epidermoid tumor, glomus jugulare tumor, infratentorial meningioma, meningioma, pituitary adenoma, pituitary tumor, Rathke's cleft cyst (Rathke's cleftcyst); metastatic cancer, including intracranial metastases and metastatic brain tumors; other brain tumors, including brain cysts, choroid plexus papilloma, CNS lymphoma, glial cysts, cystic tumors, dermoid tumors, germ cell tumors, lymphomas, nasal cancer, nasopharyngeal tumors, pineal tumors, pineoblastomas, pineocytomas, supratentorial meningiomas, and hemangiomas; spinal cord tumors, including astrocytomas, ependymomas, meningiomas, and schwannomas.

[0395] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing or treating pain in a subject. "Pain" refers to the uncomfortable feelings and / or unpleasant sensations of a subject's body. The perception of pain can range from mild and occasional to severe and continuous. 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 by or associated with a 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, pain associated with Parkinson's disease, epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and nociceptive pain, including pain associated with central nervous system trauma, strains / sprains, and burns; myocardial infarction, acute pancreatitis, postoperative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0396] The compositions and methods herein can be used to alleviate the pain level of a subject. In some cases, the pain level of a subject alleviates 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 99% or about 100%, including all ranges and sub-ranges therebetween. The pain level of a subject can be assessed by a variety of methods. In some cases, pain level (that is, a verbal report of the pain level that a human subject expresses himself experiencing) is assessed by self-report. In some cases, pain level is assessed by behavioral indicators of pain, for example, facial expression, limb movement, vocalization, restlessness and defense. For example, these types of assessments can be useful when the subject (e.g., infants, unconscious subjects, non-human subjects) is unable to self-report. Pain levels can be assessed after treatment with a composition of the present disclosure compared to the pain level experienced by the subject prior to treatment with the composition.

[0397] 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 the use of the vectors disclosed herein to modulate neuronal activity to alleviate pain in a subject.

[0398] In various embodiments, a vector encoding an engineered receptor that activates or depolarizes a neuron is applied to (or introduced into) one or more neuron cells that reduce pain sensation, such as inhibitory interneurons. In the presence of a ligand, the neuron cells expressing the engineered receptor are activated and reduce sensitivity to pain, thereby enhancing the analgesic effect of stimulating these neuron cells.

[0399] 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 increase pain sensation or sensitivity to pain, e.g., nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc. In the presence of a ligand, the neuronal cell expressing the engineered receptor is inactivated and has reduced sensitivity to pain, and analgesia is enhanced.

[0400] Targeting the expression of engineered receptors to a subset of nociceptors can be achieved by one or more of the following: selecting a 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); selecting a promoter; and a means of delivery.

[0401] In certain embodiments, the compositions and methods contemplated herein are effective in reducing pain. Illustrative examples of pain suitable for treatment with 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, postherpetic neuralgia, postoperative pain, poststroke pain, and menstrual pain.

[0402] Pain can be classified as acute or chronic. "Acute pain" refers to pain that starts suddenly and is usually sharp in quality. Acute pain may be mild and last only a few moments, or it may be severe and last for weeks or months. In most cases, acute pain does not last more than three months, and the pain disappears when the underlying cause of the pain has been treated or has been cured. However, unrelieved acute pain may lead to chronic pain. "Chronic pain" refers to pain that persists or recurs, persists beyond the normal course of an acute illness or injury, or persists for more than three to six months, and it adversely affects the health of an individual. In a specific embodiment, the term "chronic pain" refers to pain that continues when it should not continue. Chronic pain can be nociceptive pain or neuropathic pain.

[0403] In some embodiments, the pain is associated with or developed 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 a surgical procedure, such as a surgical procedure to the central core of the body. In some embodiments, the medical intervention is a surgical procedure to remove part or all of one or more tissues, tumors, or organs in the body. In some embodiments, the medical intervention is an amputation. In specific embodiments, the compositions and methods contemplated herein are effective in reducing acute pain. In specific embodiments, the compositions and methods contemplated herein are effective in reducing chronic pain.

[0404] Clinical pain is present when discomfort and abnormal sensitivity are characteristic of the patient's symptoms. Individuals may have a variety of pain symptoms. Such symptoms include: 1) spontaneous pain that may be dull, burning, or tingling; 2) excessive pain response to noxious stimuli (hyperalgesia); and 3) pain caused by normally harmless stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Although patients with various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and therefore may require different treatment strategies. Therefore, pain can also be divided into a variety of different subtypes according to different pathophysiologies, including nociceptive pain, inflammatory pain, and neuropathic pain.

[0405] 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.

[0406] Nociceptive pain is induced by tissue damage or by strong stimulation that may cause damage. Moderate to severe acute nociceptive pain is the prominent feature of pain from central nervous system trauma, strain / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical operation), post-traumatic pain, renal colic, cancer pain and back pain. Cancer pain can be chronic pain, such as tumor-related pain (for example, bone pain, headache, facial pain or visceral pain) or pain related to cancer therapy (for example, post-chemotherapy syndrome, chronic postoperative pain syndrome or post-radiation syndrome). Cancer pain can also occur in response to chemotherapy, immunotherapy, hormone therapy or radiotherapy. Back pain may be due to disc herniation or rupture or lumbar intervertebral facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament abnormalities. Back pain can naturally subside, but back pain continues in some patients more than 12 weeks, and back pain becomes a chronic condition that may make people particularly weak.

[0407] Neuropathic pain can be defined as pain caused or initiated by a primary lesion or functional disorder of the nervous system. Causes of neuropathic pain include, 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 pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0408] Neuropathic pain may be associated with a pain disorder, the term pain disorder refers to a disease, disorder or condition associated with or caused by pain. Illustrative examples of pain disorders include arthritis, allodynia, classic trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia of pain, causalgia, sciatica disorders, degenerative joint disorders, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis, or pain associated with cancer.

[0409] The inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue damage or the presence of foreign substances, which leads to swelling and pain. Arthritic pain is a common inflammatory pain.

[0410] Other types of pain suitable for treatment with the vectors, compositions and methods contemplated herein include, but are not limited to, pain caused by musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, 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 headaches, tension-type headaches, mixed headaches and headaches associated with vascular disorders; and maxillofacial pain, including dental pain, ear pain, burning mouth syndrome and temporomandibular myofascial pain.

[0411] A variety of pain scales can be used to determine the effective amount of the compositions and methods contemplated herein for reducing the amount of pain experienced by a human subject. Patient self-report can be used to assess whether pain is reduced; see, e.g., Katz and Melzack (1999) Surg. Clin. North Am. 79: 231. Alternatively, an observational pain scale can be used. The LANSS pain scale can be used to assess whether pain is reduced; see, e.g., Bennett (2001) Pain 92: 147. A visual analog pain scale can be used; see, e.g., Schmader (2002) Clin. J. Pain 18: 350. A Likert pain scale can be used; e.g., where 0 is no pain, 5 is moderate pain, and 10 is the worst possible pain. Self-report pain scales for children include, e.g., a facial pain scale; a Wong-Baker FACES pain rating scale; and a color analog scale. Self-report pain scales for adults include, for example, the visual analogue scale; the verbal numeric rating scale; the verbal descriptive scale; and the Brief Pain Inventory.Pain measurement scales include, for example, the Alder Hey Triage Pain Score (Stewart et al. (2004) Arch. Dis. Child. 89:625); Behavioral Pain Rating Scale (Payen et al. (2001) Critical Care Medicine 29:2258); Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129); Nonverbal Pain Indicator Checklist (Feldt (2000) Pain Manager. Nurs. 1:13); Critical Illness Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); COMFORT Scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); Dallas Pain Questionnaire (Ozguler et al. (2002) Spine Pain Rating Scale). 27:1783); Algometer pain index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Face Pain Scale-Revised (Hicks et al. (2001) Pain 93:173); Face Leg Movement Crying Soothing Scale; McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptor Difference Scale (Gracely and Kwilosz (1988) Pain 35:279); Numerical 11 point Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numerical Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310); Wong-Baker FACES pain rating scale; and visual analog scale (Huskisson (1982) J. Rheumatol. 9:768).

[0412] In a specific embodiment, there is provided a method for alleviating the pain of a subject, the method comprising introducing an engineered receptor into a neuronal cell and controlling the activity of the cell by providing an effective amount of a ligand for activating the engineered receptor, thereby alleviating the pain of the subject. The method provides significant analgesia for pain without off-target effects, such as general central nervous system inhibition. In certain embodiments, compared with an untreated subject, the method reduces the neuropathic pain of the subject 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 some embodiments, the method includes the step of measuring the subject's pain before and after administration of the binding agent, wherein the subject's pain 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 cases, the measurement can occur 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.

[0413] In a specific embodiment, the vector contemplated herein is administered or introduced into one or more neuronal cells. The neuronal cells can be a mixed population of neuronal cells of the same type or different types of neuronal cells. In one embodiment, the neuronal cells are nociceptors or peripheral sensory neurons. Illustrative 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.

[0414] In some cases, the vector encoding the engineered receptor is administered to a subject in need. Non-limiting examples of methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cisterna magna administration, and intraneural administration. In some cases, administration may involve a liquid formulation of an injection vector. In other cases, administration may involve a solid formulation of an oral delivery vector. In some cases, oral formulations may be administered with food. In specific embodiments, the vector is administered to a subject parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneurally, intraganglionically, intraspinally, or intraventricularly to introduce the vector into one or more neuronal cells. In various embodiments, the vector is rAAV.

[0415] In one embodiment, AAV is administered to sensory neurons or nociceptors, e.g., DRG neurons, TGG neurons, etc., by intrathecal (IT) or intraganglionic (IG) administration. The IT route delivers AAV to the cerebrospinal fluid (CSF). This route of administration 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 through the cisterna magna and advancing its caudal end to the lumbar level. In humans, IT delivery can be readily performed by lumbar puncture (LP), a conventional invasive procedure with an excellent safety profile.

[0416] In certain cases, the vector can be administered to the subject by intraganglionic administration. Intraganglionic administration can involve direct injection into one or more ganglia. The IG approach can deliver AAV directly to the DRG or TGG parenchyma. In animals, IG administration to DRG is performed by open neurosurgery, which is undesirable in humans because it will require complex and invasive procedures. In humans, minimally invasive CT imaging guidance technology can be used to safely target DRG. Customized needle assemblies for convection enhanced delivery (CED) can be used to deliver AAV to the DRG parenchyma. 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) to treat epilepsy.

[0417] In yet another specific case, the vector can be administered to the subject by intracranial administration (i.e., directly administered to the brain). In a non-limiting example of intracranial administration, the vector of the present disclosure can be delivered to the cerebral cortex to treat, for example, epileptic seizure foci, to the paraventricular hypothalamus to treat, for example, satiety disorders, or to the central nucleus of the amygdala to treat, for example, satiety disorders. In another specific case, the vector can be administered to the subject by intraneural injection (i.e., directly injected into the nerve). The nerve can be selected based on the indication to be treated, for example, injected into the sciatic nerve to treat chronic pain or injected into the vagus nerve to treat epilepsy or satiety disorders. In yet another specific case, the vector can be administered to the subject by subcutaneous injection, for example, injected into the sensory nerve terminal to treat chronic pain.

[0418] The vector dose can be expressed as the number of vector genome units delivered to the subject. As used herein, "vector genome unit" refers to the number of individual vector genomes administered in a dose. The size of a single vector genome generally depends on the type of viral vector used. The vector genome of the present disclosure can be about 1.0 kilobases, 1.5 kilobases, 2.0 kilobases, 2.5 kilobases, 3.0 kilobases, 3.5 kilobases, 4.0 kilobases, 4.5 kilobases, 5.0 kilobases, 5.5 kilobases, 6.0 kilobases, 6.5 kilobases, 7.0 kilobases, 7.5 kilobases, 8.0 kilobases, 8.5 kilobases, 9.0 kilobases, 9.5 kilobases, 10.0 kilobases to greater than 10.0 kilobases. Therefore, a single vector genome can contain up to or greater than 10,000 nucleotide base pairs. In some cases, the vector dose can be about 1×10 6 pcs, 2×10 6 pcs, 3×10 6 4×10 6 pcs, 5×10 6 pcs, 6×10 6 pcs, 7×10 6 8×10 6 pcs, 9×10 6 pcs, 1×10 7 pcs, 2×10 7 pcs, 3×10 7 pcs, 4×10 7 pcs, 5×10 7 pcs, 6×10 7 pcs, 7×10 7 8×10 7 pcs, 9×10 7 pcs, 1×10 8 pcs, 2×10 8 pcs, 3×10 8 4×10 8pcs, 5×10 8 pcs, 6×10 8 pcs, 7×10 8 8×10 8 pcs, 9×10 8 pcs, 1×10 9 pcs, 2×10 9 pcs, 3×10 9 4×10 9 pcs, 5×10 9 pcs, 6×10 9 pcs, 7×10 9 8×10 9 pcs, 9×10 9 pcs, 1×10 10 pcs, 2×10 10 pcs, 3×10 10 4×10 10 pcs, 5×10 10 pcs, 6×10 10 pcs, 7×10 10 8×10 10 pcs, 9×10 10 pcs, 1×10 11 pcs, 2×10 11 pcs, 3×10 11 4×10 11 pcs, 5×10 11 pcs, 6×10 11 pcs, 7×10 11 8×10 11 pcs, 9×10 11 pcs, 1×10 12 pcs, 2×10 12 pcs, 3×10 12 4×10 12 pcs, 5×10 12 pcs, 6×10 12 pcs, 7×10 12 8×10 12 pcs, 9×10 12 pcs, 1×10 13 pcs, 2×10 13 pcs, 3×10 13 4×10 13 pcs, 5×10 13 pcs, 6×10 13 pcs, 7×10 13 8×10 13 pcs, 9×10 13 pcs, 1×10 14 pcs, 2×10 14 pcs, 3×1014 4×10 14 pcs, 5×10 14 pcs, 6×10 14 pcs, 7×10 14 8×10 14 pcs, 9×10 14 pcs, 1×10 15 pcs, 2×10 15 pcs, 3×10 15 4×10 15 pcs, 5×10 15 pcs, 6×10 15 pcs, 7×10 15 8×10 15 pcs, 9×10 15 pcs, 1×10 16 pcs, 2×10 16 pcs, 3×10 16 4×10 16 pcs, 5×10 16 pcs, 6×10 16 pcs, 7×10 16 8×10 16 ,9×10 16 pcs, 1×10 17 pcs, 2×10 17 pcs, 3×10 17 4×10 17 pcs, 5×10 17 pcs, 6×10 17 pcs, 7×10 17 8×10 17 pcs, 9×10 17 pcs, 1×10 18 pcs, 2×10 18 pcs, 3×10 18 4×10 18 pcs, 5×10 18 pcs, 6×10 18 pcs, 7×10 18 8×10 18 pcs, 9×10 18 pcs, 1×10 19 pcs, 2×10 19 pcs, 3×10 19 4×10 19 pcs, 5×10 19 pcs, 6×10 19 pcs, 7×10 19 8×10 19 pcs, 9×10 19 pcs, 1×10 20pcs, 2×10 20 pcs, 3×10 20 4×10 20 pcs, 5×10 20 pcs, 6×10 20 pcs, 7×10 20 8×10 20 pcs, 9×10 20 or more vector genome units.

[0419] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 1×10 9 genome particles / mL, at least about 1×10 10 genome particles / mL, at least about 5×10 10 genome particles / mL, at least about 1×10 11 genome particles / mL, at least about 5×10 11 genome particles / mL, at least about 1×10 12 genome particles / mL, at least about 5×10 12 genome particles / mL, at least about 6×10 12 genome particles / mL, at least about 7×10 12 genome particles / mL, at least about 8×10 12 genome particles / mL, at least about 9×10 12 genome particles / mL, at least about 10×10 12 genome particles / mL, at least about 15×10 12 genome particles / mL, at least about 20×10 12 genome particles / mL, at least about 25×10 12 genome particles / mL, at least about 50×10 12 genome particles / mL or at least about 100×10 12 As used with reference to viral titer, the term "genomic particles (gp)" or "genomic equivalents" or "genomic copies" (gc) refers to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by methods well understood in the art, such as genomic DNA quantitative PCR or, for example, Clark et al. (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: 272-278.

[0420] The carrier of the present disclosure can be applied with a certain volume of fluid. In some cases, the carrier can be applied with the following volume: 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 more than 20.0mL. In some cases, the carrier dosage can be expressed as the concentration or titer of the carrier applied to the subject. In this case, the carrier dosage can be expressed as the number of carrier genome units in each volume (i.e., genome units / volume).

[0421] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 5×10 9 infectious units / mL, at least about 6×10 9 infectious units / mL, at least about 7×10 9 infectious units / mL, at least about 8×10 9 infectious units / mL, at least about 9×10 9 infectious units / mL, at least about 1×10 10 infectious units / mL, at least about 1.5×10 10 infectious units / mL, at least about 2×10 10 infectious units / mL, at least about 2.5×10 10 infectious units / mL, at least about 5×10 10 infectious units / mL, at least about 1×10 11 infectious units / mL, at least about 2.5×10 11 infectious units / mL, at least about 5×10 11 infectious units / mL, at least about 1×10 12 infectious units / mL, at least about 2.5×10 12 infectious units / mL, at least about 5×10 12 infectious units / mL, at least about 1×10 13 infectious units / mL, at least about 5×10 13 infectious units / mL, at least about 1×10 14Infectious units / mL. As used with reference to viral titer, the terms "infectious unit (iu)", "infectious particles" or "replication units" refer to the number of recombinant AAV vector particles that are capable of infection and replication, as measured by the infectious center assay (also called replication center assay) as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0422] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 5×10 10 transduction units / mL, at least about 1×10 11 transduction units / mL, at least about 2.5×10 11 transduction units / mL, at least about 5×10 11 transduction units / mL, at least about 1×10 12 transduction units / mL, at least about 2.5×10 12 transduction units / mL, at least about 5×10 12 transduction units / mL, at least about 1×10 13 transduction units / mL, at least about 5×10 13 transduction units / mL, at least about 1×10 14 As used with reference to viral titers, the term "transduction unit (tu)" refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay described, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0423] The dosage of the vector will generally be determined by the route of administration. In a specific example, an intraganglionic injection may contain about 1×10 9 To about 1×10 13 In another specific embodiment, the intrathecal injection may contain about 1×10 10 To about 1×10 15 In yet another specific embodiment, the intracranial injection may contain about 1×10 9 To about 1×10 13 In another specific embodiment, the intraneural injection may contain about 1×10 9 To about 1×1013 In another specific example, the intraspinal injection may contain about 1×10 9 To about 1×10 13 In yet another specific embodiment, the cisterna magna infusion may contain about 5×10 9 To about 5×10 13 In yet another specific embodiment, a subcutaneous injection may contain about 1×10 9 To about 1×10 13 vector genome.

[0424] In some cases, the vector is delivered to the subject by infusion. The vector dose delivered to the subject by infusion can be measured by the vector infusion rate. Non-limiting examples of vector infusion rates include: 1-10 microliters / minute for intraganglionic, intraspinal, intracranial, or intraneural administration; and 10-1000 microliters / minute for intrathecal or cisterna magna administration. In some cases, the vector is delivered to the subject by MRI-guided convection-enhanced delivery (CED). This technology enables increased viral diffusion and transduction throughout the large volume of the brain and reduces backflow of the vector along the needle path.

[0425] In various embodiments, a method is provided comprising administering a vector encoding an engineered receptor that inactivates or hyperpolarizes neuronal cells to one or more neuronal cells that increase pain perception or sensitivity to pain, and administering a ligand that specifically binds to neuronal cells expressing the engineered receptor to a subject, thereby inactivating the cells, reducing sensitivity to pain, and enhancing analgesia.

[0426] In various embodiments, a method is provided, comprising administering a vector encoding an engineered receptor that activates or polarizes neuronal cells to one or more neuronal cells that reduce pain perception or sensitivity to pain, and administering a ligand that specifically binds to neuronal cells expressing the engineered receptor to a subject, thereby activating the cells, reducing sensitivity to pain, and enhancing analgesia.

[0427] The formulation of the ligand can be applied to the subject by various routes. Non-limiting examples of methods of administration 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 involve a liquid formulation of an injected ligand. In some cases, administration may involve a solid formulation of an oral delivery ligand. In certain cases, the ligand is administered by oral administration (e.g., pills, tablets, capsules, etc.). In some cases, oral compositions may be administered with food. In another specific case, the ligand is administered by intrathecal injection (i.e., injection into the subarachnoid space of the spinal cord) for delivery to the cerebrospinal fluid (CSF) of the subject. In another specific case, the ligand is administered topically (e.g., skin patch, cream, lotion, ointment, etc.).

[0428] The dosage of the ligand administered to the subject is not subject to absolute restrictions, but will depend on the properties of the composition and its active ingredients and its harmful side effects (e.g., immune responses to antibodies), the subject being treated and the type of condition being treated, and the mode of administration. Typically, the dosage will be a therapeutically effective amount, such as an amount sufficient to achieve the desired biological effect, such as an amount that effectively reduces or attenuates the pain level experienced by the subject. In specific embodiments, the dosage can also be a prophylactic amount or an 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.

[0429] In one embodiment, the ligand is first applied to the subject before the vector is applied. The ligand of the therapeutically effective amount can be applied to the subject at a certain time after the delivery vector. Usually, after the delivery vector, one or more cells of the subject need a period of time to produce the protein (i.e., engineered receptor) encoded by the vector. During this period of time, applying the ligand to the subject may be unhelpful to the subject. In this case, it may be suitable to apply the ligand after a certain amount of engineered receptors are produced by one or more cells of the subject.

[0430] In one embodiment, the ligand is first administered to the subject at about the same time that the vector is administered to the subject.

[0431] In one embodiment, the ligand is first administered after the carrier is applied to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 11 hours or 12 hours, several days, weeks, months or years. In some cases, the ligand of the therapeutically effective amount can be applied to the subject for at least one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or more than 30 days after the delivery of the carrier. In a specific example, the ligand of the therapeutically effective amount is applied to the subject for at least one week after the delivery of the carrier. In another example, the ligand of the therapeutically effective amount is applied to the subject for at least three consecutive days every day.

[0432] The therapeutically effective amount or dose of the ligand disclosed herein can be expressed as mg or μg of the ligand per kg of subject body mass. In some cases, the therapeutically effective amount of the ligand can be 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 5 0μg / kg, about 60μg / kg, about 70μg / kg, about 80μg / kg, about 90μg / kg, about 100μg / kg, about 120μg / kg, about 140μg / kg, about 160μg / kg, about 180μg / kg, about 200μg / kg, about 220μg / kg, about 240μg / kg, about 260μg / kg, about 280μg / kg, about 300μg / kg, about 320μg / kg, about 340μg / kg, about 360μg / kg, about 380μg / kg, about 4 00μg / kg, about 420μg / kg, about 440μg / kg, about 460μg / kg, about 480μg / kg, about 500μg / kg, about 520μg / kg, about 540μg / kg, about 560μg / kg, about 580μg / kg, about 600μg / kg, about 620μg / kg, about 640μg / kg, about 660μg / kg, about 680μg / kg, about 700μg / kg, about 720μg / kg, about 740μg / kg, about 760μg / kg, about 780μg / kg / kg, about 800 μg / kg, 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.

[0433] In certain embodiments, the dose of the ligand administered to a subject is at least about 0.001 micrograms per kilogram (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 kilogram (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 or more mg / kg.

[0434] In certain embodiments, the dose of the 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 intermediate ranges thereof.

[0435] In some aspects, 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, 1000 nM, 2000 nM, 3000 nM, 4000 nM, 5000 nM, 6000 nM, 7000 nM, 8000 nM, 9 ... 00nM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM , 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or more.

[0436] The ligand of a therapeutically effective amount can be administered once or more than once per day. In some cases, the ligand of a therapeutically effective amount is administered as needed (e.g., when pain relief is needed). The ligand can be administered continuously (e.g., every day without interruption for the duration of the treatment regimen). In some cases, the treatment regimen can be less than a week, a week, two weeks, three weeks, a month, or more than a month. In some cases, a 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 at least more than ten consecutive days. In certain cases, a therapeutically effective amount of the ligand is administered for three consecutive days. In some cases, a therapeutically effective amount of a ligand may be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, 10 times a week, 11 times a week, 12 times a week, 13 times a week, 14 times a week, 15 times a week, 16 times a week, 17 times a week, 18 times a week, 19 times a week, 20 times a week, 25 times a week, 30 times a week, 35 times a week, 40 times a week, or more than 40 times a week. In some cases, a therapeutically effective amount of a ligand may be administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, 10 times a day, or more than 10 times a day. In some cases, a therapeutically effective amount of the ligand is administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine 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. The dose of the ligand can be administered to the subject continuously or once, twice, three times, four times, or five times a day, once, twice, three times, four times, five times, six times, or seven times a week, once, twice, three times, or four times a month, once every two months, once every three months, once every four months, once every five months, or once every six months, or once a year, or at even longer intervals. The duration of treatment can last for a 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.

[0437] The subject treated by the methods and compositions disclosed herein can be human or can be a non-human animal. The term "treatment" and its grammatical equivalents used herein generally refer to the use of a composition or method to reduce, eliminate or prevent the symptoms of a disease and include achieving a therapeutic benefit and / or a preventive benefit. Therapeutic benefit means slowing down the progression of the symptoms of the disease or condition being treated, stopping its progression, reversing its progression, or eradicating or alleviating the symptoms. The preventive benefit of treatment includes reducing the risk of a condition, delaying the progression of a condition, or reducing the likelihood of a condition occurring.

[0438] Non-limiting examples of non-human animals include non-human primates, livestock animals, domestic pets, and experimental animals. For example, non-human animals can be apes (e.g., chimpanzees, baboons, gorillas, or orangutans), old world monkeys (e.g., rhesus monkeys), new world monkeys, dogs, cats, bison, camels, cows, deer, pigs, donkeys, horses, mules, llamas, sheep, goats, buffaloes, reindeer, yaks, mice, rats, rabbits, or any other non-human animals. Compositions and methods as described herein are suitable for the treatment of 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 tissues or cell colonies with compositions includes applying the compositions to cell colonies or subjects. In some embodiments, application occurs in vitro, such as by adding the compositions to a cell culture system. In some aspects, application occurs in vivo, such as by applying via a specific route. Wherein more than one composition is to be used, the composition can be used at the same time (e.g., on the same day) by the same route or at different times by the same route. Alternatively, the composition can be used at the same time (e.g., on the same day) by different routes or at different times by different routes.

[0439] The number of times the composition is applied to a subject in need depends on the judgment of the medical professional, the severity of the disease, the disease, and the subject's response to the formulation. In some aspects, the application of the composition occurs at least once. In other aspects, the application occurs more than once, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more in a given time period. The dosage and / or frequency of application each time can be adjusted as needed based on the patient's condition and physiological response.

[0440] In some embodiments, the composition can be administered a sufficient number of times to achieve the desired physiological effect or improvement of the subject's condition. Where the subject's condition does not improve, the composition can be administered chronically, i.e., for a long period of time, including for the duration of the subject's entire life, at the discretion of the physician, to alleviate or otherwise control or limit the symptoms of the subject's disease or condition. Where the subject's condition does improve, the composition can be administered continuously at the discretion of the physician; alternatively, the administered drug dose can be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday varies between 2 days and 1 year, including, 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. The dose reduction during a drug holiday may be 10%-100%, including 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.

[0441] When the composition is applied more than once, each application can be performed by the same actor and / or in the same geographic location. Alternatively, each application can be performed by a different actor and / or in a different geographic location.

[0442] For human and veterinary treatment, the amount of the one or more specific agents administered can depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the one or more specific agents being used; the age, weight, general health, sex and diet of the patient; the time of administration, route of administration and excretion rate of the one or more specific agents being used; the duration of treatment; drugs used in combination or concomitantly with the one or more specific agents being used; the judgment of the prescribing physician or veterinarian; and similar factors known in the medical and veterinary arts. Similarly, the effective concentration of a given composition can depend on a variety of factors, including the age, sex, weight, genetic condition and overall health of the patient or subject.

[0443] All papers, publications and patents cited in this specification are incorporated herein by reference, as if each individual paper, publication or patent was expressly and individually indicated to be incorporated by reference, and incorporated by reference to disclose and describe the methods and / or materials related to the cited publication. However, any references, articles, publications, patents, patent publications and patent applications cited herein are not, and should not be taken as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0444] It is specifically intended that the various features described herein can be used in any combination, unless the context dictates otherwise.

[0445] Unless defined otherwise, 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.

[0446] It should be understood that the above description and the following examples are intended to illustrate rather than limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs.

[0447] Example

[0448] Example 1: Construction of engineered receptors comprising an ion pore domain derived from human GlyRα1, GlyRα2, GlyRα3 or GABArho receptors

[0449] To generate LGICs that conduct anion currents after exposure to non-natural small molecule agonists of human α7-nAChR, chimeric ligand-gated ion channel (LGIC) receptors were constructed according to Table 12, comprising 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.

[0450] Example 2: Characterization of Engineered Receptors Using High-Throughput Fluorescence-Based Plate Screening

[0451] The engineered LGIC receptor in Example 1 was analyzed for its potency against its natural ligand acetylcholine (Ach) and 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 Facinicline / RG3487 (researchgate.net / figure / Molecular-structure-of-RG3487_fig1_47499934).

[0452] An anion reporter assay was used to evaluate the response profile of these engineered receptors to ligands. In this assay, cells expressing a YFP reporter (whose fluorescence is quenched in the presence of anions) were transfected with DNA encoding the engineered receptor of interest. Upon exposure to the ligand, the activated channel will flux anions, which results in a dose-dependent quenching of YFP that can be detected on a plate reader.

[0453] 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 create cells that stably express a mutant YFP (H148Q / I152L) reporter that exhibits enhanced sensitivity to anions. Two days before the assay, cells were split at a density of 20,000 cells / well in 96-well tissue culture plates coated with poly-d-lysine (Thermo Scientific). The next day, cells were transiently transfected with 0.1 μg DNA per well using a standard Fugene 6 protocol (Promega). On the day of the assay, cells were washed twice in 1X extracellular solution (1X ECS: 140mM NaCl, 5mM KCl, 1mM MgCl2, 2mM CaCl2, 10mM HEPES, 10mM glucose, pH 7.2, mOsms 300). After the last wash, 100 μL of 1X ECS was added to the wells and the plates were incubated at 37°C for 30 min. While the plates were incubated, the drugs were diluted to 2X concentration in 1X ECS-NaI (the same components as 1X ECS, except that 140mM NaCl was replaced by 140mM NaI). The plates were then read on a Flexstation3 (Molecular Devices). Each well of the plate was read using a Flexstation3 (Molecular Devices) as follows with 8 wells for 2 min each time: 1) read baseline YFP fluorescence for 17 sec, 2) add 100 μL of ligand, and 3) then measure the change in YFP fluorescence every 1.3 sec for the remainder of the time.

[0454] Figure 1 Provided is a heat map of the percentage quenching of YFP fluorescence after stimulation by various doses of acetylcholine or non-natural ligands as indicated in the figure for cells expressing CODA71 (comprising a GlyRα1 ion pore domain) or CODA1237 (comprising a GlyRα2 ion pore domain). The non-natural ligands tested include AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and Facinicline / RG3487. The positive fluorescence signal indicated by the blue shaded cells indicates that the engineered receptor is activated by the non-natural ligand at this concentration. The results show that the two groups of cells show similar responsiveness to a variety of ligands. Compared with cells expressing CODA71, cells expressing CODA1237 are more responsive to ABT-126, RG3487, and AZD-0328.

[0455] Figure 2Provided are heat maps of the percent quenching of YFP fluorescence after stimulation by various doses of acetylcholine or TC-5619 for cells expressing chimeric LGIC receptors based on CODA1237 containing various amino acid mutations in the ligand binding domain derived from α7-nAChR, as summarized in Table 12. For most of these mutants, the corresponding cell lines exhibited a very rightward shift in the dose required to elicit an acetylcholine response (i.e., a lesser response to acetylcholine), while the response to TC-5619 showed little to no significant shift. CODA75 is a non-responsive chimeric receptor used here as a negative control.

[0456] Figure 3 Provided are heat maps of the percent quenching of YFP fluorescence following stimulation with the indicated doses of acetylcholine in cells expressing the indicated chimeric LGIC receptors. Four chimeric LGIC receptors (CODA1342, CODA1343, CODA1344, and CODA1345, as summarized in Table 12) containing the GlyRα3 ion pore domain were tested and their responsiveness to acetylcholine was compared 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 lower responsiveness to acetylcholine, and cells expressing CODA1343 have higher responsiveness to acetylcholine.

[0457] Table 13 below provides EC50 measurements of various ligands for CODA 1316 using either the fluorescent plate screening method or the manual patch clamp method (as described below in Example 4). CODA 1316 is able to bind to TC-5619 with a low nanomolar EC50.

[0458] Table 13: EC50 of engineered receptors

[0459]

[0460]

[0461] Example 3: Localization of engineered receptors

[0462] Fluorescently labeled α-bungarotoxin, which specifically binds to the engineered receptors, was used to evaluate the efficiency of the engineered receptors disclosed herein being localized to the cell surface, and the surface localization of the engineered receptors was then evaluated by flow cytometry.

[0463] Alpha-bungarotoxin, Alexa Fluor647 conjugated to biotin are purchased from Thermo / Fisher (WalthamMA). In short, HEK-293T or HEK293T cells transduced with Ric3, Nacho and Gcamp6s were plated at a density of 200,00 cells per well, and transfected with Fugene 6 at a ratio of 1:3 DNA to Fugene on the second day. Ric3 and Nacho are chaperone proteins of human α7 nicotinic acetylcholine receptors, and Gcamp6s is a calcium reporter. Flow cytometry was used to analyze cells the next day after transfection. For flow cytometry analysis, transfected HEK293T cells were manually floated, washed and incubated in alpha-bungarotoxin (1:1000) in FACS buffer (2% BSA, 1XPBS without Ca+ and Mg+, and 1X penicillin-streptomycin) for 1 hour. The 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).

[0464] Figure 4A The percentage of HEK293T cells transiently transfected with CODA71, CODA829, CODA1237, CODA1238, CODA1298, CODA1239, CODA1342, CODA1343, CODA1344, or CODA1345 that stained positive for α-bungarotoxin is shown. The design of the LGIC constructs is summarized in Table 12. Figure 4B Shows the Figure 4A The mean fluorescence intensity of α-bungarotoxin-positive cells.

[0465] Figure 4C 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 GCAMPB6s is shown. The design of the LGIC constructs is summarized in Table 12. Figure 4D Shows the Figure 4C The mean fluorescence intensity of α-bungarotoxin-positive cells.

[0466] Figures 4A-4DThe results showed that for cells transfected with chimeric LGICs comprising GlyRα1IPD, according to the comparison between CODA71 and CODA829, the T225I mutation can improve the surface expression level of LGIC in α-bungarotoxin staining-positive cells. For cells transfected with chimeric LGICs comprising GlyRα2IPD, according to the comparison between CODA1237 and CODA1298, the T225I mutation improves the surface expression level of LGIC in α-bungarotoxin staining-positive cells. In addition, chimeric LGIC receptors (CODA1343 and CODA1345) comprising the ion pore domain of GlyRα3 isoform K have a higher percentage of surface expression cells and higher surface expression than the corresponding LGIC receptors (CODA1342 and CODA1344) comprising the ion pore domain of GlyRα3 isoform L.

[0467] Figure 5A The percentage of HEK293T cells transiently transfected with CODA71, CODA409, CODA1237, CODA1297, CODA1298, CODA1299, CODA1300, CODA1301, CODA1302, CODA1303, CODA1304, CODA1305, CODA1306, CODA1308, CODA1309 or CODA1310 that stained positive for α-bungarotoxin is shown. The design of the LGIC constructs is summarized in Table 12. Figure 5B Shows the Figure 5A The mean fluorescence intensity of α-bungarotoxin-positive cells.

[0468] The results showed that CODA1237 had higher surface expression than CODA71, and the T202I mutation further increased the surface expression of the chimeric engineered receptor. Mutations in the ligand binding domain also affected the surface expression level of the chimeric engineered receptor.

[0469] Example 4. Peak current measurements of engineered receptors using IonFlux and manual patch clamp

[0470] Peak current and dose response measurements using IonFlux:

[0471] HEK 293 cells were routinely subcultured in DMEM with 10% fetal bovine serum (ThermoFisher Scientific). 24 hours before transfection with Fugene 6 (Promega, E2691) and plasmid DNA (20 μg DNA in total), cells were plated in 15 cm dishes. 24 hours after transfection, cells were floated with Accutase (Sigma, A6964), then resuspended in serum-free medium (CHO-S-SFMII, ThermoFisher Scientific 12052 has 25mM HEPES, ThermoFisherScientific, 15630080), left to stand for 30-90 min, then loaded onto IonFlux (FluxionBiosciences) and experimented. Recordings were performed in an extracellular solution (ECS) containing (in mM): 140 sodium chloride (S7653), 4 potassium chloride (P9333), 1 magnesium chloride (M2670), 2 calcium chloride (C3881), 10 HEPES (54457) and 10 D-(+)-glucose (G7528) (pH=7.3, using sodium hydroxide, osmolarity=305-315 mOsm). The intracellular solution (ICS) contained (in mM): 140 cesium chloride (289329), 5 potassium fluoride (60238), 2 calcium chloride (P9333), 2 magnesium chloride (M2670), 10 HEPES (54457) and 10 EGTA (3777) (pH=7.2, using cesium hydroxide, osmolarity=290-295 mOsm).

[0472] Drug-induced currents were recorded in overall mode (20 cells were attached to each recording channel). Cells were kept at -60mV and transmembrane currents were recorded at a sampling rate of 1kHz. Tested drugs included acetylcholine chloride (A6625) and other synthetic compounds. Unless otherwise stated, drug doses were applied for 5sec, with 2min washing between each dose application. The holding current was subtracted from the peak to find the current amplitude. The dose was applied in semi-log increments, and the concentration increased until the peak current amplitude stabilized. For each overall channel, the amplitude was normalized to the maximum current. The normalized current amplitude across the channel was averaged, and the data were fitted to the dose-response curve using a nonlinear regression model (Y = bottom + (top-bottom) / (1 + EC50 / X) ^ Hillslope). The maximum peak current (absolute amplitude) from all overall channels was averaged for each receptor, and normalized to the average maximum peak current from CODA71HA transfected and experimented in the same batch.

[0473] Peak current and dose response measurements using manual patch clamp:

[0474] Cell culture: HEK 293 cells were routinely subcultured in DMEM with 10% fetal bovine serum (ThermoFisher Scientific). Cells were plated in six-well dishes 24 hours before transfection with Fugene 6 (Promega, E2691) and plasmid DNA (1 μg DNA in total). 24 hours after transfection, cells were re-plated onto 10 mm PDL and collagen-coated coverslips and then used for patch clamp experiments 24-48 hours later. For dorsal root ganglion neuron experiments, adult rat lumbar ganglia were harvested and dissociated according to standard protocols, cultured on glass coverslips coated with poly-L-lysine and mouse laminin, transduced with lentivirus or AAV6 vectors containing ires-GFP at DIV1, and processed for immunocytochemistry on DIV7. For hippocampal neuron experiments, hippocampi were harvested from embryonic day 18 rat pups or postnatal day 0 mouse pups and isolated according to standard protocols. Cells were cultured on glass coverslips coated with poly-L-lysine, transduced with lentivirus or AAV6 vectors containing ires-GFP at DIV3, and processed for immunocytochemistry at DIV14 (surface expression experiments) or calcein violet staining and imaging at DIV16 (neuronal viability assays). 5-Fluorodeoxyuridine 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 lentiviral vectors driven by the human synapsin promoter at MOI=20 (DRG) or MOI=5 (hippocampus). For lentiviral viability assays, cultures were transduced with lentiviral vectors driven by the ubiquitin promoter at MOI=2 (DRG) or MOI=1 (hippocampus). For all experiments employing AAV6 expression, cultures were transduced with AAV6 vectors driven by the human synapsin promoter at MOI = 2e5 (rat DRG and E18 hippocampus) or MOI = 3e8 (mouse p0 hippocampus).

[0475] Whole-cell patch clamp recording protocol: Cells plated on coverslips were visualized at 10x and 40x on an inverted fluorescence microscope (Olympus). Recordings were performed at RT using a 3-6 MOhm glass patch electrode (Sutter, BF150-86-10) with an Axopatch 200B amplifier and a Digidata 1550B (Molecular Devices). Chemicals were obtained from Sigma except for TC-5619 manufactured by Attenua / Targacept. Unless otherwise stated, recordings were made in an extracellular solution (ECS) containing (in mM): 140 sodium chloride (S7653), 4 potassium chloride (P9333), 1 magnesium chloride (M2670), 2 calcium chloride (C3881), 10 HEPES (54457) and 10 D-(+)-glucose (G7528) (pH = 7.3, using sodium hydroxide, osmolarity = 305-315 mOsm). Perfusion was performed using an 8-line reservoir (AutoMate Scientific) in combination with an 8-channel zero dead volume perfusion pen (AutoMate Scientific). Data were acquired using pClamp software (Molecular Devices). Traces were analyzed in Clampfit (Molecular Devices) or using custom Python code. Plots and statistics were ...

Claims

1. An engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor and comprises (a) a ligand binding domain derived from the first wild-type Cys loop LGIC receptor, and (b) Ion pore domain of the LGIC receptor derived from the second wild-type Cys loop.

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

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

4. The engineered receptor of any one of claims 1-3, wherein the second wild-type Cys-loop LGIC receptor is a chloride-permeable Cys-loop ligand-gated ion channel receptor.

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. 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. The engineered receptor of any one of claims 1-6, wherein the second wild-type Cys-loop LGIC receptor is not glycine receptor alpha 1.

8. The engineered receptor of any one of claims 1-7, 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. The engineered receptor of any one of claims 1-8, 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. The engineered receptor of any one of claims 1-9, wherein the engineered receptor comprises a pre-M1 linker derived from the first wild-type Cys-loop LGIC receptor or the second wild-type Cys-loop LGIC receptor.

11. The engineered receptor of any one of claims 1-9, wherein the engineered receptor comprises 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. The engineered receptor according to 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. The engineered receptor of any one of claims 1-12, wherein part or all of the M2-M3 linker of the ion pore domain is derived from the first wild-type Cys loop LGIC receptor.

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

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

4.

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

17. The engineered receptor of claim 16, wherein greater than 50% of the engineered receptor expressed on the cell surface forms a homomeric ion channel.

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

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

2.

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 to amino acids 166-180 of SEQ ID NO:

2.

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

2.

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

2.

23. The engineered receptor of any one of claims 18-21, 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. The engineered receptor of any one of claims 18-23, 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 human GlyRα1.

25. The engineered receptor of any one of claims 18-24, wherein the engineered receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16 or 33.

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

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

59.

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 to amino acids 172-186 of SEQ ID NO:

59.

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

59.

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

31. The engineered receptor of any one of claims 26-30, wherein the engineered receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 62 or 65.

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

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

69.

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

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

36. The engineered receptor of any one of claims 32-35, 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.

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

39. The engineered receptor of any one of claims 32-38, wherein the engineered receptor comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 63, 66, 70, and 71.

40. The engineered receptor of any one of claims 1-17, wherein the second wild-type Cys-loop LGIC receptor is the human GABA-A receptor ρ1 subunit (GABA-Aρ1).

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

10.

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 to amino acids 198-212 of SEQ ID NO:

10.

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

10.

44. The engineered receptor of any one of claims 40-43, wherein the engineered receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 64 or 67.

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

46. ​​An engineered receptor according to claim 45, wherein 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 human α7-nAChR.

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

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

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

50. An engineered receptor according to any one of claims 3-49, wherein the ligand binding domain comprises a sequence that is 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% identical to amino acids 23-220 of SEQ ID NO:

4.

51. An engineered receptor according to any one of claims 3-50, wherein the ligand binding domain comprises an amino acid substitution at one or more residues of human α7-nAChR, wherein the one or more residues include 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.

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

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

4.

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

4.

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

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

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

65.

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

59. The engineered receptor of claim 58, wherein the engineered receptor is at least 2-fold less potent toward the native ligand than the first wild-type Cys-loop LGIC receptor is toward the native ligand.

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

61. The engineered receptor of any one of claims 1-59, wherein the engineered receptor has a higher potency for a non-natural ligand than the first wild-type Cys loop LGIC receptor has for the non-natural ligand.

62. The engineered receptor of claim 61, wherein the engineered receptor has at least 2-fold greater potency toward the non-natural ligand than the first wild-type Cys-loop LGIC receptor has toward the non-natural ligand.

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

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

65. The engineered receptor of claim 64, wherein the engineered receptor has at least 2-fold greater potency in the presence of a non-natural ligand than the first wild-type Cys-loop LGIC receptor in the presence of the non-natural ligand.

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

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

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

71. The polynucleotide of claim 70, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the engineered receptor.

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

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

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

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

76. A vector comprising the polynucleotide of any one of claims 70-75.

77. The vector of claim 76, wherein the vector is a plasmid or a viral vector.

78. The vector of claim 77, wherein the vector 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 viral vector (HSV-1).

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

80. A composition comprising the engineered receptor of any one of claims 1-69, the polynucleotide of any one of claims 70-75, or the vector of any one of claims 76-79.

81. A pharmaceutical composition comprising the engineered receptor of any one of claims 1-69, the polynucleotide of any one of claims 70-75, or the vector of any one of claims 76-79; and a pharmaceutically acceptable carrier.

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

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

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

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

87. The method of any one of claims 82-86 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. The method of any one of claims 82-87 or the polynucleotide of claim 75, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers.

89. The method of claim 88 or the polynucleotide of claim 75, wherein the neurons are A[beta] afferent fibers.

90. The method of claim 89 or the polynucleotide of claim 75, wherein the A[beta] afferent fibers are damaged A[beta] afferent fibers.

91. The method of claim 89 or the polynucleotide of claim 75, wherein the A[beta] afferent fibers are undamaged A[beta] afferent fibers.

92. The method of any one of claims 82-91 or the polynucleotide of claim 75, wherein the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5.

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

1.

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

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

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. The method of claim 94, wherein the contacting step is performed in vitro or ex vivo.

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

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

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

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

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

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

104. The method of any one of claims 100-102, wherein the neuron is a non-nociceptive neuron.

105. The method of any one of claims 100-104, 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. The method of any one of claims 100-105, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers.

107. The method of claim 106, wherein the neurons are A[beta] afferent fibers.

108. The method of claim 107, wherein the A[beta] afferent fibers are damaged A[beta] afferent fibers.

109. The method of claim 107, wherein the A[beta] afferent fibers are intact A[beta] afferent fibers.

110. The method of any one of claims 100-109, wherein the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5.

111. The method of any one of claims 100-110, wherein the neurons do not express TrpV1, prostatic acid phosphatase, NaV1.

1.

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

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

114. The method of any one of claims 100-113, 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. The method of any one of claims 100-115, wherein the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection.

117. The method of any one of claims 100-116, wherein the engineered receptor is capable of localizing to the cell surface of a neuron.

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

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

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

121. The method of any one of claims 118-120, wherein the nervous system disorder is epilepsy, movement disorders, eating disorders, spinal cord injury, neurogenic bladder, allodynia, spastic disorders, 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. The method of claim 121, wherein the nervous system disorder is allodynia.

123. The method of any one of claims 118-122, 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. 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. The method of any one of claims 118-125, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or to the cisterna magna.

127. The method of any one of claims 118-126, 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 118-127, wherein the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

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

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

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

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

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

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

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

136. The method of any one of claims 131-135, wherein the symptom of the nervous system disorder is pain.

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

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

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

140. The method of any one of claims 137-139, 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. The method of any one of claims 137-140, 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. The method of any one of claims 137-142, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or to the cisterna magna.

144. The method of any one of claims 137-143, 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 137-144, wherein the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

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

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

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

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

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

151. The method of any one of claims 137-150, wherein the subject suffers from allodynia.

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

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

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

155. The method of any one of claims 137-154, 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. The method of any one of claims 137-155, wherein steps (a) and (b) are performed prior to the manifestation of pain in the subject.

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