Compositions and methods for neurological diseases

Abstract

To provide compositions and methods for neurological diseases.SOLUTION: Compositions and methods are provided for modulating the activity of cells using engineered receptors, polynucleotide encoding the engineered receptors, and gene therapy vectors comprising polynucleotides encoding the engineered receptors. These compositions and methods find particular use in modulating the activity of neurons, e.g., in the treatment of disease or in the study of neuronal circuits.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 889,963, filed Aug. 21, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Description of electronically submitted text files The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is "SWCH02901WO-Sequence_Listing". The text file is 200kb, was created on August 21, 2020, and has been submitted electronically via EFS-Web.

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

[0004] Intractable neurological diseases are often associated with abnormally functioning neurons. Attempts to develop therapies to treat these conditions are hampered by the lack of tractable target proteins associated with the disease. For example, unrelieved chronic pain is a significant health problem in the United States and worldwide. According to an Institute of Medicine report, 116 million Americans suffer from pain that lasts for weeks to years, resulting in an estimated annual cost of over $560 million. There is a lack of adequate long-term treatment for chronic pain patients, leading to a significant burden for both society and individuals. Pain is often disabling and, even in the absence of disability, has a significant impact on quality of life. Even when the care delivery context is optimal, including attentive, well-trained physicians, immediate access to opioids, use of adjuvant analgesics, availability of patient-controlled pain medications, and evidence-based use of procedures such as nerve blocks and IT pumps, pain treatment often fails.

[0005] The most commonly used therapy for chronic pain is the application 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 impairment, hallucinations, and other systemic side effects. Despite the widespread use of pharmaceutical drugs, the success rate of their effectiveness in pain relief is remarkably low. In a large randomized trial with various drugs, it was found that only one in two or three patients achieved at least 50% pain relief (Finnerup et al., 2005). A follow-up study using the most developed pharmacological treatments found the same results, indicating that there was no improvement in the effectiveness of drugs for pain (Finnerup et al., Pain, 150(3):573-81, 2010).

[0006] More invasive options for treating pain include nerve blocks and electrical stimulation. Nerve blocks involve local anesthetic injections, usually into the spinal cord, to interrupt pain signals to the brain, with effects lasting only for weeks to months. Nerve blocks are not a recommended treatment option in most cases (Mailis and Taenzer, Pain Res Manag. 17(3):150-158, 2012). Electrical stimulation involves delivering an electrical current to interrupt pain signals. The effects can last longer than nerve blocks, but there are issues with the leads themselves dislocating, becoming infected, breaking, or the batteries running out. According to one review, 40% of patients who received electrical stimulation for neuropathy experienced one or more of these problems with the device (Wolter, 2014).

[0007] The most invasive and least desirable method of managing pain is the complete surgical removal of the nerve or its parts causing the pain. This option is only recommended when the patient has exhausted the former and other less invasive treatments and they are no longer effective. Radiofrequency nerve ablation uses heat to destroy the problematic nerve and provides longer pain relief than nerve blocks. However, one study found no differences between control and treatment groups in partial radiofrequency lesions of the DRG for chronic lumbosacral radicular pain (Geurts et al., 2003). Other surgical methods for surgically removing pain nerves suffer from similar drawbacks and have serious long-term side effects, including sensory or motor impairments, or cause pain elsewhere. Methods for treating neurological disorders should be safe, efficient, and cost-effective. Gene therapy may provide a non-invasive treatment option for various neurological diseases, including pain management. However, gene therapy methods have not been widely used in the treatment of neurological diseases to date. The present disclosure addresses these needs. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Finnerup et al.,Pain,150(3):573-81,2010 [Non-Patent Document 2] Mailis and Taenzer,Pain Res Manag.17(3):150-158,2012 [Brief description of the drawings]

[0009] The disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawings will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0010] [Figure 1A-B] Figures 1A-G show heat maps of the percentage quenching of YFP fluorescence in mutants of engineered chimeric receptor SEQ ID NO:33 containing the indicated double amino acid substitutions following stimulation with various doses of either acetylcholine or the indicated non-natural ligand. Ligand doses are listed across the top of each chart. Numbers in boxes indicate the absolute amount of quenching observed. Dark blue = 80% maximal quenching of the YFP reporter. Light blue = 30-80% quenching. White = 10-30%. Orange = 0-10% quenching. Negative values ​​represent non-responsive with negative quenching due to stimulation artifact. SEQ ID NO:29 is a non-responsive chimera used as a negative control. Abbreviations for non-natural ligand names: abt: ABT-126; ach: acetylcholine; apn: APN-1125; azd: AZD-0328; brd: TC-5619; fac: RG3487; tc6: TC-6987. [Figure 1C-D] Same as above. [Figure 1E-F] Same as above. [Figure 1G] Same as above.

[0011] [Diagram 2] Figures 2A-B show concentration-response curves of CR-11 (chemically elicited receptor-11, an engineered receptor containing an amino acid sequence with amino acid substitutions Y115D and L131Q of SEQ ID NO: 33) expressed in HEK293 cells to acetylcholine and the non-natural ligand RG-3487 (SA-2, synthetic agonist-2). Responses were assessed using manual patch clamp electrophysiology. Currents were normalized to unity relative to the maximal response. The solid lines through the data points are best fits obtained with the Hill equation, and the EC50 for each ligand is estimated from the concentration-response curves. Figure 2A shows the concentration-response curves of wild-type and CR-11 receptors to acetylcholine. Figure 2B shows the concentration-response curves of wild-type and CR-11 receptors to RG-3487 (SA-2).

[0012] [Diagram 3] FIG. 3 shows an exemplary chloride current induced by RG-3487 (SA-2) in adult rat DRG neurons transduced with a lentivirus expressing CR-11 (chemical-induced receptor-11, an engineered receptor comprising an amino acid sequence having amino acid substitutions Y115D and L131Q of SEQ ID NO: 33).

[0013] [Figure 4A] Figure 4A shows evoked action potentials of transduced DRG neurons expressing CR-11 (an engineered receptor containing an amino acid sequence with amino acid substitutions of Y115D and L131Q of SEQ ID NO: 33), or control DRG neurons (no CR-11 expression), upon injection of different currents (50 pA-700 pA). The top panel shows evoked action potentials of control DRG neurons. The bottom left panel shows evoked action potentials of transduced DRG neurons expressing CR-11 in the presence of 3 μM RG-3487 (SA-2). The bottom right panel shows evoked action potentials of transduced DRG neurons expressing CR-11 after RG-3487 (SA-2) has been washed out. [Figure 4B]FIG. 4B shows rheobase values ​​(current required to elicit an action potential) of control and transduced DRG neurons expressing CR-11 in the absence or presence of the indicated ligands.

[0014] [Diagram 5] Figure 5 shows the percentage of HA tag positive cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33 ("Average HA Tag %), and the percentage of α-bungarotoxin positive cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33 ("Normalized AB %). Figure 5 also shows the median fluorescence intensity (MFI) of cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33, assessed using an anti-HA antibody ("Average HA MFI") or fluorescently labeled α-bungarotoxin conjugated to Alexa Fluor 647 ("Normalized AB MFI"). Summary of the Invention [Means for solving the problem]

[0015] The present disclosure provides an engineered receptor derived from human α7 nicotinic acetylcholine receptor (α7-nAChR), comprising a ligand-binding domain comprising a Cys loop domain derived from human glycine receptor α1 subunit, and an ion pore domain derived from human glycine receptor α1 subunit. In some embodiments, the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor. In some embodiments, the ligand-binding domain comprises (i) two amino acid substitutions in a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131, or (ii) an amino acid substitution of L131E, wherein the amino acid residues correspond to those of α7-nAChR. In some embodiments, the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, wherein the amino acid sequence further comprises 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, or an amino acid substitution of L131E, wherein the amino acid residues correspond to amino acid residues in α7-nAChR.

[0016] 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 engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, wherein the amino acid sequence further 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.

[0017] In some embodiments, the ligand binding domain comprises an amino acid substitution of L131E. In some embodiments, the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, wherein the amino acid sequence further comprises an amino acid substitution of L131E.

[0018] In some embodiments, the Cys-loop domain comprises amino acids 166-172 of SEQ ID NO:2. In some embodiments, the Cys-loop domain comprises amino acids 166-180 of SEQ ID NO:2. In some embodiments, the receptor comprises a β1-2 loop domain from the human glycine receptor α1 subunit. In some embodiments, the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:2. In some embodiments, the engineered receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO:58-63. In some embodiments, the engineered receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63.

[0019] In some embodiments, the potency of the engineered receptor for acetylcholine is lower than the potency of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is at least 2 times lower than the potency of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for non-natural ligand is approximately the same as the potency of human α7 nicotinic acetylcholine receptor (α7-nAChR) for non-natural ligand.

[0020] In some embodiments, the potency of the engineered receptor for the non-natural ligand is higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least 2-fold higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, determining the potency comprises determining the EC50.

[0021] In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least 2-fold greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, determining efficacy comprises determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand.

[0022] In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and facinicline / RG3487. In some embodiments, the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and APN-1125. In some embodiments, the non-natural ligand is TC-5619.

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

[0024] The present disclosure provides a vector comprising any one of the polynucleotides disclosed herein. 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 viral (AAV) vector, and a herpes simplex viral vector-1 (HSV-1). In some embodiments, the viral vector is an AVV vector, and the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof.

[0025] The present disclosure provides a composition comprising any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, or any one of the vectors disclosed herein. The present disclosure further provides a pharmaceutical composition comprising any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, or any one of the vectors disclosed herein, and a pharma- ceutically acceptable carrier.

[0026] The present disclosure provides a method of producing an engineered receptor in a neuron, comprising contacting the neuron with any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an injured Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an uninjured Aβ afferent fiber. In some embodiments, the neurons express neurofilament 200 (NF200), Piezo2, and TLR-5. In some embodiments, the neurons do not express TrpV1, prostatic acid phosphatase, NaV1.1.

[0027] 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 the polynucleotide, vector, composition, or pharmaceutical composition to a 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 can be localized to the cell surface of a neuron.

[0028] The present disclosure provides a method of inhibiting activity of a neuron, comprising: (a) contacting the neuron with any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein; and (b) contacting the neuron with a non-native ligand of the engineered receptor. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an injured Aβ afferent fiber. In some embodiments, the Aβ afferent fibers are uninjured Aβ afferent fibers. In some embodiments, the neurons express neurofilament 200 (NF200), Piezo2, and TLR-5. In some embodiments, the neurons do not express TrpV1, prostatic acid phosphatase, NaV1.1.

[0029] 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 step (a) and / or (b) are performed in vivo in a subject. In some embodiments, the contacting step (a) comprises administering an engineered receptor, polynucleotide, vector, or pharmaceutical composition to a subject, and / or the contacting step (b) comprises administering a non-natural ligand to a subject. In some embodiments, the contacting step (a) and / or (b) comprises lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor can be localized to the cell surface of a neuron.

[0030] The present disclosure provides a method of treating and / or delaying the onset of a neurological disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein, and administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand simultaneously with step (a).

[0031] In some embodiments, the neurological disorder is a seizure disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spasticity disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation. In some embodiments, the neurological disorder is allodynia. In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and fascinicline / RG3487.

[0032] In some embodiments, the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionously, intraneurally, intracranially, intraspinal, or cisternal. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally. In some embodiments, the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. In some embodiments, the subject is a human.

[0033] In some embodiments, the therapeutically effective amount reduces the severity of the signs and / or symptoms of a neurological disorder. In some embodiments, the therapeutically effective amount delays the onset of the signs and / or symptoms of a neurological disorder. In some embodiments, the therapeutically effective amount removes the signs and / or symptoms of a neurological disorder. In some embodiments, the signs of a neurological disorder are nerve damage, nerve atrophy, and / or seizures. In some embodiments, the nerve damage is peripheral nerve damage. In some embodiments, the symptoms of a neurological disorder are pain.

[0034] The present disclosure provides a method of treating and / or delaying the onset of pain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein, and administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand simultaneously with step (a). In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487.

[0035] In some embodiments, the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinal, or cisternal. In some embodiments, the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally.

[0036] In some embodiments, the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject.

[0037] In some embodiments, the subject is a human. In some embodiments, the pain is neuropathic pain. In some embodiments, the pain is associated with, caused by, or resulting from chemotherapy. In some embodiments, the pain is associated with, caused by, or resulting from trauma. In some embodiments, the subject suffers from allodynia. In some embodiments, the pain appears after a medical procedure. In some embodiments, the pain is associated with, caused by, or resulting from childbirth or Caesarean section. In some embodiments, the pain is associated with, caused by, or resulting from migraine headaches. In some embodiments, the therapeutically effective amount temporarily relieves the subject's pain, permanently relieves the subject's pain, prevents the onset of the subject's pain, and / or eliminates the subject's pain. In some embodiments, steps (a) and (b) are performed before the subject's pain appears. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] A. Overview Compositions and methods are provided for regulating the activity of cells using engineered ligand-gated ion channel (LGIC) receptors, polynucleotides encoding engineered LGIC receptors, and gene therapy vectors that include polynucleotides encoding engineered LGIC receptors.These compositions and methods find particular use in regulating the activity of neurons, for example, in treating disease or studying neural circuits.In addition, reagents, devices, and kits for use in practicing the subject methods are provided.

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

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

[0041] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which either or both limits are included or not included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any method and material similar or equivalent to those described herein can be used to carry out or test this disclosure, but some potential and preferred methods and materials will now be described.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material related to the one in which the publication is cited.It is understood that this disclosure takes precedence over any disclosure of the incorporated publication to the extent of any discrepancy.

[0043] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0044] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0045] B. Definition 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 a "peptide" includes one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art.

[0046] As used herein, unless otherwise indicated, the term "and / or" is used in this disclosure as either "and" or "or."

[0047] Throughout this specification, unless the context requires otherwise, the term "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of a specified element or integer, or group of elements or integers, but not to the exclusion of other elements or integers, or group of elements or integers. Moreover, throughout this specification, recitations of numerical ranges specifically include all integers and decimal points therebetween.

[0048] Throughout this specification, unless the context requires otherwise, the phrase "consisting essentially of" refers to limitations in scope of the described compositions, methods, or kits to the specified materials or steps that do not materially affect the basic and novel characteristics of the subject disclosure. For example, a ligand binding domain "consisting essentially of" a disclosed sequence has an amino acid sequence of ± about 5 residues of the amino acid sequence of the disclosed sequence, e.g., about 5, 4, 3, 2, or about 1 residue less than the recited binding amino acid residues, or about 1, 2, 3, 4, or 5 residues more than the recited binding amino acid residues, at the boundaries of the sequence.

[0049] Throughout this specification, unless the context requires otherwise, the phrase "consisting of" is meant to exclude from a 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 solely of the disclosed amino acid sequence.

[0050] As used in this application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this application, whether about / approximately or not, are meant to cover any normal variations recognized by those of ordinary skill in the art. In certain embodiments, the term "about" or "approximately" refers to a value in the range of 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 (greater or less) of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such value may exceed 100%).

[0051] As used herein, the term "isolated" refers to material that is substantially or essentially free from components that normally accompany it and that is found in its natural state. In some embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0052] The terms "subject", "individual", and "patient" are used interchangeably herein to refer to vertebrates, such as mammals. A mammal may be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject may be an adult, a teenager, a child (2-14 years), an infant (1 month-24 months), or a newborn (less than 1 month old). In some embodiments, an adult is about 65 years of age or older, or about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman contemplating pregnancy.

[0053] The term "sample" refers to a volume and / or mass of biological material subjected to analysis. In some embodiments, a sample includes a tissue sample, a cell sample, a fluid sample, and the like. In some embodiments, a sample is taken from or provided by a subject (e.g., a human subject). In some embodiments, a sample includes some of the tissue taken from any internal organ, cancerous, precancerous, or noncancerous tumor, brain, skin, hair (including hair roots), eye, muscle, bone marrow, cartilage, white adipose tissue, and / or brown adipose tissue. In some embodiments, a fluid sample includes oral swabs, blood, umbilical cord blood, saliva, semen, urine, peritoneal fluid, pleural fluid, spinal fluid, lung lavage, tears, sweat, and the like. One of skill in the art will recognize that in some embodiments, a "sample" is a "primary sample" in that it is obtained directly from a source (e.g., a subject). In some embodiments, a "sample" is the result of processing a primary sample, for example, to remove certain potential contaminating components, to isolate certain components, and / or to purify certain components of interest. In some embodiments, the sample is a cell or cell population (e.g., neural cells). The cell sample may be derived directly from a subject (e.g., a primary sample) or may be a cell line. The cell line may include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0054] As used herein, "treating" or "treatment" refers to the delivery of a composition (e.g., engineered receptor and / or binding agent) to a subject and / or cell population to affect a physiological outcome. In certain embodiments, treatment results in an improvement (e.g., reduction, improvement, or treatment) of one or more disease symptoms. The improvement may be an observable or measurable improvement, or an improvement in the subject's general sense of well-being. Treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to a reduction in the severity of disease symptoms to a level equivalent to the level 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., persistent or chronic) reduction in disease symptoms. In some embodiments, treatment may refer to the amelioration of disease symptoms. In some embodiments, treatment may refer to a prophylactic treatment administered to a subject at risk of developing a particular disease to prevent the onset of the disease. Preventing the onset of a disease can refer to preventing symptoms of the disease altogether, delaying the onset of the disease, reducing the severity of symptoms of the disease that subsequently develops, or reducing the likelihood of developing the disease.

[0055] As used herein, "management" or "control" refers to the use of the compositions or methods contemplated herein to improve the quality of life of an individual suffering from a particular disease. In certain embodiments, the compositions and methods described herein provide analgesia to a subject suffering from pain.

[0056] A "therapeutically effective amount" is the amount of a composition necessary to achieve a desired therapeutic result. The therapeutically effective amount may vary depending on factors such as, but not limited to, the condition and the age, sex, and weight of the subject. In general, a therapeutically effective amount is one in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes an amount of a composition that is effective to treat a subject.

[0057] "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. Increase also refers to an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (e.g., 500, 1000 times) higher than a reference or control level.

[0058] "Reduction", "reduction", "diminishment" or synonyms thereof refer to a decrease in value (e.g., a decrease in binding affinity, a decrease in physiological response, a decrease in therapeutic effect, a decrease in pain in a subject, etc.) of at least 5% compared to a reference or control level. For example, a decrease 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 can also mean a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (e.g., 500, 1000 times) lower than a reference or control level.

[0059] "Maintained" or "retained" or "sustained" or "no change" or "no substantial change" or "no substantial decrease" generally refers to a physiological and / or therapeutic effect that is comparable to that caused by either a vehicle or a control molecule / composition. A comparable response is one that is not significantly or measurably different from the reference response.

[0060] The terms "reference" or "control" are used interchangeably herein and refer to the value of a particular physiological and / or therapeutic effect in a subject or sample not treated with a composition described herein, or in a subject or sample 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 described herein (e.g., a baseline level).

[0061] As used herein, "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 herein and refer to the physical interaction between a receptor (e.g., LGIC) and a ligand. As used herein, the term "ligand" may refer to an endogenous or naturally occurring ligand. For example, in some embodiments, a ligand refers to a neurotransmitter (e.g., λ-aminobutyric acid (GABA), acetylcholine, serotonin, etc.), and a signal transduction intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., synthetic or non-naturally occurring ligand (e.g., a binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by various methods known in the art (e.g., detection of association with a radiolabeled ligand).

[0063] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a receptor and a ligand. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d Affinity can be measured by common methods known in the art, including those described herein.

[0064] The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims and refer to the binding that occurs between a pair of molecular species, such as, for example, a receptor and a ligand. When the interaction of the two species produces a non-covalent complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. 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 the background binding (non-specific binding), about 5 times the background binding, about 10 times the background binding, about 20 times the background binding, about 50 times the background binding, about 100 times the background binding, or about 1000 times or more the background binding.

[0065] "Signaling" refers to the production of a biochemical or physiological response as a result of ligand binding to a receptor (e.g., as a result of binding of a binding agent to an engineered receptor described herein).

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

[0067] The terms "non-naturally occurring," "variant," and "mutant" are used interchangeably throughout the specification and claims to refer to variants of a native or wild-type composition, e.g., variant polypeptides having less than 100% sequence identity to the native or wild-type sequence.

[0068] The amino acid modification may be an amino acid substitution, an amino acid deletion, and / or an amino acid insertion. The amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative change) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used herein, a "conservative change" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative changes include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another, or the replacement of one polar residue with another, such as the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid, or the replacement of glutamine with asparagine. 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 praline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, etc.

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

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

[0071] Generally, "sequence identity" or "sequence homology" refers to the nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health. The BLAST program is based on the alignment method as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percent identity over the entire length of the proteins being compared. Default parameters are provided, for example, in the blastp program to optimize searches of short query sequences.The program can also use a SEG filter to mask off 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%, with intervening integer values. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0072] As used herein, "substantially identical" refers to having 85% or more, e.g., 90% or more, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity, where the activity of the composition is not altered by modifications of the sequence that result in differences in sequence identity.

[0073] As used herein, the term "promoter" refers to one or more nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the 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 linkage between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0074] As used herein, the term "virus vector", "viral vector", or "gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors of the present disclosure include adenovirus vectors, adeno-associated virus vectors (AAV), lentivirus vectors, and retrovirus vectors.

[0075] As used herein, "neuronal activity," "neuronal activity," "neuronal firing," and variations and synonyms thereof, refer to electrical activity resulting from stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch clamp techniques. In some embodiments, determining neuronal activity includes determining excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), and / or neuronal action potentials. In some embodiments, the level of neuronal activity is dependent on or influenced by the excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), and / or action potentials.

[0076] As used herein, "neurological disease" or "neurological disorder" refers to a disease or disorder of the nervous system. In some embodiments, a neurological disease is associated with, caused by, or results from structural, biochemical, and / or electrical abnormalities in the brain, spinal cord, nerves, or any component of the nervous system.

[0077] As used herein, a "sign" of a disease refers to a physical or mental characteristic that is considered to be indicative of the pathology of the disease. In some embodiments, the sign is an objective sign of the disease. In some embodiments, the sign is objectively assessed, examined, observed, or measured by someone other than the patient, such as a physician.

[0078] As used herein, a "symptom" of a disease refers to a physical or mental characteristic that is considered to be indicative of the pathology of the disease, particularly such characteristic that is evident to the patient. In some embodiments, a symptom is subjectively assessed by the patient. For example, in some embodiments, a symptom is pain.

[0079] As used herein, "potency" refers to the amount of ligand required to produce a particular level of activity of a protein, such as LGIC. In some embodiments, the activity of a protein, such as LGIC, refers to the opening and closing of an ion channel. In some embodiments, determining the potency includes determining the half-maximal effective concentration (EC50) of a protein, such as LGIC, for a ligand under particular conditions. EC50 refers to the concentration of ligand that induces a response halfway between baseline and maximum after a particular exposure time.

[0080] As used herein, "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 passing through the LGIC under certain conditions, such as in the presence of a particular concentration of a ligand. In some embodiments, determining efficacy includes determining the amount of current passing through a receptor and / or the rheobase of the receptor.

[0081] As used herein, "responsiveness" refers to a measure of the overall function of a protein, such as a LGIC, in the presence of a ligand. Determining responsiveness may include determining and considering one or more factors, such as potency, efficacy, and intracellular localization of the protein.

[0082] C. Engineered Receptors The present disclosure is directed to engineered receptors, engineered receptor variants, and methods for their use. As used herein, the term "receptor" refers to any protein located on the surface of a cell and capable of mediating signaling to and / or from a cell. The term "engineered receptor" is used herein to refer to a receptor that has been experimentally altered to be physically and / or functionally different from the corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. The term "wild-type receptor" is used herein to refer to a receptor that has a polypeptide sequence identical to that of a naturally occurring protein. Wild-type receptors include receptors that naturally occur in humans, as well as orthologs that naturally occur in other eukaryotes, e.g., protists, fungi, plants, or animals, e.g., yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates. In some embodiments, the parent receptor is a non-natural receptor, i.e., a receptor that does not occur in nature, e.g., a receptor that is engineered from a wild-type receptor. For example, the parent receptor may be an engineered receptor that includes one or more subunits from one wild-type receptor and one or more subunits from a second wild-type receptor. Thus, the resulting protein is composed of 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 receptors, parent receptor mutants, and switch receptors.

[0083] In some aspects, the engineered receptors of the present disclosure include at least one amino acid mutation relative to the corresponding parent receptor, e.g., one or more mutations in one or more domains of the wild-type receptor. By "amino acid mutation" is meant any difference in amino acid sequence relative to the corresponding parent sequence, e.g., amino acid substitution, deletion, and / or insertion. In some embodiments, 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 corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the parent receptor mutant has 85% or more, e.g., 90% or more or 95% or more, e.g., about 96%, about 97%, about 98%, or about 99% sequence identity with the corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the engineered receptor (e.g., parent receptor mutant) is generated by error-prone PCR.

[0084] 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 compared to the parent receptor, for example, by reducing the binding of an endogenous ligand to the engineered receptor compared to the binding of the endogenous ligand to the parent receptor, or by reducing the activity of a downstream signaling pathway of the engineered receptor that is typically activated in response to binding of a binding agent to the corresponding parent receptor.

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

[0086] In some embodiments, the loss-of-function mutation and the gain-of-function mutation are at the same residue, i.e., 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, a subject engineered receptor comprising a loss-of-function mutation and / or a gain-of-function mutation shares about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity with a corresponding parent receptor, e.g., a wild-type receptor or a non-natural receptor. In some embodiments, a subject engineered receptor shares 85% or more sequence identity, e.g., 85%, 90%, or 95% or more sequence identity, in some cases 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity, with a corresponding parent receptor, including all values ​​and subranges therebetween.

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

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

[0089] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGICs refer to a large group of transmembrane proteins that allow the passage of ions upon activation by a specific ligand (e.g., a chemical or binding agent). LGICs are composed of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Ligand binding to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding produces an abrupt change in the permeability of the channel to a specific ion or ions. Effectively, ions cannot pass through the channel when it is in the inactive or closed state, but can increase in permeability by up to 10 ions upon ligand binding. 7 ions / sec can pass through. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and promote the influx of ions into the cytosol. In some embodiments, LGICs respond to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and promote the efflux of ions from the cytosol to the extracellular environment. Importantly, activation of LGICs promotes the passage of ions (e.g., Ca) across the cell membrane. 2+ , Na + , K + , Cl - etc.), but not the ligand itself.

[0090] LGIC receptors are composed of multiple subunits and can be either homomeric or heteromeric receptors. Homomeric receptors are composed of subunits that are all of the same type. Heteromeric receptors are composed of at least one subunit that is different from at least one other subunit contained within the receptor. For example, glycine receptors are composed of five subunits and come in two types: α-subunits, which have four isoforms (α1-α4), and β-subunits, which have one known isoform. An exemplary homomeric GlyR is a GlyR composed of five α1-GlyR subunits. Similarly, homomeric GABA receptors are composed of two types: α-subunits, which have four isoforms (α1-α4), and β-subunits, which have one known isoform. A The receptor is β3-GABA ASubunits, nAchR receptors may be composed of α7-nAchR subunits. Exemplary heteromeric GlyRs may be composed of one or more α-subunits and one or more β-subunits (e.g., α1β-GlyR). Examples of LGIC receptor subunits are shown in Table 1. [Table 1-1] [Table 1-2]

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

[0092] Specific non-limiting examples of LGICs suitable for use 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;GRIA4;GRID1;GRID2; GRIK1;GRIK2;GRIK3;GRIK4;GRIK5;GRIN1;GRIN2A;GRIN2B;GRIN2C;GRIN2D;GRIN3A;GRIN3B;ITPR1;ITPR2;ITPR3;CHRNA1;CHRNA2;CHRNA3;CHRNA4;CHRNA5;CHRNA6;CHRNA7;CHRNA9;CHRNA10;CHRNB1;CHRNB2;CHRNB3;CHRNB4;CHRNG;CHRND;CHRNE;P2RX1;P2RX2;P2RX3;P2RX4;P2RX5;P2RX6;P2RX7;RYR1;RYR2;RYR3; and ZACN.

[0093] TRPV1, TRPM8, and P2X2 are members of the large LGIC family that share structural features and gating principles.For example, TRPV4, like TRPV1, is also triggered by heat, but not by capsaicin, and P2X3 is triggered by ATP, but desensitizes more rapidly than P2X2.Therefore, TRPV1, TRPM8, and P2X2 are non-limiting examples of LGICs suitable for use in certain embodiments.

[0094] In one embodiment, the engineered receptor is the TRPV1 or TRPM8 receptor or a mutant protein thereof. TRPV1 and TRPM8 are vanilloid and menthol receptors expressed by nociceptive neurons in the peripheral nervous system. Both channels are believed to function as nonselective, sodium- and calcium-permeable homotetramers. Furthermore, both channels and their main agonists, cold-sensing compounds such as capsaicin and menthol, respectively, are virtually absent from the central nervous system. Some cold-sensing compounds, including capsaicin and menthol and icilin, contain potential acceptor sites for photosensitive blocking groups. The association of photosensitive blocking groups with such acceptors results in ligand-gated ion channels in which light acts as an indirect trigger by releasing the active ligand.

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

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

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

[0098] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor alpha 7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 4) encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5 (SEQ ID NO: 3). [ka]

[0099] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3A (5HT3A, GenBank Accession No. NP_998786.2, SEQ ID NO: 6), encoded by the HTR3A gene (GenBank Accession No. NM_213621.3 (SEQ ID NO: 5). [ka]

[0100] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3B (5HT3B GenBank Accession No. NP_006019.1, SEQ ID NO: 57) encoded by the HTR3B gene (GenBank Accession No. NM_006028.4 (SEQ ID NO: 56). [ka]

[0101] In some embodiments, the wild-type LGIC receptor is the human gamma-aminobutyric acid receptor A (GABA-A), subunit beta-3 (GABA-A β3) (GenBank Accession No. NP_000805.1, SEQ ID NO: 8) encoded by the GABRB3 gene (GenBank Accession No. NM_000814.5 (SEQ ID NO: 7)). [ka]

[0102] In some embodiments, the wild-type LGIC receptor is human GABA-A encoded by the GABRR1 gene (GenBank Accession No. NM_002042.4 (SEQ ID NO: 9) subunit rho1 (ρ1) (GABA-A ρ1) (GenBank Accession No. NP_002033.2, SEQ ID NO: 10). [ka]

[0103] In some embodiments, the wild-type LGIC receptor is human GABA-A encoded by the GABRR2 gene (GenBank Accession No. NM_002043.4 (SEQ ID NO: 11), subunit rho2 (ρ2) (GABA-A ρ2) (GenBank Accession No. NP_002034.3, SEQ ID NO: 12). [ka]

[0104] In some embodiments, the wild-type LGIC receptor is human GABA-A encoded by the GABRR3 gene (GenBank Accession No. NM_001105580.2 (SEQ ID NO: 13), subunit rho3 (ρ3) (GABA-A ρ3) (GenBank Accession No. NP_001099050.1, SEQ ID NO: 14). [ka]

[0105] In some aspects, the subject engineered receptor is a chimeric receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence from at least a first LGIC and an ion pore conduction domain sequence from at least a second LGIC, or more simply, an "ion pore domain sequence". In some embodiments, the first and second LGIC are Cys-loop receptors. The ligand binding domain sequence and the ion pore domain sequence of Cys-loop receptors are well known in the art and can be easily identified from the literature by using public software such as PubMed, Genbank, Uniprot, etc. In the above sequences, the ligand binding domain is shown in bold and the ion pore domain is underlined.

[0106] In some embodiments, the ligand binding domain of the chimeric receptor comprises 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 approximately amino acids 29-235 of GlyRα1, e.g., amino acids 29-235, amino acids 29-240, amino acids 29-246, amino acids 29-248, amino acids 29-250, or amino acids 29-252 of SEQ ID NO:2. In certain such embodiments, the ligand binding domain consists essentially of amino acids 29-235 of SEQ ID NO:2, essentially of amino acids 29-240 of SEQ ID NO:2, essentially of amino acids 29-246 of SEQ ID NO:2, essentially of amino acids 29-248 of SEQ ID NO:2, essentially of amino acids 29-250 of SEQ ID NO:2, or essentially of amino acids 29-252 of SEQ ID NO:2. In some embodiments, the ion pore domain sequence is from a Cys-loop receptor other than human GlyRα1.

[0107] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-AChR. In some such embodiments, the ligand binding domain comprises approximately amino acids 23-220 of α7-nAChR (SEQ ID NO: 4), e.g., amino acids 23-220, amino acids 23-226, amino acids 23-229, amino acids 23-230, and in some cases, amino acids 23-231 of SEQ ID NO: 4. In certain such embodiments, the ligand binding domain consists essentially of amino acids 23-220 of SEQ ID NO: 4, essentially of amino acids 23-226 of SEQ ID NO: 4, essentially of amino acids 23-229 of SEQ ID NO: 4, essentially of amino acids 23-230 of SEQ ID NO: 4, or essentially of amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ion pore domain sequence is from a Cys-loop receptor other than the human α7-nAChR.

[0108] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ligand binding domain comprises approximately 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, essentially of amino acids 23-245 of SEQ ID NO:6, essentially of amino acids 30-247 of SEQ ID NO:6, essentially of amino acids 23-250 of SEQ ID NO:6, and 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 particular 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, or consists essentially of amino acids 21-245 of SEQ ID NO:57. In some embodiments, the ion pore domain sequence is derived from a Cys-loop receptor other than the human 5-hydroxytryptamine receptor 3.

[0109] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-A β3. In some such embodiments, the ligand binding domain comprises approximately amino acids 26-245 of GABA-A β3 (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, essentially of amino acids 26-245 of SEQ ID NO:8, essentially of amino acids 26-248 of SEQ ID NO:8, essentially of amino acids 26-250 of SEQ ID NO:8, or essentially of amino acids 26-255 of SEQ ID NO:8. In some embodiments, the ion pore domain sequence is from a Cys-loop receptor other than human GABA-A.

[0110] In some embodiments, the ion pore domain to which the ligand binding domain is fused conducts anions, e.g., it comprises the ion pore domain sequence of the human glycine receptor or the human serotonin receptor, In other embodiments, the ion conducting pore domain to which the ligand binding domain is fused conducts cations, e.g., it comprises the ion pore domain sequence of the human acetylcholine receptor or the human gamma-aminobutyric acid receptor A.

[0111] In some embodiments, the ion pore domain comprises an ion pore domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some such embodiments, the ion pore domain comprises approximately amino acids 245-457 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 240-457, amino acids 245-457, amino acids 248-457, amino acids 249-457, amino acids 250-457, amino acids 255-457, or amino acids 260-457 of SEQ ID NO:2. In certain such 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.

[0112] In some embodiments, the ion pore domain comprises an ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-AChR. In some such embodiments, the ion pore domain comprises approximately amino acids 230-502 of α7-nAChR (SEQ ID NO:4), e.g., 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, essentially of amino acids 230-502 of SEQ ID NO:4, essentially of amino acids 231-502 of SEQ ID NO:4, essentially of amino acids 232-502 of SEQ ID NO:4, or essentially of amino acids 235-502 of SEQ ID NO:4.

[0113] In some embodiments, the ion pore domain comprises an ion pore domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ion pore domain comprises approximately 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, essentially of amino acids 245-516 of SEQ ID NO:6, essentially of amino acids 248-516 of SEQ ID NO:6, essentially of amino acids 250-516 of SEQ ID NO:6, or essentially of amino acids 253-516. In some such embodiments, the ion pore domain comprises approximately 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.

[0114] In some embodiments, the ion pore domain comprises an ion pore domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-A β3. In some such embodiments, the ion pore domain comprises approximately amino acids 246-473 of GABA-A β3 (SEQ ID NO:8), e.g., amino acids 240-473, amino acids 245-473, amino acids 247-473, amino acids 250-473, or amino acids 253-473 of SEQ ID NO:8. In certain such embodiments, the ion pore domain consists essentially of amino acids 240-473 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.

[0115] 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 adjacent at its amino (N) terminus to the C-terminus of the transmembrane domain 2 (M2) of the receptor and adjacent at its carboxy (C) terminus to the N-terminus of the transmembrane domain 3 (M3) of the receptor. The M2-M3 linker of the LGIC can be easily determined by using any protein analysis tool known in the art and / or published, such as Expasy, uniProt, etc. Typically, 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, where a subject ligand-gated ion channel comprises a ligand binding domain from AChR and an ion pore domain from GlyR, the subject ligand-gated ion channel can comprise the ion pore domain sequence from GlyR, except for the M2-M3 linker, which would instead be from AChR. In some embodiments, the ion pore domain is from GlyRα1 and the M2-M3 linker is from α7-nAChR. In some embodiments, the M2-M3 linker sequence removed from GlyRα1 is approximately amino acids 293-311 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 304-310, 293-306, 298-310, 305-311, etc. In some such embodiments, the inserted M2-M3 linker is approximately amino acids 281-295 of the α7-nAChR (SEQ ID NO:4), e.g., amino acids 290-295, 281-290, 281-295, 287-292, etc., or a sequence having about 95% or greater identity to amino acids 281-295 of the α7-nAChR.

[0116] 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 loop structure flanked by cysteines at the N-terminus and C-terminus. Without intending to be bound by theory, it is believed that upon binding of a ligand to the ligand-binding domain, the Cys-loop conformationally moves closer to the M2-M3 loop, which mediates the biophysical translation of ligand binding in the extracellular domain to signal in the ion pore domain (reviewed in Miller and Smart, Trends in Pharmacological Sci 2009:31(4)). Substitution of the endogenous Cys-loop sequence with a heterologous Cys-loop sequence may increase the conductivity of the LGIC by 1.5-fold or more, e.g., at least 2-fold, 3-fold, or 4-fold, in some cases at least 5-fold or 6-fold, and at certain doses at least 7-fold, 8-fold, 9-fold, or 10-fold. The Cys-loop domain of a Cys-loop receptor can be easily determined by using any protein analysis tool known in the art and / or published, e.g., Expasy, uniProt, etc. Typically, when the ligand-binding domain of a chimeric receptor comprises a heterologous Cys-loop sequence, the Cys-loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when a subject chimeric ligand-gated ion channel comprises a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the subject ligand-gated ion channel can comprise the ligand-binding domain sequence derived from AChR, except for the sequence of the Cys-loop domain, which is instead derived from GlyR. In some embodiments, the ligand binding domain is from α7-nAChR and the Cys-loop sequence is from GlyRα1.In some such embodiments, the Cys-loop sequence removed from α7-AChR is about amino acids 150-164 of α7-nAChR (SEQ ID NO:4), e.g., amino acids 150-157 of α7-nAChR. In some such embodiments, the Cys-loop sequence inserted is about amino acids 166-180 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 166-172 of GlyRα1, or a sequence having about 95% or greater identity to amino acids 166-180 of GlyRα1.

[0117] 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 that is flanked at its N-terminus by the C-terminus of the β1 sheet and at its C-terminus by the N-terminus of the β2 sheet. Without intending to be bound by theory, it is believed that the β1-2 loop serves to mediate the biophysical translation of ligand binding to the ion pore domain in the extracellular domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). Upon ligand binding, it is believed that the β1-2 loop, together with the Cys-loop, in close proximity to the M2-M3 loop, mediates the biophysical translation of ligand binding in the extracellular domain and signal transduction in the ion pore domain in which the M2-M3 loop resides (reviewed in Miller and Smart, supra). Substitution of the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence may increase the conductivity of the LGIC by 1.5-fold or more, e.g., at least 2-fold, 3-fold, or 4-fold, in some cases at least 5-fold or 6-fold, and at certain doses at least 7-fold, 8-fold, 9-fold, or 10-fold. The β1-2 loop of a Cys-loop receptor can be easily determined by using any protein analysis tool known in the art and / or published, e.g., Expasy, uniProt, and the like. Typically, when the ligand binding domain of a chimeric receptor comprises a heterologous β1-2 loop sequence, the β1-2 loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when a subject chimeric ligand-gated ion channel comprises a ligand binding domain derived from AChR and an ion pore domain derived from GlyR, the subject ligand-gated ion channel can comprise the ligand binding domain sequence from AChR, except for the sequence of the β1-2 loop domain, which is instead derived from GlyR. In some embodiments, the ligand binding domain is from α7-nAChR and the β1-2 loop sequence is from GlyRα1.In some embodiments, the β1-2 loop sequence removed from α7-AChR is about amino acids 67-70 of α7-nAChR (SEQ ID NO:4), e.g., amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the β1-2 loop sequence inserted is about amino acids 79-85 of GlyRα1 (SEQ ID NO:2), e.g., amino acids 81-84, 79-85, or 81-84 of GlyRα1, or a sequence with about 95% or greater identity to amino acids 79-85 of GlyRα1.

[0118] Non-limiting examples of sequences of chimeric LGIC receptors of the present disclosure include the sequences disclosed herein as SEQ ID NO: 15 to SEQ ID NO: 52. In some embodiments, the chimeric LGIC receptor or a polynucleotide encoding it has 85% or more sequence identity to the sequences provided herein in SEQ ID NO: 15 to SEQ ID NO: 52, e.g., 90% or more, 93% or more, or 95% or more, i.e., about 96%, about 97%, about 98%, about 98%, about 99%, or about 100% sequence identity to the sequences provided herein in SEQ ID NO: 15 to SEQ ID NO: 52. In the sequences, the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is underlined.

[0119] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 junction) that contains the human α7-AChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0120] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (R228 junction) chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0121] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (V224 junction) chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0122] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (Y233 junction) chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0123] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 junction) that contains the human α7-AChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined) containing the α7-nAChR M2-M3 linker (lowercase): [ka] [ka]

[0124] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-AChR signal peptide (italics) and a ligand binding domain (bold) containing the 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 SEQ ID NO:33.

[0125] [ka]

[0126] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) containing the GlyRα1 β1-2 loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0127] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) containing the GlyRα1 β1-2 loop sequence (lowercase) and Cys-loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0128] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) containing the GlyRα1 β1-2 loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined) containing the human α7-nAChR M2-M3 linker (lowercase): [ka]

[0129] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, comprising the human α7-AChR signal peptide (italics) and ligand binding domain (bold) containing the GlyRα1 Cys-loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined) containing the human α7-nAChR M2-M3 linker (lowercase): [ka]

[0130] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (R241 junction) that contains the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0131] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (V236 junction) that contains the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0132] In some embodiments, the chimeric LGIC receptor is a GABRB3 / GLRA1 chimera (Y245 junction) that contains the human GABA-A β3 signal peptide (italics) and the ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0133] As noted above, in some aspects, a subject engineered receptor comprises at least one amino acid mutation that alters the potency of a ligand for the engineered receptor compared to that of the non-mutated parent receptor. In other words, the one or more amino acid mutations, e.g., loss-of-function or gain-of-function mutations, alter the responsiveness of the engineered receptor to a ligand compared to the responsiveness of the non-mutated parent receptor. In some such embodiments, the one or more mutations are within the ligand-binding domain of the engineered receptor. In some embodiments, as in the case where the ligand-binding domain of the engineered receptor is a Cys-loop receptor protein, the one or more amino acid mutations are substitutions at a residue corresponding to a residue in α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213, and Y217. In some embodiments, one residue is substituted. In some embodiments, 2, 3, 4, or 5 or more residues are substituted, for example, 6, 7, 8, 9, or 10 residues are substituted. In certain embodiments, the residue corresponds to a residue of α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence.

[0134] In some embodiments, the one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine and non-natural ligands, for example, by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In certain embodiments, the one or more substitutions correspond to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of the α7-nAChR. In other cases, the one or more substitutions selectively reduce the potency of acetylcholine at the engineered receptor. In other words, the one or more substitutions reduce the responsiveness of the engineered receptor to acetylcholine while essentially maintaining the responsiveness to the non-natural ligand, or reduce the responsiveness of the engineered receptor to acetylcholine by more than two-fold, e.g., three-fold, four-fold, five-fold, or even ten-fold, twenty-fold, fifty-fold, or one hundred-fold, more than it otherwise reduces the responsiveness of the engineered receptor to the non-natural ligand. Exemplary substitutions include those corresponding to L131E, L131S, L131T, L131D, or S172D of the α7-nAChR. In yet other embodiments, the one or more substitutions selectively reduce the potency of the non-natural ligand on the engineered receptor. In other words, the substitution or substitutions reduce the responsiveness of the engineered receptor to a non-natural ligand while essentially maintaining responsiveness to acetylcholine, or reduce the responsiveness of the engineered receptor to a non-natural ligand by more than two-fold, e.g., more than three-fold, more than five-fold, and in some cases more than ten-fold, more than twenty-fold, or more than fifty-fold, than it would otherwise reduce the responsiveness of the engineered receptor to acetylcholine. Exemplary substitutions include those corresponding to W77M, Y115W, S172T, or S172C of the α7-nAChR.In certain embodiments, the one or more substitutions are in the α7-nAChR sequence, hi certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126, and facinicline / RG3487.

[0135] In other embodiments, the substitution or substitutions increase the responsiveness of the engineered receptor to acetylcholine and / or the non-natural ligand, 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. Exemplary substitutions include those corresponding to L131N, L141W, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S172C, S188I, S188V, S188F, S188M, S188Q, S188T, S188P, or S188W. In some cases, the substitution or substitutions increase the potency of both acetylcholine and the non-natural ligand, 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 the α7-nAChR. In other cases, the substitution or substitutions selectively increase the potency of acetylcholine for the engineered receptor. In other words, one or more substitutions, such as those corresponding to L141W, S172T, S172C, S188P, or S188W of the α7-nAChR, increase the responsiveness of the engineered receptor to acetylcholine by more than two-fold, such as three-fold, four-fold, or five-fold, and in some cases, ten-fold, twenty-fold, fifty-fold, or one hundred-fold, more than it increases the responsiveness of the engineered receptor to a non-natural ligand. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126, and facinicline / RG3487. In still other cases, the one or more substitutions selectively increase the potency of the non-natural ligand for the engineered receptor. In other words, the substitution or substitutions increase the responsiveness of the engineered receptor to a non-native ligand by more than two-fold, e.g., three-fold, five-fold, or more, and in some cases, ten-fold, twenty-fold, or even fifty-fold, more than it increases the responsiveness of the engineered receptor to acetylcholine.

[0136] In some embodiments, the amino acid residue mutated in a subject engineered receptor is not an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7 nAChR (SEQ ID NO: 4). In some embodiments, the amino acid residue mutated in a subject engineered receptor is an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7 nAChR (SEQ ID NO: 4). In some embodiments, the substitution is not corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution corresponds to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, such substitutions, when present in an engineered receptor, are present in combination with one or more of the amino acid mutations described herein.

[0137] For example, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 4) have been found to mediate the binding of natural ligand acetylcholine. Mutations at these residues reduce the binding of acetylcholine and are therefore loss-of-function mutations. In contrast, residues W77, Y115, N129, V130, L131, Q139, L141, S170, Y210, C212, C213, and Y217 of α7-nAChR mediate the binding of non-natural ligand AZD0328 to this receptor, and mutations at these residues may increase the affinity of AZD0328 and / or other ligands to this receptor and therefore may be gain-of-function mutations. In some embodiments, a subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain region of the α7-nAChR (SEQ ID NO: 4), or in the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of the α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213, and Y217. In certain embodiments, the mutation in one or more amino acid residues in the ligand binding domain region of α7-nAChR (SEQ ID NO: 4), or in the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of α7-nAChR, is a substitution with one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213, and Y217.

[0138] As another example, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of the α7-nAChR (SEQ ID NO: 4) mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues have been found to reduce the binding of acetylcholine and / or nicotine. R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of the α7-nAChR mediate the binding of the non-natural ligand ABT126, and mutations at one or more of these residues are expected to increase the affinity of ABT126 and / or other ligands for the α7-nAChR. R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR mediate binding of the non-natural ligand TC6987, and mutation of one or more of these residues is expected to increase the affinity of TC6987 and / or other ligands for the α7-nAChR. R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR mediate binding of the non-natural ligand facinicline / RG3487, and mutation of one or more of these residues is expected to increase the affinity of facinicline / RG3487 and / or other ligands for the α7-nAChR. In some embodiments, a subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain region of an a7-nAChR or in the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of an a7-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 binding of acetylcholine and / or nicotine to the a7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212, and Y217 of the a7-nAChR.In certain such embodiments, the amino acid is selected from C212 and S170. In some embodiments, the mutation in one or more amino acid residues alters the binding of ABT126 to the α7-nAChR, and 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 the α7-nAChR. In certain such embodiments, the amino acid is selected from R101, S188, and Y210. In some embodiments, the mutation in one or more amino acid residues alters the binding of TC6987 to the α7-nAChR, and the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210 and C213. In some embodiments, the mutation in one or more amino acid residues alters the binding of fascinicline / RG3487 to the a7-nAChR, and the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the a7-nAChR. In certain such embodiments, the amino acid is selected from Y210, R101, and N129.

[0139] As another example, it has been found that residues W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3 (SEQ ID NO: 6) mediate the binding of serotonin, and mutations at one or more of these residues reduce the binding of serotonin to 5HT3. It is predicted that D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3 mediate the binding of the non-natural ligand silansetron, and mutations at one or more of these residues increase the affinity of silansetron and / or other ligands for 5HT3. In some embodiments, a subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain region of 5HT3A, or in the ligand binding domain of a chimeric receptor comprising a 5HT3 ligand binding domain region, 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 E231. In some embodiments, the mutation in one or more amino acid residues alters the binding of serotonin to 5HT3, and the 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, and the one or more amino acid residues are selected from the group consisting of D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3A.In certain such embodiments, the amino acids are selected from D64, I66, Y89, N123, T176, W190, R191, F221, E224, and E231.

[0140] In some embodiments, one or more mutations that affect the ability of a ligand to regulate the activity of a LGIC are located within the ion pore domain of the LGIC. For example, residue T279 of the serotonin receptor 5HT3A mediates how ligands regulate the activity of the channel, such that mutation of this residue, for example to serine (T279S), converts its effect from antagonistic (i.e., reducing the activity of the LGIC) to agonistic (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 ion pore domain of 5HT3A, where the substitution is within the amino acid corresponding to 279 of SEQ ID NO: 6. In certain embodiments, the substitution is a T279S substitution for SEQ ID NO: 6.

[0141] The present disclosure provides engineered receptors that have 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 that comprises the amino acid sequence of SEQ ID NO: 33.

[0142] In some embodiments, the two amino acid substitutions are at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of L131S and S172D, L131T and S172D, L131D and S172D, Y115D and S170T, Y115D and L131Q, and Y115D and L131E. In some embodiments, the ligand binding domain comprises an amino acid substitution at L131E.

[0143] In some embodiments, the potency of the engineered receptor for acetylcholine is lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is at least about 1.5 times (e.g., about 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) lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine.

[0144] In some embodiments, the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least about 1.5 times higher (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) than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, determining the efficacy comprises determining the EC50.

[0145] In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least about 1.5 times (e.g., about 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) greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, determining efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand.

[0146] In some embodiments, the subject ligand-gated ion channel comprises one or more non-desensitizing mutations. When used in the context of a ligand-gated ion channel, "desensitization" refers to a progressive decrease in ion flux in the prolonged presence of an agonist. This results in a gradual loss of neuronal responsiveness to the ligand. A non-desensitizing mutation refers to an amino acid mutation that prevents the LGIC from being desensitized to the ligand, thereby making the neuron less responsive or unresponsive to the ligand. A non-desensitizing mutation can be easily identified by introducing a LGIC carrying the mutation into a neuron and analyzing the current flow over time during prolonged exposure to the ligand. If the LGIC does not contain a non-desensitizing mutation, the current recovers from the peak to a steady state during prolonged exposure, whereas if the LGIC contains a non-desensitizing mutation, the current remains at the peak flux during the period of exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (V294L post-processing of the proprotein to remove the signal peptide) and the L321V mutation in human GABA-A receptor GABRB3 (L296V post-processing of the proprotein to remove the signal peptide). In some embodiments, the desensitizing mutation is a substitution of amino acid residues at or near the C-terminus of LGIC with a sequence having 90% or greater identity to a desensitizing sequence, e.g., IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53) from the C-terminus of the 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.

[0147] In some embodiments, the subject ligand-gated ion channels include one or more converting mutations. By converting mutation is meant a mutation that changes the permeability of the ion pore domain of the LGIC such that it allows the conduction of non-native ions, i.e., ions that do not allow natural passage. In some cases, the mutation converts the permeability from cations to anions, e.g., by substituting amino acid residues 260-281 in human α7-nAChR (CHRNA7) (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 54), or the corresponding amino acids in another cation-permeable LGIC, with the peptide sequence PAKIGLGITVLLSLTFMSGVAN (SEQ ID NO: 55). In some cases, the mutation converts permeability from anions to cations, for example, substitution of amino acid residue 279 of GLRA1 or a corresponding amino acid in another anion-permeable LGIC with glutamic acid (E) (A293E substitution in GLRA1 converts the LGIC from anion-permeable to calcium-permeable), or deletion of amino acid residue 278 of GLRA1 or a corresponding amino acid in another anion-permeable LGIC, substitution of amino acid residue 279 of GLRA1 or a corresponding amino acid in another anion-permeable LGIC with glutamic acid (E), and substitution of amino acid residue 293 of GLRA1 or a corresponding amino acid in another anion-permeable LGIC with valine (V) (P278Δ, A279E, T293V in GLRA1 converts the LGIC from anion-permeable to cation-permeable).

[0148] Additional engineered receptors beyond those described herein can be readily identified by in vitro screening and validation methods. In some embodiments, a library of parent receptor mutants is generated from a limited number of parent receptors. The parent receptors can be mutated using methods known in the art, including error-prone PCR. In some embodiments, the library of parent receptor mutants is then transfected into yeast or mammalian cells and screened in high throughput to identify functional receptors (e.g., to identify parent receptor mutants that can signal in response to a binding agent or ligand). In some embodiments, functional parent receptor mutants identified in this primary screen are then expressed in mammalian cells and screened for responsiveness to a binding agent or ligand, for example, by a plate reader and / or electrophysiology assays as described herein. Any parent receptor mutants that show increased binding affinity to agonist binders or that allow the use of antagonist or modulator binders as agonists in secondary screens can then be selected and further validation and characterization assays in vitro and / or in vivo can be performed. Such screening assays are known in the art and are described, for example, in Armbruster, BNet 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, JMet al. (2009) PNAS 106, 19197-19202; Ellefson JWet 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 DFet 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.

[0149] D. Binder The terms "binding agent" or "drug" are used interchangeably herein and refer to an exogenous drug or compound that has a known mechanism of action on mammalian cells (e.g., known to act as an agonist, antagonist, or modulator of a receptor). Binding agents may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the binding agent includes a drug or compound that has been approved by the U.S. Food and Drug Administration (FDA) for clinical use in the treatment of a particular disease (e.g., a neurological disease). In some embodiments, the binding agent includes a drug or compound that has not been approved for clinical use by the FDA but has been tested in one or more clinical trials, is currently being tested in one or more clinical trials, and / or is expected to be tested in one or more clinical trials. In some embodiments, the binding agent includes a drug or compound that has not been approved by the FDA for clinical use but is routinely used in laboratory research. In some embodiments, the binding agent is an analog of one of the aforementioned agents. In certain embodiments, the binding agent is selected from any one of the agents in Tables 2-9. In some embodiments, the binding agent is selected from the group consisting of AZD0328, ABT-126, AQW-051, cannabidiol, cilansetron, PH-399733, facinicline / RG3487 / MEM-3454, TC-6987, APN-1125, and TC-5619 / AT-101. In some embodiments, the binding agent is selected from the group consisting of ABT-126, AZD-0328, APN-1125, RG3487, TC-6987, and TC-5619.

[0150] In certain embodiments, the binding agent is an analog of cilansetron, for example, as described by one of compound formulas 2-7 in either its R or S enantiomer. [ka]

[0151] 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. The term antagonist is used herein to refer to an agent that inhibits a signaling response.

[0152] In some embodiments, the binding agent is an anti-anxiety agent, an anti-convulsant, an anti-depressant, an anti-psychotic agent, an anti-emetic agent, a nootropic agent, an antibiotic, an anti-fungal agent, an anti-viral agent, or an anti-parasitic agent. [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 5-3] [Table 6] [Table 7] [Table 8] [Table 9]

[0153] E. Polynucleotides In various exemplary embodiments, the present disclosure contemplates, in part, polynucleotides encoding engineered receptor polypeptides comprising LGICs, and subunits and mutant proteins thereof, as well as fusion polypeptides, viral vector polynucleotides, and compositions comprising the same.

[0154] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence", or "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (single locus) defined from binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. Polynucleotides may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.Polynucleotide refers to polymeric forms 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 nucleotides in length, as well as all intermediate lengths, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. It will be readily understood that "intermediate lengths" in this context means 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 certain embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence described herein or known in the art, and typically the variant retains at least one biological activity of the reference sequence, unless otherwise specified.

[0155] As used herein, the term "gene" can refer to a polynucleotide sequence that includes enhancers, promoters, introns, exons, etc. In certain embodiments, the term "gene" refers to a polynucleotide sequence that encodes a polypeptide, regardless of whether the polynucleotide sequence is identical to a genomic sequence that encodes the polypeptide.

[0156] As used herein, "cis-acting sequence", "cis-acting regulatory sequence", or "cis-acting nucleotide sequence", or equivalents, refers to a polynucleotide sequence associated with the expression, e.g., transcription and / or translation, of a gene. In one embodiment, a cis-acting sequence is a polynucleotide sequence associated with a binding site for a polypeptide that represses or reduces transcription, or a transcription factor binding site that contributes to transcription repression, and thus regulates transcription. Examples of cis-acting sequences that can be operably linked to the polynucleotides of the present disclosure to regulate expression of a polynucleotide sequence and regulate 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.

[0157] As one non-limiting example, a promoter sequence is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, a promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence, the transcription initiation site is found, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. Various promoters may be used to drive the various vectors of the present invention. For example, the promoter may be a constitutively active promoter, i.e., a promoter that is active in the absence of an externally applied agent, such as the CMV IE1 promoter, the SV40 promoter, the GAPDH promoter, the actin promoter. The promoter may be an inducible promoter, i.e., a promoter whose activity is controlled upon application of an agent to the cell, such as the doxycycline, tet-on or tet-off promoter, the estrogen receptor promoter, and the like. The promoter may be a tissue-specific promoter, ie, a promoter that is active on a particular type of cell.

[0158] In some embodiments, the promoter is active in excitable cells. "Excitable cells" refers to cells that are activated by changes in membrane potential, such as neurons or muscle cells, such as dorsal root ganglia, motor neurons, excitatory neurons, inhibitory neurons, or sensory neurons. Promoters active in excitable cells that find use in the polynucleotide compositions of the present invention include neuronal promoters, such as synapsin (SYN), TRPV1, Na v 1.7, Na v 1.8, Na v1.9, CamKII, NSE, and advillin promoters; muscle cell promoters such as desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), and cardiac troponin C (cTnC) promoters; and ubiquitous acting promoters such as CAG, CBA, E1Fa, Ubc, CMV, and SV40 promoters.

[0159] As used herein, "regulatory element for inducible expression" refers to a polynucleotide sequence that is a promoter, enhancer, or functional fragment thereof, operably linked to a polynucleotide to be expressed and responds to the presence or absence of a molecule that binds the element to increase (on) or decrease (off) the expression of the polynucleotide operably linked thereto. Exemplary 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-gamma promoters, and peroxide-inducible promoters.

[0160] "Regulatory elements for transient expression" refer to polynucleotide sequences that can be used to express a polynucleotide sequence for short periods or temporarily. In certain embodiments, one or more regulatory elements for transient expression can be used to limit the duration of the polynucleotide. In certain embodiments, the preferred duration of polynucleotide expression is on the order of minutes, hours, or days. Exemplary regulatory elements for transient expression include, but are not limited to, nuclease target sites, recombinase recognition sites, and inhibitory RNA target sites. Furthermore, to some extent, in certain embodiments, regulatory elements for inducible expression can also contribute to controlling the duration of polynucleotide expression.

[0161] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added or deleted, or modified, or replaced with different nucleotides. In this regard, it is well known in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the modified polynucleotide retains the biological function or activity of the reference polynucleotide. In certain embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence described herein or known in the art, and typically the variant retains at least one biological activity of the reference sequence, unless otherwise specified.

[0162] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. To hybridize under "stringent conditions", a hybridization protocol is described in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Typically, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes that are complementary to the target sequence hybridize to the target sequence at equilibrium. The target sequence is typically present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0163] As used herein, "sequence identity" or statements including, for example, "50% identical sequences" refer to the extent to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over 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) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is at least 12, often 15-18, and often at least 25 monomeric units in length, including nucleotides and amino acid residues. Because two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only some of the complete polynucleotide sequence), and (2) sequences that are dispersed between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150, in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. A comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences to align the comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by examining and selecting the optimal alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of the various methods. Reference can also be made to the BLAST family of programs, for example, as disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Unit 19.3 of Chapter 15.

[0164] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been created artificially.

[0165] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in the 5' to 3' direction or the 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus", or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus", or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0166] The term "flanking" refers to upstream and / or downstream polynucleotide sequences, i.e., polynucleotide sequences that are 5' and / or 3' to a sequence. For example, a sequence "flanking" two other elements (e.g., ITRs) indicates that one element is located 5' to the sequence and the other is located 3' to the sequence, although there may be intervening sequences between them.

[0167] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a series of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often described as the reverse complement of 5'CATGACT3', with the 5' end on the left and the 3' end on the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only some of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids.

[0168] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a polynucleotide sequence within a larger polynucleotide, such as a vector, that is sufficient to express one or more RNAs from a polynucleotide. The expressed RNA may be translated into a protein or may function as a guide RNA or an inhibitory RNA to target other polynucleotide sequences for cleavage and / or degradation. In one embodiment, the nucleic acid cassette comprises one or more polynucleotides of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences operably linked to one or more polynucleotides of interest. Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or a polynucleotide that serves as a template for transcription of the inhibitory polynucleotides contemplated herein, such as LGIC, and its subunits and mutant proteins. In certain embodiments, the polynucleotide of interest encodes a polypeptide or fusion polypeptide that has one or more enzymatic activities, such as nuclease activity and / or chromatin remodeling or epigenetic modification activity.

[0169] A vector may contain 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 comprises one or more expression control sequences (e.g., promoters or enhancers operable in neuronal cells) operably linked to a polynucleotide encoding an engineered receptor, e.g., LGIC, or a subunit or mutant protein thereof. The cassette may be removed or inserted as a single unit from another polynucleotide sequence, e.g., a plasmid or viral vector.

[0170] In one embodiment, a polynucleotide contemplated herein comprises 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.

[0171] Moreover, as a result of the degeneracy of the genetic code, those skilled in the art will understand that there are many nucleotide sequences that can code for fragments of the polypeptides or variants thereof contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure, for example, polynucleotides optimized for human and / or primate codon preferences. In one embodiment, a polynucleotide is provided that comprises a specific allele sequence. An allele is an endogenous polynucleotide sequence that is altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides.

[0172] F. Vector In some aspects of the disclosure, a nucleic acid molecule, i.e., a polynucleotide encoding an engineered receptor, is delivered to a subject. In some cases, the nucleic acid molecule encoding an engineered receptor is delivered to a subject by a vector. In various embodiments, the vector comprises one or more polynucleotide sequences contemplated herein. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred polynucleotide is generally linked, e.g., inserted, into a vector nucleic acid molecule. The vector may contain sequences directing autonomous replication within a cell or may contain sequences sufficient to allow integration into a host cell DNA. The vector can deliver a target polynucleotide to an organism, cell, or cellular component. In some cases, the vector is an expression vector. As used herein, "expression vector" refers to a vector, e.g., a plasmid, that can promote expression and replication of a polynucleotide incorporated therein. Typically, the nucleic acid sequence to be expressed is operably linked to cis-acting regulatory sequences, e.g., promoter and / or enhancer sequences, and depends on transcriptional regulatory control by the promoter and / or enhancer. In certain cases, a vector is used to deliver a nucleic acid molecule encoding an engineered receptor of the present disclosure to a subject.

[0173] In certain embodiments, any vector suitable for introducing an expression cassette or polynucleotide encoding an engineered receptor into a neuronal cell can be used. Examples of suitable vectors include plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some cases, the vector is a circular nucleic acid, for example, a plasmid, BAC, PAC, YAC, cosmid, fosmid, etc. In some cases, a circular nucleic acid molecule can be utilized to deliver a nucleic acid molecule encoding an engineered receptor to a subject. For example, a plasmid DNA molecule encoding an engineered receptor can be introduced into a cell of a subject, whereby the DNA sequence encoding the engineered receptor is transcribed into mRNA, and the "message" of the mRNA is translated into a protein product. A circular nucleic acid vector generally includes a regulatory element that regulates the expression of a target protein. For example, a circular nucleic acid vector may include any number of promoters, enhancers, terminators, splice signals, origins of replication, initiation signals, etc.

[0174] In some cases, the vector may include a replicon. The replicon may be any nucleic acid molecule capable of self-replication. In some cases, the replicon is an RNA replicon derived from a virus. A variety of suitable viruses (e.g., RNA viruses) are available, including, but not limited to, alphaviruses, picornaviruses, flaviviruses, coronaviruses, pestiviruses, rubiviruses, calciviruses, and hepaciviruses.

[0175] In some embodiments, the vector is a non-viral vector. By "non-viral vector" is meant any delivery vehicle that does not contain a viral capsid or envelope, such as lipid nanoparticles (anionic (negative charge), neutral, or cationic (positive charge)), heavy metal nanoparticles, polymer-based particles, plasmid DNA, minicircle DNA, minivector DNA, ccDNA, synthetic RNA, exosomes, etc. Non-viral vectors can be delivered by any suitable method well understood in the art, including, for example, nanoparticle delivery, 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): 65.

[0176] In other embodiments, the vector is a viral vector. "Viral vector" refers to a delivery vehicle that includes a viral capsid or envelope that surrounds a polynucleotide that encodes an RNA or polypeptide of interest. In some cases, the viral vector is derived from a replication-defective virus. Non-limiting examples of viral vectors suitable for delivering the nucleic acid molecules of the present disclosure to a subject include those derived from adenovirus, retrovirus (e.g., lentivirus), adeno-associated virus (AAV), and herpes simplex-1 (HSV-1). Examples of suitable viral vectors include, but are not limited to, retrovirus vectors (e.g., lentivirus vectors), herpesvirus-based vectors, and parvovirus-based vectors (e.g., adeno-associated virus (AAV)-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors).

[0177] As used herein, the term "parvovirus" encompasses all parvoviruses, including autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other 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).

[0178] The Dependovirus genus contains adeno-associated viruses (AAV), including, but not limited to, AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type rh10, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

[0179] In a preferred embodiment, the vector is an AAV vector. In certain cases, the viral vector is an AAV-6 or AAV-9 vector.

[0180] The genome structure of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for nonstructural replication (Rep) proteins and structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid and contribute to the tropism of the virus. The terminal 145 nt ITRs are self-complementary and are organized such that energetically stable intramolecular duplexes can form that form T-shaped hairpins. These hairpin structures serve as origins of viral DNA replication and as primers for the cellular DNA polymerase complex. Following wild-type (wt) AAV infection in mammalian cells, the Rep genes are expressed and function in the replication of the viral genome.

[0181] In some cases, the outer protein "capsid" of the viral vector occurs in nature, e.g., 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 characteristics not present in nature, such as altered tropism, increased transduction efficiency, or immune evasion. An example of a rationally designed capsid is the mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid protein. Non-limiting examples of viral vectors whose VP3 capsid protein has been synthetically engineered and suitable for use in 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 engineered through directed evolution and suitable for use in the compositions and methods provided herein include AAV-7m8 and AAV-ShH10.

[0182] A "recombinant parvovirus or AAV vector" (or "rAAV vector") herein refers to a vector comprising one or more polynucleotides contemplated herein flanked by one or more AAV ITRs. Such polynucleotides are said to be "heterologous" to the ITRs because such combinations do not normally occur in nature. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger nucleic acid construct (e.g., within a chromosome or within another vector such as a plasmid or baculovirus used for cloning or transfection), the rAAV vector is typically referred to as a "pro-vector" and can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

[0183] In certain embodiments, any AAV ITR may be used in the AAV vector, including ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. In a preferred embodiment, the AAV vectors contemplated herein contain one or more AAV2 ITRs.

[0184] A rAAV vector containing two ITRs has a payload capacity of about 4.4 kB. A self-complementary rAAV vector contains a third ITR and packages the double strand 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.

[0185] The expanded packaging capacity, which is about twice that of rAAV (about 9 kB), was achieved using a double rAAV vector strategy. The double vector strategies useful for generating rAAV contemplated herein include, but are not limited to, splicing (trans-splicing), homologous recombination (overlap), or a combination of the two (hybrid). In the double AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' end of the 5' half vector, and a splice acceptor (SA) signal is placed at the 5' end of the 3' half vector. Upon coinfection of the same cell with the double AAV vectors and inverted terminal repeat (ITR)-mediated head-to-tail concatenation of the two halves, trans-splicing results in the production of mature mRNA and full-sized protein (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the two halves of a large transgene expression cassette contained within a double AAV vector may contain homologous overlapping sequences (double AAV overlap at the 3' end of the 5' half vector and the 5' end of the 3' half vector) that mediate the reconstitution of a single large genome by homologous recombination (Duan et al., 2001). This strategy relies on the recombination properties of the transgene overlapping sequences (Ghosh et al., 2006). The third double AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al., 2008, Ghosh et al., 2011) to the trans-splicing vector. The added region is placed downstream of the SD signal of the 5' half vector and upstream of the SA signal of the 3' half vector to increase recombination between the double AAVs.

[0186] "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 one or more AAV ITRs), or may otherwise be referred to as a pseudotyped rAAV. For example, a rAAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 genome can be encapsidated within an AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 capsid or a variant thereof, provided that the AAV capsid and genome (and preferably one or more AAV ITRs) are of different serotypes. In certain embodiments, pseudotyped rAAV particles may be referred to as "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.

[0187] "Host cells" include cells that have been transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present disclosure. Host cells can include virus-producing cells and cells infected with a viral vector. In certain embodiments, in vivo host cells are infected with a viral vector 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.

[0188] 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, US 2006 / 0188484, WO 98 / 22607, WO 2005 / 072364, and WO / 1999 / 011764, and in 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; Ilnoue et al., (1998) J. Virol. 72, 7024. Methods for producing pseudotyped AAV vectors (e.g., WO00 / 28004), as well as various modifications or formulations of AAV vectors have also been reported to reduce their immunogenicity upon in vivo administration (see, e.g., WO01 / 23001, WO00 / 73316, WO04 / 112727, WO05 / 005610, WO99 / 06562).

[0189] Pharmaceutical Compositions Also provided are pharmaceutical preparations, including pharmaceutical preparations of vectors and pharmaceutical preparations of binding agents. The pharmaceutical preparations include the subject polynucleotides (RNA or DNA) encoding the engineered receptors, the vector carrying the polynucleotides (RNA or DNA) encoding the subject engineered receptors, or the binding agent present in a pharmaceutically acceptable vehicle. A "pharmaceutically acceptable vehicle" can be a vehicle approved by a federal or state regulatory agency for use in mammals, such as humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier in which the compounds of the present disclosure are formulated for administration to a mammal. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants and coloring agents can be used.When administered to mammals, the compounds and compositions of the present disclosure and pharma-ceutically acceptable vehicles, excipients or diluents can be sterilized.In some instances, when the compounds of the present disclosure are administered intravenously, aqueous media such as water, saline solution, and aqueous dextrose and glycerol solution are used as vehicles.

[0190] The pharmaceutical composition may take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained release formulations thereof, or any other form suitable for administration to mammals.In some instances, the pharmaceutical composition is formulated for administration according to routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans.Examples of suitable pharmaceutical vehicles and methods of their formulation are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, which are incorporated herein by reference.

[0191] The choice of excipient will be determined in part by the particular vector, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the present disclosure.

[0192] For example, the vectors can be formulated into a preparation for injection by dissolving, suspending or emulsifying 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, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0193] As another example, the vectors can be formulated into preparations suitable for oral administration, including (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent such as water or saline, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient as a solid or granules, (c) suspensions in a suitable liquid, and (d) suitable emulsions. Tablet forms can contain one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can contain a flavoring, usually the active ingredient in sucrose and acacia or tragacanth, and pastilles containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, etc., containing the active ingredient in addition to excipients as described herein.

[0194] As another example, the formulations of the presently disclosed subject matter can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. They can also be formulated as pharmaceuticals in non-pressurized preparations, such as for use in nebulizers or atomizers.

[0195] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a frozen and dried (lyophilized) state, requiring only the addition of a sterile liquid excipient for injection, such as water, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described.

[0196] Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams containing, in addition to the active ingredient, suitable carriers, etc. In some embodiments, topical formulations contain one or more components selected from structuring agents, thickening or gelling agents, and emollients or lubricants. Frequently used structuring agents include long-chain alcohols, such as stearyl alcohol, and their glyceryl ethers or esters, and oligo(ethylene oxide) ethers or esters. Thickening and gelling agents include, for example, acrylic or methacrylic acid polymers and their esters, polyacrylamides, and naturally derived thickeners, such as agar, carrageenan, gelatin, and guar gum. Examples of emollients include triglyceride esters, fatty acid esters and amides, waxes, such as beeswax, spermaceti, or carnauba wax, phospholipids, such as lecithin, and their sterols and fatty acid esters. Topical formulations may further include other ingredients, such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (i.e., sunblocks), etc.

[0197] The compounds of the present disclosure may be formulated for topical administration. The vehicle for topical application may be one of various forms, such as lotion, cream, gel, ointment, stick, spray, or paste. They may contain various types of carriers, including, but not limited to, solutions, aerosols, emulsions, gels, and liposomes. The carriers may be formulated, for example, as emulsions with a water-in-oil or water-in-oil base. Suitable hydrophobic (oily) components used in emulsions include, for example, vegetable oils, animal fats and oils, synthetic hydrocarbons, and their esters, including polyesters, and alcohols, and organopolysiloxane oils. Such emulsions also include emulsifiers and / or surfactants, such as nonionic surfactants, to disperse and suspend the discontinuous phase within the continuous phase.

[0198] Suppository formulations can also be provided by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes or foams.

[0199] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor in a composition as a solution in sterile water, normal saline, or another pharma- ceutically acceptable carrier.

[0200] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary doses for human and animal subjects, each unit containing a predetermined amount of the compound of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharma- ceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0201] Dosage levels can vary as a function of the particular compound, the nature of the delivery vehicle, etc. The desired dose for a given compound can be readily determined by a variety of means.

[0202] In the context of this disclosure, the dose administered to an animal, particularly a human, should be sufficient to produce a prophylactic or therapeutic response in the animal over a reasonable time frame, for example, as described in more detail below. The dose depends on a variety of factors, including the strength of the particular compound used, the condition of the animal, and the weight of the animal, as well as the severity and stage of the disease. The size of the dose is also determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular compound.

[0203] In pharmaceutical dosage forms, the ASC inducer compounds may be administered in the form of a free base, a pharma- ceutically acceptable salt thereof, or may be used alone or in appropriate association, as well as in combination with other pharma- ceutically active compounds.

[0204] G. Clinical Applications and Treatment Methods The compositions and methods disclosed herein can be used to treat neurological diseases or disorders.In some aspects of the present disclosure, a method for treating a neurological disease or disorder in a subject is provided, the method comprising: introducing an engineered receptor into a neuronal cell; providing an effective amount of a ligand that activates the engineered receptor to control the activity of the cell; and thereby relieving the pain of the subject.In some aspects, the vector or composition disclosed herein is used to manufacture a medicament for treating a neurological disease or disorder.

[0205] In some cases, the disclosed methods and compositions are utilized to treat epilepsy. The compositions described herein can be used to prevent or control epileptic seizures. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonic, absence, or atonic seizures. In some cases, the compositions and methods herein can prevent or reduce the number of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%.

[0206] In some cases, the disclosed methods and compositions are utilized to treat eating disorders. An eating disorder can be a mental disorder defined by abnormal eating behavior that negatively impacts the subject's physical or mental health. In some cases, the eating disorder is anorexia nervosa. In other cases, the eating disorder is bulimia nervosa. In some cases, the eating disorder is pica, rumination disorder, avoidant / restrictive food intake disorder, binge eating disorder (BED), other specified eating and eating disorders (OSFED), compulsive binge eating disorder, diabulimia, orthorexia, selective eating disorder, drunkorexia, anorexia during pregnancy, or Gruman syndrome. In some cases, the composition comprises a G protein-coupled receptor that increases or decreases the production of one or more molecules associated with an eating disorder. In other cases, the composition comprises a ligand-gated ion channel that alters the production of one or more molecules associated with an eating disorder. The one or more molecules associated with eating disorders may include, but are not limited to, molecules of the hypothalamic-pituitary-adrenal (HPA) axis, including vasopressin, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, epinephrine, or norepinephrine, as well as serotonin, dopamine, neuropeptide Y, leptin, or ghrelin.

[0207] In some cases, the compositions and methods are utilized to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumors), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that may be treated by the compositions and methods herein include abulia, agraphia, alcoholism, dyslexia, aneurysm, amaurosis fugax, memory loss, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, attention deficit hyperactivity disorder, auditory perceptual disorders, autism spectrum disorder, bipolar disorder, facial paralysis, brachial plexus injury, brain tumors, and the like. Injury, brain injury, brain tumor, Canavan disease, Capgras syndrome, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, cephalic disorders, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, autosomal dominant cerebral arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, Correa, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic Pain, Coffin-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital ophthalmoplegia, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cyclothymic disorder, cytomegalic inclusion body disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, Dermatomyositis, developmental coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, dysarthria, autonomic imbalance, dyscalculia, dysgraphia, dyskinesia, dyslexia, dystonia, empty sella syndrome, encephalitis, brain herniation, hemangioma of the trigeminal nerve region, encopresis, nocturnal enuresis, epilepsy, intellectual disability in women, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry disease, Fahr's syndrome, syncope, familial spastic paraplegia,Febrile convulsions, Fisher syndrome, Friedreich's ataxia, fibromyalgia, Fauville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy febrile convulsions plus, Gerstmann syndrome, giant cell arteritis, giant cell inclusion body disease, globoid cell leukodystrophy, heterotopic grey matter, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, Hallervorden-Spatz syndrome, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary ataxia of the polyneuropathy type, herpes zoster oticus, herpes zoster, Hirayama syndrome, Hirschsprung's disease, Holmes-Ardie syndrome, holoprosencephaly, Huntington's disease, hydrocephalus, hydrocephalus, hypercortisolism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, achromatopsia ataxia, infantile Refsum's disease, infantile spasms, inflammatory myopathy, intracranial cysts, intracranial hypertension, isodicentric 15, Joubert syndrome, Karak's syndrome, Cairns-Sayre syndrome, Kinsbourne syndrome, Kleine-Lewin syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral bulbar (Wallenberg) syndrome, learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, white matter dystrophies, leukoencephalopathy with loss of white matter, dementia with Lewy bodies, gyri, locked-in syndrome, lumbar discopathy, lumbar spinal stenosis, Lyme disease - neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephalopathy, macropsia, post-landing syndrome (Mal de debarquement), macrocephalic leukoencephalopathy with subcortical cysts, megalencephaly, Melkersson-Rosenthal syndrome, Meniere's disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, micropsia, migraine, Miller-Fisher syndrome, petit mal (transient ischemic attack), misophonia, mitochondrial myopathy, Moebius syndrome, unilateral upper limb muscular atrophy, motor skills disorder, Moyamoya disease, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelin-destructive diffuse sclerosis, infantile myoclonus encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy,Neuro-Behçet's disease, neurofibromatosis, neuroleptic malignant syndrome, neurological symptoms of AIDS, neurological sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neuropathy, Niemann-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, sequelae of latent spinal neural tube defects, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus ataxia, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, recurrent vision, abnormal sensations, Parkinson's disease, congenital paramyotonia , paraneoplastic disease, paroxysmal seizures, Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, pervasive developmental disorder, photic sneeze reflex, phytanic acid storage disease, Pick's disease, shrunken nerve, pituitary tumor, PMG, polyneuropathy, polio, polymicrogyria, polymyositis, porosis, post-polio syndrome, post-herpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemiatrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrantal blindness, quadriplegia, rabies, nerve root Paralysis, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen's encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett's syndrome, Rayet syndrome, rhythmic movement disorder, Romberg's syndrome, chorea, Sandhoff's disease, Schilder's disease, schizencephaly, sensory processing disorder, septo-optic dysplasia, shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, sneezing after meals (Sn atiation), Sotos syndrome, spasticity, vertebral rupture, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinal-bulbar muscular atrophy, spinocerebellar ataxia, split brain, Steele-Richardson-Olszewski syndrome, rigid-body syndrome, seizures, Sturge-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, Sydenham chorea, syncope, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, tardive dysphrenia, Tarlov cyst, Tisachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered cord syndrome, Thomsen's disease, thoracic outlet syndrome,These conditions include trigeminal neuralgia, Todd's palsy, Tourette's syndrome, toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, von Hippel-Lindau disease (VHL), Villius encephalomyelitis (VE), Wallenberg complex, West syndrome, whiplash, Williams syndrome, Wilson's disease, or Zellweger syndrome.

[0208] 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 suitable for treatment with the vectors and compositions described herein include gliomas, including malignant astrocytoma (grade III glioma), astrocytoma (grade II glioma), brain stem glioma, ependymoma, ganglioglioma, ganglioneuroma, glioblastoma (grade IV glioma), glioma, juvenile pilocytic astrocytoma (JPA), low grade astrocytoma (LGA), medulloblastoma, mixed glioma, oligodendroglioma, optic nerve glioma, pilocytic astrocytoma (grade I glioma), and primitive neuroectodermal (PNET). tumors of the skull base, including acoustic neuroma (vestibular schwannoma), acromegaly, adenoma, chondrosarcoma, chordoma, craniopharyngioma, epidermoid tumor, jugular body tumor, infratentorial meningioma, meningioma, pituitary adenoma, pituitary tumor, Rathke's cleft cyst; metastatic carcinomas, including brain metastases, metastatic brain tumors; other brain tumors, including brain cyst, choroid plexus papilloma, CNS lymphoma, colloid cyst, cystic tumor, dermoid tumor, germinoma, lymphoma, nasal carcinoma, nasopharyngeal tumor, pineal gland tumor, pineoblastoma, pineocytoma, supratentorial meningioma, and vascular tumor; tumors of the spinal cord, including astrocytoma, ependymoma, meningioma, and schwannoma.

[0209] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating pain in a subject. "Pain" refers to discomfort and / or unpleasant sensations in the body of a subject. Pain sensations can range from mild and occasional to severe and constant. Pain can be classified as acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain, or psychogenic pain. In some cases, pain is caused or associated with disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, lower back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, brachial 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, post-operative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with cartilage tunnel syndrome, and lower back pain.

[0210] The compositions and methods herein can be utilized to improve the level of pain in a subject. In some cases, the level of pain in a subject is improved by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100%. The level of pain in a subject can be evaluated by various methods. In some cases, the level of pain is evaluated by self-report (i.e., the human subject verbally reports the level of pain that he or she is experiencing). In some cases, the level of pain is evaluated by behavioral indicators of pain, such as facial expression, limb movement, vocalization, restlessness, and alert behavior. These types of assessments can be useful, for example, when a subject is unable to self-report (e.g., young children, unconscious subjects, non-human subjects). Pain levels can be assessed after treatment with a composition of the present disclosure compared to the level of pain experienced by the subject prior to treatment with the composition.

[0211] 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 intending to be bound by any particular theory, the present disclosure contemplates using the vectors disclosed herein to modulate neuronal activity to alleviate pain in a subject.

[0212] In various embodiments, a vector encoding an engineered receptor that activates or depolarizes a neuron is administered (or introduced) into one or more neurons, e.g., inhibitory interneurons, that reduce pain sensation. In the presence of a ligand, the neurons expressing the engineered receptor are activated, reducing sensitivity to pain enhancing the analgesic effect of stimulating these neurons.

[0213] In various embodiments, a vector encoding an engineered receptor that inactivates or hyperpolarizes a neuronal cell is administered (or introduced) into one or more neuronal cells that increase pain sensation or sensitivity to pain, such as nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aδ fibers, DRG neurons, TGG neurons, etc. In the presence of ligand, the neuronal cells expressing the engineered receptor are inactivated, decreasing sensitivity to pain and enhancing the analgesic effect.

[0214] Targeting expression of the engineered receptor to subpopulations of nociceptors can be achieved by one or more of the following: choice of vector (e.g., AAV1, AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10(Y733F), and AAV-ShH10), choice of promoter, and delivery means.

[0215] 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, lower back pain, eye pain, visceral pain, cancer pain (e.g. bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, post-herpetic neuralgia, post-operative pain, post-stroke pain, and menstrual pain.

[0216] Pain can be classified as acute or chronic. "Acute pain" refers to pain that begins suddenly and is usually sharp in quality. Acute pain may be mild and last only a moment, or severe and last for weeks or months. In most cases, acute pain does not last more than three months and disappears when the underlying cause of the pain is treated or cured. However, unrelieved acute pain can lead to chronic pain. "Chronic pain" refers to ongoing or recurrent pain that lasts beyond the usual course of an acute illness or injury, or lasts for more than three to six months and adversely affects the health of an individual. In certain embodiments, the term "chronic pain" refers to pain that continues when it should not. Chronic pain can be nociceptive pain or neuropathic pain.

[0217] In some embodiments, the pain is expected or predicted to occur in association with or as a result of an injury, infection, or medical intervention. In some embodiments, the infection causes nerve damage. In some embodiments, the medical intervention is a surgical procedure, such as surgery on the 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 within the body. In some embodiments, the medical intervention is an amputation. In certain embodiments, the compositions and methods contemplated herein are effective in reducing acute pain. In certain embodiments, the compositions and methods contemplated herein are effective in reducing chronic pain.

[0218] Clinical pain is present when discomfort and abnormal sensitivity are characteristic of the patient's symptoms. Individuals may present with a variety of pain symptoms. Such symptoms include: 1) spontaneous pain that is dull, burning, or stabbing; 2) exaggerated pain response to noxious stimuli (hyperalgesia); and 3) pain caused by normally innocuous stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Patients suffering from various forms of acute and chronic pain may have similar symptoms, but the underlying mechanisms may differ and therefore require different treatment strategies. Thus, pain can also be divided into a number of different subtypes according to different pathophysiology, including nociceptive pain, inflammatory pain, and neuropathic pain.

[0219] 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.

[0220] Nociceptive pain is induced by tissue injury or by strong stimuli that may cause injury. Moderate to severe acute nociceptive pain is a hallmark of pain from central nervous system trauma, strain / sprain, burns, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and low back pain. Cancer pain can be tumor-related pain (e.g., bone pain, headache, facial pain, or visceral pain), or chronic pain such as pain associated with cancer therapy (e.g., postchemotherapy syndrome, chronic postoperative pain syndrome, or postradiation syndrome). Cancer pain can also occur in response to chemotherapy, immunotherapy, hormonal therapy, or radiation therapy. Low back pain may be due to herniated or ruptured intervertebral discs, or abnormalities of the lumbar facet joints, sacroiliac joints, paraspinal muscles, or posterior longitudinal ligament. Low back pain may resolve spontaneously, but it becomes a chronic condition that is particularly debilitating in patients who persist for more than 12 weeks.

[0221] Neuropathic pain can be defined as the pain that is initiated or caused by the primary lesion or dysfunction of the nervous system.The etiology of neuropathic pain includes, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, low back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, post-stroke central pain, and chronic alcoholism-related pain, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0222] Neuropathic pain can be related to pain disorder, a term referring to diseases, disorders, or conditions associated with or caused by pain. Examples of pain disorders include arthritis, allodynia, classic trigeminal neuralgia, trigeminal neuralgia, somatoform disorder, hypoesthesia, hyperesthesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia, causlagia, sciatica disorder, degenerative joint disorder, fibromyalgia, visceral disease, chronic pain disorder, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurological dystrophy, plantar fasciitis, or cancer-related pain.

[0223] The inflammatory process is a complex series of biochemical and cellular events that are activated in response to tissue injury or the presence of a foreign body, resulting in swelling and pain. Joint pain is a common form of inflammatory pain.

[0224] Other types of pain suitable for treatment with the vectors, compositions, and methods contemplated herein include, but are not limited to, pain resulting from musculoskeletal disorders including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-rheumatoid arthritis, dystrophinopathy, glycogenolysis, polymyositis, and pyomyositis; cardiac and vascular pain including tonsillitis, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma, and skeletal muscle ischemia; headaches, e.g., migraine (including migraine with aura and migraine without aura), cluster headaches, mixed tension-type headaches, headaches associated with vascular disorders; and orofacial pain including toothache, earache, burning mouth syndrome, and temporomandibular joint myofascial pain.

[0225] The effective amount of the compositions and methods contemplated herein for reducing the amount of pain experienced by a human subject can be determined using various pain scales. Patient self-report can be used to assess whether pain is reduced. See, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231. Alternatively, observational pain scales can be used. LANSS pain scale can be used to assess whether pain is reduced. See, for example, Bennett (2001) Pain 92:147. Visual analog pain scales can be used. See, for example, Schmader (2002) Clin. J. Pain 18:350. Likert pain scales can be used. For example, 0 is no pain, 5 is moderate pain, and 10 is the worst pain possible. Self-report pain scales for children include, for example, facial pain scales, Wong-Baker facial pain rating scales, and color analog scales. Self-report pain scales for adults include, for example, visual analog scales, verbal numerical rating scales, verbal descriptor scales, 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 scales (Payen et al. (2001) Critical Care Medicine 29:2258); the Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129); the Nonverbal Pain Indicator Checklist (Feldt (2000) Pain Manag. Nurse. 1:13); the Critical Care Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); the Comfort Scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); the Dallas Pain Questionnaire (Ozguler et al. (2002) Spine 27:1783); algometer pain index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Revised Face Pain Scale (Hicks et al. (2001) Pain 93:173); Face Legs Activity Cry Consolability Scale (Face Legs Activity Cry Consolability Scale); McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptor Differential Scale (Gracely and Kwilosz (1988) Pain 35:279); Numeric 11-point box (Jensen et al. (1989) Clin. J. Pain 5:153); Numeric Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310); the Wong-Baker Facial Pain Rating Scale; and the visual analog scale (Huskisson (1982) J. Rheumatol. 9:768).

[0226] In certain embodiments, a method of alleviating pain in a subject is provided, the method comprising introducing an engineered receptor into a neuronal cell, controlling the activity of the cell by providing an effective amount of a ligand that activates the engineered receptor, and thereby alleviating pain in the subject. The method provides significant analgesia for pain without off-target effects, such as general central nervous system depression. In certain embodiments, the method provides a 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 reduction in neuropathic pain in the subject compared to untreated subjects. In some embodiments, the method includes measuring pain in the subject before and after administration of the binding agent, and the pain in the subject is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In such cases, the measurement may be performed 4 hours or more after administration of the binding agent, e.g., 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.

[0227] In certain embodiments, the vector contemplated herein is administered or introduced into one or more neural cells. The neural cells may be the same type of neural cells or may be a mixed population of different types of neural cells. In one embodiment, the neural cells are nociceptors or peripheral sensory neurons. Examples of sensory neurons include, but are not limited to, dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In one embodiment, the neural cells are inhibitory interneurons that are involved in neuronal pain circuits.

[0228] In some cases, the vector encoding the engineered receptor is administered to a subject in need thereof. Non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, injection, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cisternal administration, and intraneuronal administration. In some cases, administration can include injection of a liquid formulation of the vector. In other cases, administration can include oral delivery of a solid formulation of the vector. In some cases, the oral formulation can be administered with food. In certain embodiments, the vector is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneurally, intraganglionously, intraspinally, or intracerebroventricularly to introduce the vector into one or more neural cells. In various embodiments, the vector is rAAV.

[0229] In one embodiment, AAV is administered to sensory neurons or nociceptors, such as DRG neurons, TGG neurons, etc., by intrathecal (IT) or intraganglionic (IG) administration. The IT route delivers AAV to 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 it caudally to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a routine clinical procedure with an excellent safety profile.

[0230] In certain cases, the vector may be administered to a subject by intraganglionic administration. Intraganglionic administration may involve direct injection into one or more ganglia. The IG route may deliver AAV directly to the DRG or TGG parenchyma. In animals, IG administration to the DRG is performed by open neurosurgical procedures, which are undesirable in humans because it requires complex and invasive procedures. In humans, minimally invasive CT imaging guided techniques to safely target the DRG may be used. AAV may be delivered into the DRG parenchyma using a customized needle assembly for convection enhanced delivery (CED). In a non-limiting example, the vector of the present disclosure may 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 may be delivered to the supragangial ganglion (vagus nerve) for the treatment of epilepsy.

[0231] In yet another specific case, the vector may be administered to the subject by intracranial administration (i.e., directly into the brain). In a non-limiting example of intracranial administration, the vector of the present disclosure may be delivered to the cortex of the brain, for example, to treat epileptic seizure focus, to the paraventricular hypothalamus, for example, to treat satiety disorder, or to the central amygdala, for example, to treat satiety disorder. In another specific case, the vector may be administered to the subject by intraneural injection (i.e., directly into the nerve). The nerve may be selected based on the indication to be treated, for example, injection into the sciatic nerve to treat chronic pain, or injection into the vagus nerve to treat epilepsy or satiety disorder. In yet another specific case, the vector may be administered to the subject by subcutaneous injection into the sensory nerve ending, for example, to treat chronic pain.

[0232] A vector dose can be expressed as the number of vector genome units delivered to a subject. As used herein, "vector genome unit" refers to the number of individual vector genomes administered in a dose. The size of an individual vector genome generally depends on the type of viral vector used. The vector genomes of the present disclosure can be about 1.0 kilobase to 1.5 kilobase, 2.0 kilobase, 2.5 kilobase, 3.0 kilobase, 3.5 kilobase, 4.0 kilobase, 4.5 kilobase, 5.0 kilobase, 5.5 kilobase, 6.0 kilobase, 6.5 kilobase, 7.0 kilobase, 7.5 kilobase, 8.0 kilobase, 8.5 kilobase, 9.0 kilobase, 9.5 kilobase, 10.0 kilobase, more than 10.0 kilobase. Thus, a single vector genome can contain up to 10,000 base pairs of nucleotides, or more. In some cases, a vector dose can be about 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x109 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、8x10 11 、9x10 11 、1x10 12 、2x10 12 、3x10 12 、4x10 12 、5x10 12 、6x10 12 、7x10 12 、8x10 12 、9x10 12 、1x10 13 、2x10 13 、3x10 13 、4x10 13 、5x10 13 、6x10 13 、7x10 13 、8x10 13 、9x10 13 、1x10 14 、2x10 14 、3x10 14 、4x10 14 、5x10 14 、6x10 14 、7x10 14 、8x10 14 、9x10 14 、1x10 15 、2x10 15 、3x10 15 、4x10 15 、5x10 15 、6x10 15 、7x10 15 、8x10 15 、9x10 15, 1x10 16 , 2x10 16 , 3x10 16 , 4x10 16 , 5x10 16 , 6x10 16 , 7x10 16 , 8x10 16 , 9x10 16 , 1x10 17 , 2x10 17 , 3x10 17 , 4x10 17 , 5x10 17 , 6x10 17 , 7x10 17 , 8x10 17 , 9x10 17 , 1x10 18 , 2x10 18 , 3x10 18 , 4x10 18 , 5x10 18 , 6x10 18 , 7x10 18 , 8x10 18 , 9x10 18 , 1x10 19 , 2x10 19 , 3x10 19 , 4x10 19 , 5x10 19 , 6x10 19 , 7x10 19 , 8x10 19 , 9x10 19 , 1x10 20 , 2x10 20 , 3x10 20 , 4x10 20 , 5x10 20 , 6x10 20 , 7x10 20 , 8x10 20 , 9x10 20 or more vector genome units.

[0233] In certain embodiments, the vectors contemplated herein are at least about 1 x 10 9 Genomic particles / mL, at least approximately 1x10 10 Genomic particles / mL, at least approximately 5x10 10Genomic particles / mL, at least approximately 1x10 11 Genomic particles / mL, at least approximately 5x10 11 Genomic particles / mL, at least approximately 1x10 12 Genomic particles / mL, at least approximately 5x10 12 Genomic particles / mL, at least approximately 6x10 12 Genomic particles / mL, at least approximately 7x10 12 Genomic particles / mL, at least approximately 8x10 12 Genomic particles / mL, at least approximately 9x10 12 Genomic particles / mL, at least approximately 10x10 12 Genomic particles / mL, at least approximately 15x10 12 Genomic particles / mL, at least approximately 20x10 12 Genomic particles / mL, at least approximately 25x10 12 Genomic particles / mL, at least approximately 50x10 12 Genomic particles / mL, or at least about 100x10 12 The titer of genome particles / mL is administered to the subject. When used in relation to virus titer, the term "genomic particle (gp)" or "genomic equivalent" or "genomic copy" (gc) refers to the number of virions that contain recombinant AAV DNA genome, regardless of whether they are infectious or functional. The number of genome particles in a particular vector preparation can be measured by methods well understood in the art, such as quantitative PCR of genome DNA, or as described in, for example, Clark et al. (1999) Hum.Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol.Ther., 6:272-278.

[0234] The vector of the present disclosure may be administered in a fluid volume. In some cases, the vector may be administered in a volume of about 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, 6.0mL, 7.0mL, 8.0mL, 9.0mL, 10.0mL, 11.0mL, 12.0mL, 13.0mL, 14.0mL, 15.0mL, 16.0mL, 17.0mL, 18.0mL, 19.0mL, 20.0mL, or more than 20.0mL. In some cases, the vector dose may be expressed as the concentration or titer of the vector administered to the subject. In this case, the vector dose may be expressed as the number of vector genome units per volume (i.e., genome units / volume).

[0235] In certain embodiments, the vectors contemplated herein contain at least about 5×10 9 Infectious units / mL, at least approximately 6x10 9 Infectious units / mL, at least approximately 7x10 9 Infectious units / mL, at least approximately 8x10 9 Infectious units / mL, at least approximately 9x10 9 Infectious units / mL, at least approximately 1x10 10 Infectious units / mL, at least approximately 1.5x10 10 Infectious units / mL, at least approximately 2x10 10 Infectious units / mL, at least approximately 2.5x10 10 Infectious units / mL, at least approximately 5x10 10 Infectious units / mL, at least approximately 1x10 11 Infectious units / mL, at least approximately 2.5x10 11 Infectious units / mL, at least approximately 5x10 11 Infectious units / mL, at least approximately 1x10 12 Infectious units / mL, at least approximately 2.5x10 12 Infectious units / mL, at least approximately 5x10 12 Infectious units / mL, at least approximately 1x10 13 Infectious units / mL, at least approximately 5x10 13 Infectious units / mL, at least approximately 1x1014 It is administered to subjects in a titer of infectious unit / mL. When used in relation to virus titer, the term "infectious unit (iu)", "infectious particle" or "replication unit" refers to the number of infectious and replicative recombinant AAV vector particles, as measured by infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J.Virol., 62:1963-1973.

[0236] In certain embodiments, the vectors contemplated herein contain at least about 5×10 10 Transducing units / mL, at least approximately 1x10 11 Transducing units / mL, at least approximately 2.5x10 11 Transducing units / mL, at least approximately 5x10 11 Transducing units / mL, at least approximately 1x10 12 Transducing units / mL, at least approximately 2.5x10 12 Transducing units / mL, at least approximately 5x10 12 Transducing units / mL, at least approximately 1x10 13 Transducing units / mL, at least approximately 5x10 13 Transducing units / mL, at least approximately 1x10 14 It is administered to subjects in a titer of transducing unit / mL.When used in relation to viral titer, the term "transducing unit" (tu) refers to the number of infectious recombinant AAV vector particles that results in the production of functional transgene product, as measured in functional assays such as those described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0237] Vector dosage is generally determined by the route of administration. In a particular embodiment, intraganglionic injections are administered in a volume of about 0.1 mL to about 1.0 mL, with a dose of about 1 x 10 9 ~ approx. 1x10 13In another example, the intrathecal injection may contain about 1x10 vector genomes in a volume of about 1.0 mL to about 12.0 mL. 10 ~ approx. 1x10 15 In yet another particular example, the intracranial injection may comprise about 1x10 vector genomes in a volume of about 0.1 mL to about 1.0 mL. 9 ~ approx. 1x10 13 In another particular example, the intraneural injection may contain about 1x10 vector genomes in a volume of about 0.1 mL to about 1.0 mL. 9 ~ approx. 1x10 13 In another specific embodiment, the intrathecal injection can include about 1x10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~ approx. 1x10 13 In yet another particular example, the cisterna magna injection can include about 5x10 vector genomes in a volume of about 0.5 mL to about 5.0 mL. 9 ~about 5x10 13 In yet another particular example, the subcutaneous injection can contain about 1x10 vector genomes in a volume of about 0.1 mL to about 1.0 mL. 9 ~ approx. 1x10 13 It may contain a vector genome.

[0238] In some cases, the vector is delivered to the subject by injection. The vector dose delivered to the subject by injection can be measured as the vector infusion rate. Non-limiting examples of vector infusion rates include: 1-10 μL / min for intraganglionic, intraspinal, intracranial, or intraneural administration, and 10-1000 μL / min for intrathecal or intracisternal administration. In some cases, the vector is delivered to the subject by MRI-guided convection-enhanced delivery (CED). This technique allows for increased viral diffusion and transduction across a larger portion of the brain, and reduces vector reflux along the needle track.

[0239] In various embodiments, a method is provided that includes administering to one or more neural cells that increase pain sensation or sensitivity to pain a vector encoding an engineered receptor that inactivates or hyperpolarizes the neural cell, and administering to a subject a ligand that specifically binds to the neural cell expressing the engineered receptor, thereby inactivating the cell, decreasing sensitivity to pain, and enhancing the analgesic effect.

[0240] In various embodiments, a method is provided that includes administering to one or more neural cells that reduces pain sensation or sensitivity to pain a vector encoding an engineered receptor that activates or polarizes the neural cells, and administering to a subject a ligand that specifically binds to the neural cells expressing the engineered receptor, thereby activating the cells, reducing sensitivity to pain, and enhancing the analgesic effect.

[0241] The formulation of the ligand may be administered to the subject by various routes. Non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, transdermal administration, intradermal administration, intraperitoneal administration, oral administration, injection, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, and intraneural administration. In some cases, administration can include injection of a liquid formulation of the ligand. In other cases, administration can include oral delivery of a solid formulation of the ligand. In certain cases, the ligand is administered by oral administration (e.g., pills, tablets, capsules, etc.). In some cases, the oral composition can be administered with food. In another particular case, the ligand is administered by intrathecal injection (i.e., intrathecal space of the spinal cord) for delivery to the cerebrospinal fluid (CSF) of the subject. In another particular case, the ligand is administered locally (e.g., skin patch, cream, lotion, ointment, etc.).

[0242] The dose of the ligand administered to the subject is not subject to absolute limitations, but depends on the nature of the composition and its active ingredients, as well as its undesirable side effects (e.g., immune response to antibodies), the subject being treated, and the type of condition being treated and the method of administration. In general, the dose is a therapeutically effective amount, such as an amount sufficient to achieve a desired biological effect, for example, an amount effective to reduce or alleviate the level of pain experienced by the subject. In certain embodiments, the dose may also be a prophylactic or effective amount. The therapeutically effective amount of the ligand may depend on the route of administration, the indication being treated, and / or the ligand selected for use.

[0243] In one embodiment, the ligand is first administered to the subject before the administration of the vector. A therapeutically effective amount of the ligand may be administered to the subject at some point after the delivery of the vector. Generally, after the delivery of the vector, there is a period of time that is required for one or more cells of the subject to produce the protein (i.e., the engineered receptor) encoded by the vector. During this period, administration of the ligand to the subject may not be beneficial to the subject. In this situation, it may be appropriate to administer the ligand after a certain amount of the engineered receptor is produced by one or more cells of the subject.

[0244] In one embodiment, the ligand is administered first, at approximately the same time that the vector is administered to the subject.

[0245] In one embodiment, the ligand is first administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 hours, days, weeks, months, or years after the subject is administered the vector. In some cases, the therapeutically effective amount of the ligand may be administered to the subject at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 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 delivery of the vector. In certain examples, the therapeutically effective amount of the ligand is administered to the subject at least one week after delivery of the vector. In further examples, the therapeutically effective amount of the ligand is administered to the subject daily for at least three consecutive days.

[0246] The therapeutically effective amount or dose of the ligand of the present disclosure may be expressed as mg or μg of ligand per kg of the subject's body weight. In some cases, the therapeutically effective amount of the ligand is about 0.001 μg / kg, about 0.005 μg / kg, about 0.01 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 ...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 μ 0μg / kg, approximately 60μg / kg, approximately 70μg / kg, approximately 80μg / kg, approximately 90μg / kg, approximately 100μg / kg, approximately 120μg / kg, approximately 140μg / kg, approximately 160μg / kg, approximately 180μg / kg, approximately 200μg / kg, approximately 220μg / kg, approximately 240μg / kg, approximately 260μg / kg, approximately 280μg / kg, approximately 300μg / kg, approximately 320μg / kg, approximately 340μg / kg, approximately 360μg / kg, approximately 380μg / kg, approximately 400μ g / kg, approximately 420μg / kg, approximately 440μg / kg, approximately 460μg / kg, approximately 480μg / kg, approximately 500μg / kg, approximately 520μg / kg, approximately 540μg / kg, approximately 560μg / kg, approximately 580μg / kg, approximately 600 μg / kg, approximately 620 μg / kg, approximately 640 μg / kg, approximately 660 μg / kg, approximately 680 μg / kg, approximately 700 μg / kg, approximately 720 μg / kg, approximately 740 μg / kg, approximately 760 μg / kg, approximately 780 μg / kg, approximately 80 The dose can be about 0 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 greater than 10 mg / kg.

[0247] In certain embodiments, the dose of the ligand administered to the subject is at least about 0.001 micrograms / 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 / 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 mg / kg or more.

[0248] In certain embodiments, the dose of ligand administered to the subject is at least about 0.001 μg / kg to at least about 10 mg / kg, at least about 0.01 μg / kg to at least about 10 mg / kg, at least about 0.1 μg / kg to at least about 10 mg / kg, at least about 1 μg / kg to at least about 10 mg / kg, at least about 0.01 mg / kg to at least about 10 mg / kg, at least about 0.1 mg / kg / at least about 10 mg / kg, or at least about 1 mg / kg to at least about 10 mg / kg, or any intervening ranges thereof.

[0249] In some embodiments, a therapeutically effective amount of a ligand may be expressed as a molar concentration (i.e., M or mol / L). In some cases, a therapeutically effective amount of a ligand may 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, M, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM or more.

[0250] The therapeutically effective amount of the ligand can be administered one or more times a day. In some cases, the therapeutically effective amount of the ligand is administered as needed (e.g., when pain relief is required). The ligand may be administered continuously (e.g., daily without interruption during the treatment regimen). In some cases, the treatment regimen can be less than one week, one week, two weeks, three weeks, one month, or more than one month. In some cases, the therapeutically effective amount of the ligand is administered for one day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or more than at least ten consecutive days. In certain cases, the therapeutically effective amount of the ligand is administered for three consecutive days. In some cases, a therapeutically effective amount of the ligand can be administered once per week, twice per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per week, nine times per week, ten times per week, eleven times per week, twelve times per week, thirteen times per week, fourteen times per week, fifteen times per week, sixteen times per week, seventeen times per week, eighteen times per week, nineteen times per week, twenty times per week, twenty-five times per week, thirty-five times per week, thirty-five times per week, forty times per week, or more than forty times per week. In some cases, a therapeutically effective amount of the ligand can be administered once per day, twice per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, ten times per day, or more than ten times per day. In some cases, a therapeutically effective amount of the ligand is administered at least every 1 hour, at least every 2 hours, at least every 3 hours, at least every 4 hours, at least every 5 hours, at least every 6 hours, at least every 7 hours, at least every 8 hours, at least every 9 hours, at least every 10 hours, at least every 11 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.Doses of the ligand may be administered to the subject continuously, or 1, 2, 3, 4, or 5 times per day, 1, 2, 3, 4, 5, 6, or 7 times per week, 1, 2, 3, or 4 times per month, once every 2, 3, 4, 5, or 6 months, or once per year, or even at longer intervals. Treatment periods may last for 1 day, 1, 2, or 3 weeks, 1, 2, 3, 4, 5, 7, 8, 9, 10, or 11 months, 1, 2, 3, 4, 5 years or more, or longer.

[0251] The subject treated by the methods and compositions disclosed herein may be a human or a non-human animal. The term "treat" and its grammatical equivalents as used herein generally refer to the use of a composition or method to reduce, eliminate, or prevent the symptoms of disease, including achieving therapeutic benefit and / or prophylactic benefit. Therapeutic benefit means slowing, stopping, reversing, or eradicating or improving the progression of the symptoms of the disorder or condition being treated. Prophylactic benefit of treatment includes reducing the risk of a condition, slowing the progression of a condition, or reducing the likelihood of the occurrence of a condition.

[0252] Non-limiting examples of non-human animals include non-human primates, livestock animals, household pets, and laboratory animals. For example, the non-human animal may be a hominoid (e.g., chimpanzee, baboon, gorilla, or orangutan), an old world monkey (e.g., rhesus monkey), a new world monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a llama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal. The compositions and methods described herein are suitable for treating veterinary animals. Veterinary animals may include, but are not limited to, dogs, cats, horses, cows, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some aspects, contacting a tissue or cell population with the composition includes administering the composition to the cell population or subject. In some embodiments, the administration is performed in vitro, for example, by adding the composition to a cell culture system. In some embodiments, administration is performed in vivo, for example, by administration via a particular route. When two or more compositions are administered, the compositions can be administered via the same route at the same time (e.g., on the same day), or via the same route at different times. Alternatively, the compositions can be administered via different routes at the same time (e.g., on the same day), or via different routes at different times.

[0253] The number of times that the composition is administered to the subject in need thereof depends on the medical professional's discretion, the disorder, the severity of the disorder, and the subject's response to the formulation.In some embodiments, the composition is administered at least once.In further embodiments, the administration is, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times during a given period.The dosage and / or frequency of each administration can be adjusted as necessary based on the patient's condition and physiological response.

[0254] In some embodiments, the composition may be administered a sufficient number of times to achieve a desired physiological effect or improvement in the subject's condition. If the subject's condition does not improve, at the physician's discretion, the composition may be administered chronically, i.e., for an extended period of time, including the subject's entire life, to improve or otherwise control or limit the symptoms of the subject's disease or condition. If the subject's condition improves, at the physician's discretion, the composition may be administered continuously, or the dose of the administered drug may be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug holiday"). The drug holiday period may vary 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. Dose reductions during drug holidays may be from 10% to 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%.

[0255] When a composition is administered multiple times, each administration may be performed by the same practitioner and / or in the same geographic location, or each administration may be performed by a different practitioner and / or in a different geographic location.

[0256] For human and veterinary treatments, the amount of a particular agent administered may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular agent used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular agent used; the duration of treatment; drugs used in combination with or incidentally with the particular agent used; the judgment of the prescribing physician or veterinarian; as well as factors known in the medical and veterinary arts. Similarly, the effective concentration of a given composition may depend on a variety of factors, including the age, sex, weight, genetic status, and overall health of the patient or subject.

[0257] Tables 2-8 below list the rate of quenching of YFP fluorescence following stimulation of the indicated engineered receptors with various doses of either acetylcholine or non-natural ligands. [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0258] Tables 9-11 below list the EC50 for the indicated engineered receptors, calculated by different techniques as indicated. [Table 17] [Table 18] [Table 19] Table A below lists the double mutants disclosed herein. [Table 20]

[0259] All articles, publications, and patents cited herein are incorporated by reference as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not an admission, nor should it be construed as any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0260] Unless the context indicates otherwise, it is specifically contemplated that the various features described herein can be used in any combination.

[0261] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0262] It is to be understood that the foregoing description and the following examples are intended to illustrate, but not limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. EXAMPLES

[0263] Example 1. Discovery of engineered receptors containing mutations in the ligand-binding domain To generate LGICs that conduct anion currents after exposure to non-natural small molecule agonists of human α7-nAChR, a chimeric ligand-gated ion channel (LGIC) receptor was engineered, containing the ligand-binding domain from human α7 nicotinic acetylcholine receptor (α7-nAChR) and the chloride-conducting ion pore domain from human GlyR1α. An engineered receptor with the amino acid sequence of SEQ ID NO:33 was identified, which was nearly as sensitive to acetylcholine, ABT-126, and TC-6987 as wild-type α7-nAChR, and TC-6987 showed partial agonist activity to SEQ ID NO:33 as wild-type. SEQ ID NO:33 was approximately 2-fold less sensitive to nicotine and approximately 3-fold and 10-fold more sensitive to AZD-0328 and facinicline / RG3487, respectively, compared to wild-type.

[0264] Amino acid substitutions were introduced into the ligand-binding domain of the engineered receptor having the amino acid sequence of SEQ ID NO: 33. The binding pocket of each ligand of the α7-nAChR was modeled and the amino acid residues forming the binding pocket were mapped. A library of single, double and triple mutant chimeric LGICs was then generated, each mutant chimeric LGIC containing a substitution in one or more amino acids of the ligand-binding pocket of SEQ ID NO: 33. The parent chimeric receptor (SEQ ID NO: 33) was cloned into pcDNA3.1(+) (Invitrogen) using standard molecular biology techniques using the BamHI and EcoRI sites. The amino acid substitutions were introduced by site-directed mutagenesis.

[0265] All the resulting engineered receptors were analyzed for their potency against their 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).

[0266] Example 2: Characterization of engineered receptors using high-throughput fluorescence-based plate screening To screen these mutant LGICs for those with novel response profiles to ligands, we developed an anion reporter assay to assess channel function in a high-throughput format. In this assay, cells expressing a YFP reporter whose fluorescence is quenched in the presence of anions are transfected with DNA encoding the channel of interest. Upon exposure to ligand, activated channels flux anions, resulting in a dose-dependent quenching of YFP that is detectable on a plate reader.

[0267] Lenti-X 293T cells (LX293T, Clontech) were maintained in DMEM containing 10% FBS and 1% penicillin / streptomycin (Invitrogen). For plate reader assays, LX293T cells were infected with lentivirus to generate cells stably expressing a mutant YFP (H148Q / I152L) reporter that exhibits enhanced sensitivity to anions. Two days prior to 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 of DNA per well using the standard Fugene 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, mOsms300). After the final wash, 100μL of 1X ECS was added to the wells and the plate was incubated at 37°C for 30 minutes. While the plate was incubating, drugs were diluted to 2x concentration in 1X ECS-NaI (same ingredients as 1X ECS except 140mM NaCl was replaced with 140mM NaI). The plate was then read on a Flexstation3 (Molecular Devices). Each well of the plate, 8 wells at a time, is read for 2 minutes using the Flexstation3 (Molecular Devices) as follows: 1) baseline YFP fluorescence is read for 17 seconds, 2) 100μL of ligand is added, and 3) the change in YFP fluorescence is then measured every 1.3 seconds for the remainder of the time.

[0268] Figure 1 provides a heat map of the percent quenching of YFP fluorescence following stimulation with various doses of either acetylcholine or the non-natural ligands, as indicated. A positive fluorescent signal, as indicated by the blue filled cells, indicates that the engineered receptor is activated by the non-natural ligand at that concentration. The results show that the engineered receptors have various potencies against the non-natural ligands tested. Table 12 lists the EC50 values ​​of the indicated double mutants for acetylcholine (Ach) and TC-5619, as determined from YFP fluorescence plate reader experiments. [Table 21]

[0269] Example 3: Characterization of engineered receptors using high-throughput electrophysiology EC determined by plate reader 50To confirm and better understand maximal current flow, the engineered receptors were subjected to a high-throughput electrophysiology system, as described below. For HEK293T studies, cDNAs encoding ion channels were cloned into pcDNA3.1 using standard recombinant techniques. HEK293T cells (Lenti-X™ 293T cell line from Clontech) were cultured in DMEM supplemented with 10% FBS and 1% Pen / Strep to 40-50% confluence using standard cell culture protocols, transfected with ion channel plasmids at a concentration of 18 μg per 15 cm dish using Fugene 6, and grown for an additional 24 hours. Cells may then be assayed on electrophysiological systems (IonFluxHT and / or Mercury, Fluxion Biosciences) to assess dose-response relationships via a microfluidics-based platform to establish whole-cell configurations. Ensemble plates were primed with extracellular buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.2 with NaOH, mOsm 310), intracellular buffer (145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM EGTA, pH 7.2 with CsOH, mOsm 305), and test compounds (freshly prepared stocks) were diluted in extracellular buffer, as adapted from Lynagh and Lynch. Cells were then released from the plate using Accutase, centrifuged, resuspended in extracellular buffer, and loaded into the ensemble plate. Cells were then subjected to standard protocols for priming, trapping, breaking, and establishing the whole-cell configuration, and cells were held at -60 mV throughout the entire recording. After recording a baseline, progressive doses of test compound were applied using IonFlux software to assess dose-response relationships.The data was then analyzed offline using a custom Python script to convert the data to .csv format, replot the traces, and apply QC measures to reject unstable recordings (i.e., thresholding based on baseline access resistance and / or standard deviation, and artifact rejection). Peak currents were then calculated, and the population data was fitted using a four-parameter logistic equation described by the Hill equation. After adding drugs for 1 second, currents were measured with an automated patch clamp system (Fluxion Biosciences), and the calculated EC50 values ​​are tabulated in Table 13 below. [Table 22]

[0270] These results show that all engineered receptors have reduced potency for acetylcholine compared to wild-type nAchRa7. For example, some engineered receptors have EC50 values ​​that are several orders of magnitude higher than the EC50 of wild-type nAchRa7. Furthermore, the results show that some of the engineered receptors have increased potency for certain non-natural ligands compared to wild-type receptors. For example, engineered receptors containing the amino acid sequence L131D, S172D of SEQ ID NO: 33 show at least 10-fold increased potency for AZD-0328 and RG-3487 compared to wild-type control receptors. These results show that engineered receptors can be used to reduce potency for acetylcholine while retaining or increasing potency for synthetic small molecule nAChα7 receptor agonists that are recognized as safe and well tolerated in humans.

[0271] These results from the electrophysiological methods provide confirmation of the EC50 values ​​from the plate reader and further confirm the decoupling of the acetylcholine and non-native ligand responses of the engineered receptors disclosed herein.

[0272] Example 4: Characterization of engineered receptors using manual patch clamp electrophysiology Whole-cell manual patch clamp electrophysiology in HEK293 cells and rat DRG neurons was used to measure the EC50 of various engineered receptors disclosed herein for Ach ligands and non-natural ligands. Rheobase shift was also measured in rat DRG neurons, which reflects the efficacy of the receptor. To calculate the rheobase, first the amount of current capable of generating an action potential was determined. Ligand was then added, followed by a stepwise increase in the injected current to 700 pA. The current injected after the addition of the ligand was divided by the original current injected to obtain the action potential to calculate the rheobase. The EC50 was calculated by measuring the peak current from increasing doses of the ligand and using the Hill equation.

[0273] The results are summarized in Table 14 below. These results confirm that the engineered receptors disclosed herein have very low potency against Ach, but high potency and efficacy against non-natural ligands such as RG3487 and TC5619. See also Example 6. [Table 23]

[0274] Example 5: Localization of Engineered Receptors The efficiency of the engineered receptor disclosed herein localizing to the cell surface was evaluated using two methods. First, the HA-tagged engineered receptor was expressed in HEK293T cells, and its surface expression was monitored using a fluorescently tagged HA antibody. Second, the engineered receptor was bound using fluorescently labeled α-bungarotoxin, which specifically binds to an amino acid on the engineered receptor (CHNRA7). Both of these methods, followed by the use of flow cytometry, allowed confirmation of the surface localization of the engineered receptor.

[0275] Monoclonal antibody anti-HA-PeCy7 (16B12) was purchased from Biolegend (San Diego, CA). The clone names of the monoclonal antibodies are listed in parentheses. Alpha-bungarotoxin conjugated to biotin, Alexa Fluor 647 were all purchased from Thermo / Fisher (Waltham MA). Briefly, HEK-293T were plated at 200,00 cells per well and transfected the next day with Fugene 6 at a DNA to Fugene ratio of 1:3. Cells were analyzed the day after transfection using flow cytometry. For flow cytometry analysis, transfected HEK293T cells were lifted using 0.05% trypsin and 0.02% EDTA (Thermo / Fisher), washed, and incubated with antibodies (30 min, 1:100) or α-bungarotoxin (1 h, 1:1000) in FACS buffer (2% BSA, 1X PBS without Ca+ and Mg+, and 1X penicillin-streptomycin). 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). Data presented are normalized to the percentage of cells positive for HA-tag fluorescence or α-bungarotoxin staining, and the median fluorescence intensity of the parental chimeric receptor containing the amino acid sequence of SEQ ID NO: 33. See Table 1.

[0276] FIG. 5 and Table 1 show the percentage of HA tag positive cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO:33 ("Average HA Tag %), and the percentage of α-bungarotoxin positive cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO:33 ("Normalized AB %").

[0277] Figure 5 and Table 15 also show the median fluorescence intensity (MFI) of cells expressing the engineered receptor normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33, assessed using an anti-HA antibody ("mean HA MFI") or fluorescently labeled alpha-bungarotoxin conjugated to Alexa Fluor 647 ("normalized AB MFI"). Different point mutations can affect the detection of both HA and alpha-bungarotoxin by flow cytometry. [Table 24]

[0278] The results show that the mutations of engineered receptors disclosed herein affect cell surface localization. The localization of some double mutants is comparable to that of the parent chimera (SEQ ID NO: 33), while others have reduced cell surface localization compared to the parent chimera (SEQ ID NO: 33). For example, engineered receptors with L131T, S172D mutations show comparable localization to the parent chimera as assessed by HA tag. Furthermore, engineered receptors with Y115D, L131E mutations show comparable localization to the parent chimera as assessed by both techniques, i.e., HA tag and α-bungarotoxin.

[0279] Example 6: Characterization of CR-11 engineered receptors A high-throughput electrophysiology platform demonstrated that an engineered receptor containing an amino acid sequence with the amino acid substitutions Y115D and L131Q of SEQ ID NO:33 had a greater than 500-fold decrease in potency towards acetylcholine compared to the wild-type receptor, and a greater than 10-fold increase in its potency towards RG-3487.

[0280] Whole-cell manual patch clamp electrophysiology was used to confirm that engineered receptors containing amino acid sequences with amino acid substitutions Y115D and L131Q of SEQ ID NO:33 were essentially insensitive to acetylcholine but much more sensitive to RG-3487 compared to wild-type nAchRα7 receptors in both HEK293 cells and cultured rat dorsal root ganglion (DRG) sensory neurons (see FIG. 2).

[0281] In HEK293 cells, the EC for ACh of an engineered receptor containing an amino acid sequence with the amino acid substitutions Y115D and L131Q of SEQ ID NO:33 was 50 The EC value of the wild-type nAchRα7 receptor for ACh could not be determined because ACh concentrations up to 100 mM barely generated any current. 50 Further confirming the high-throughput data, manual patch clamp electrophysiology results demonstrated that the engineered receptor containing an amino acid sequence with the amino acid substitutions Y115D and L131Q of SEQ ID NO:33 inhibited the wild-type nAchα7 receptor (EC 50 = 2.9 μM), compared with RG-3487 (SA-2) (EC 50 = 0.3 μM) (Figure 2B).

[0282] In cultured adult rat DRG neurons expressing an engineered receptor containing an amino acid sequence with the Y115D and L131Q amino acid substitutions of SEQ ID NO:33, application of RG-3487 (SA-2) induced chloride currents in a dose-dependent manner (Figure 3), whereas no such currents were observed in non-transduced cells.

[0283] Furthermore, in transduced rat DRG neurons, activation of an engineered receptor containing an amino acid sequence with the Y115D and L131Q amino acid substitutions of SEQ ID NO:33 with RG-3487 (SA-2) at a concentration of 3 μM reversibly inhibited action potentials evoked by current injection (FIG. 4A) and increased the current required to evoke an action potential by approximately 3-fold (n=7). Acetylcholine at 3.0 mM did not affect evoked action potentials in transduced neurons (n=4) (FIG. 4B). These results indicate that expression of an engineered receptor containing an amino acid sequence with the Y115D and L131Q amino acid substitutions of SEQ ID NO:33 in DRG neurons can inhibit evoked action potentials.

[0284] Example 7. Characterization of engineered receptors in IPSC-derived neurons (predictive) Differentiation protocols have been developed to generate IPSC-derived Aβ neurons. The following markers are used to define cells as Aβ neurons: expression of neurofilament 200 (NF200), which delineates myelinated primary afferent neurons (Basbaum et al., 2009), Piezo2, a marker of low-threshold mechanoreceptor sensory neurons (LTMRs) (Ranade et al., 2014), and TLR5, a Toll-like receptor that has been reported to also mark Aβ fibers (Xu et al., 2015). Characterization also includes evaluation for the absence of the nociceptor-specific marker TrpV1, expressed in many C and Aβ fibers (Caterina et al., 1997), prostatic acid phosphatase, which delineates nonpeptidergic unmyelinated afferents (Zylka et al., 2009), and NaV1.1, a marker of Aβ nociceptive neurons. IPSC-derived neurons fulfilling the above criteria would be further characterized as either rapidly adapting LTMRs based on c-Ret and MafA / c-Maf expression, or slowly adapting LTMRs based on TrkB and Shox2 expression, in the absence of c-Ret expression ( Koch et al., 2018 ).

[0285] (1) Confirm that cells that meet the above expression marker criteria for Aβ neurons have electrophysiological properties characteristic of non-nociceptive sensory neurons, including the ability to generate electrical currents in response to ligands and to elicit action potentials upon direct current injection.

[0286] (2) Confirm the presence of chloride currents in neurons transduced with selective chemogenic receptors (engineered receptors). Cells are transduced with lentiviral vectors encoding HA-tagged chemogenic receptors with IRES-GFP and transduced cells are identified via GFP fluorescence. Chloride currents in response to synthetic agonists are detected in voltage clamp mode with NMDG+ as the internal and external fluid cation. Since NMDG+ is impermeable to cation channels, its inclusion removes any endogenous nAchRα7 cation currents in response to the studied synthetic agonist. The EC50 of the chemogenic receptor-synthetic agonist pair is then determined. After recording, receptor expression and cell surface localization are assessed by fluorescence microscopy in permeabilized and non-permeabilized cells using antibodies directed against the HA tag.

[0287] (3) Assess the ability to inhibit current injection-evoked action potentials. Cells are transduced with a lentiviral vector encoding an HA-tagged chemogenic receptor with an IRES GFP and transduced cells are identified via GFP fluorescence. In current clamp mode, the rheobase in the presence and absence of synthetic agonist is determined by continuing to apply a current until the cell membrane depolarizes. Results are compared to cells transduced with GFP alone.

[0288] (4) Evaluate the effect on input resistance. Cells are transduced with a lentiviral vector encoding an HA-tagged chemogenic receptor with an IRES-GFP and cells are identified via GFP fluorescence. In current clamp mode, subthreshold currents are injected and changes in membrane voltage are determined to calculate input resistance. Results are compared to cells transduced with GFP only.

[0289] (5) Evaluate the effect on the resting membrane potential in the presence and absence of a synthetic agonist. Cells are transduced with a lentiviral vector encoding an HA-tagged chemogenic receptor with an IRES GFP and transduced cells are identified via GFP fluorescence. In voltage clamp mode, the resting membrane potential is determined in the presence and absence of a synthetic agonist. Results are compared to cells transduced with GFP alone.

[0290] The above electrophysiological properties in IPSC-derived Aβ neurons are compared with those in IPSC-derived C-fiber neurons as well as adult rat DRG neurons. Furthermore, because biochemical changes in injured Aβ fiber afferents may contribute to spontaneous pain in neuropathic conditions, the electrophysiological properties of IPSC-derived Aβ neurons are investigated after injury in vitro. To produce injury in vitro, cells are harvested and reseeded after extending the processes in culture. The reseeding process severs the processes, mimicking axonal injury. At various time points after injury, cells are evaluated for various electrophysiological properties, including spontaneous action potential generation, changes in resting membrane potential, and changes in rheobase. The effects of chemogenic receptors are also evaluated in injury conditions.

[0291] Example 8. Evaluating the efficacy of engineered receptors to treat disease in animal models The engineered receptors disclosed herein are evaluated for their ability to provide analgesia in a rat model of neuropathic pain following administration of a small molecule ligand. AAV expression cassettes containing the human synapsin-1 (hSYN) promoter linked to a polynucleotide encoding either the wild-type α7-nAChR or the engineered chimeric receptors disclosed herein are constructed using standard molecular biology techniques.

[0292] These AAV expression cassettes are subcloned into AAV bacmids, purified, and transfected into Sf9 insect cells to produce recombinant baculoviruses that are then amplified. Sf9 cells are dually infected with the amplified recombinant baculoviruses containing the wild-type α7-nAChR or any one of the engineered chimeric receptor cassettes described above, and another recombinant baculovirus containing Rep and AAV6 (Y705+731F+T492V) cap genes to produce recombinant AAV vectors. The viral vectors are purified and viral titers are determined using qPCR, and AAV vector purity is verified using SDS-PAGE.

[0293] Behavioral experiments and pain models: To induce mechanical hypersensitivity in a model that mimics neuropathic pain states, the Spared Nerve Injury (SNI) model (a validated model of mechanical allodynia) is used (Shields et al., 2003, The Journal of Pain, 4, 465-470). This model is created by transection of the common peroneal and sural nerves and isolation of the tibial branch. Mechanical withdrawal thresholds are assessed by placing rats on an elevated wire mesh grid and stimulating the plantar surface of the hind paw with von Frey filaments.

[0294] AAV injection into the rat spinal cord: A dorsal hemilaminectomy is performed at the level of the lumbar enlargement to expose two segments of the lumbar spinal cord (approximately 1.5-2 mm), after which the dura is incised and reflected. The virus solution is loaded into a glass micropipette (pre-filled with mineral oil). The micropipette is connected to a manual microinjector attached to a stereotaxic device. The virus solution targets the dorsal horn (left side). Six injections of 240 nl each are performed equidistantly linearly along the rostral-caudal axis within the exposed area. After each injection, a 1 min rest period is observed, then the muscle layer is sutured, the skin is closed with staples, and the animal is allowed to recover on a heating pad before being returned to its home cage. The animal is perfused for histological analysis after the last behavioral test.

[0295] Intraganglionic injection of AAV into the dorsal root ganglion (DRG) of rats: Injections are performed with a borosilicate glass capillary (inner / outer diameter 0.78 / 1 mm) drawn to a fine tip, attached by polyethylene tubing (inner / outer diameter 0.4 / 0.8 mm) to a syringe attached to a microinjection pump. The needle is mounted on an extension arm of an outwardly swung stereotaxic frame (used only to hold and manipulate the needle). The tubing, syringe, and needle are all filled with water. One microliter of air is taken up into the needle, followed by 3 µL of viral vector solution. For each injection, fill the needle with this volume separately. Animals are anesthetized prior to surgery. After an incision along the dorsal midline, the L4 and L5 DRG are exposed by removal of the lateral processes of the vertebrae. The epineurium overlying the DRG is opened and a glass needle is inserted into the ganglion to a depth of 400 µm from the surface of the exposed ganglion. After a 3 min delay to allow for sealing of the tissue around the glass capillary tip, 1.1 μL of virus solution is injected at a rate of 0.2 μL / min. After a further 2 min delay, the needle is removed. The L4 ganglion is injected first, followed by the L5 ganglion. The muscles overlying the spinal cord are loosely sutured with 5-0 sutures and the wound is closed. The animals are allowed to recover at 37 °C and receive post-operative analgesia.

[0296] AAV intrathecal injection in rats: The rat is first anesthetized and then positioned vertically with its head fixed in a stereotaxic frame. An incision is made at the base of the neck to expose the nuchal crest groove. An incision (1-2 mm) is made in the cisternal membrane to a depth at which cerebrospinal fluid leaks out. A 4 cm 32 G intrathecal catheter is then slowly inserted in the direction of the lumbar spinal cord and the skin is closed by sutures around the catheter. The rat is then allowed to recover. The rat is then anesthetized and the vector (6 µL) is administered. The catheter is flushed with 6 µL of PBS and then removed, and the rat is allowed to recover.

[0297] Effects of Administration: This SNI model is created by transection of the common peroneal and sural nerves in rats and isolation of the tibial branch. Using the Chaplan & Yaksh up-down method, mechanical thresholds are determined before injection of AAV.hSYN-α7-nAChR / GlyRα1 into the spinal cord, DRG, or intrathecal space. Three weeks after unilateral vector injection, animals are tested again to ensure that their mechanical withdrawal thresholds remain unchanged. Motor coordination is also tested before and after injection using an accelerating rotarod (Stoelting, USA) with a maximum speed of 33 rpm. The time the rats spend on the rotarod is recorded, with a cutoff at 300 s. Each rat undergoes three training trials and is tested two hours later.

[0298] Half of the rats in each chimeric cohort are then administered a single IP injection of AZD-0328 or facinicline and mechanical thresholds are tested using the up-down method 1, 2, 5, 7, and 13 days after IP injection. On the third day, when thresholds have returned to post-injury baseline, AZD-0328 is again IP injected and again a return to pre-injury baseline thresholds is observed. These animals are followed for 48 hours. Animals are then perfused for histology.

[0299] Example 9: Treatment of patients suffering from chronic pain In a non-limiting example, a patient suffering from chronic radicular pain is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV.hSYN operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to one or more dorsal root ganglia (i.e., intraganglionic convection-enhanced delivery to lumbar, cervical, or thoracic DRG). 13The patient is treated with a vector genome. In this example, the AAV vector encodes any one of the engineered receptors disclosed herein under the control of the human synapsin-1 (SYN1) promoter for selective neuronal expression. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.1 mg / kg of AZD-0328 or another non-natural ligand as needed (i.e., during pain episodes).

[0300] Example 10. Treatment of patients suffering from chronic pain In a non-limiting example, a patient suffering from chronic craniofacial pain (e.g., trigeminal neuralgia or temporomandibular joint dysfunction) is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV.hSYN operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 0.150 mL delivered directly to the trigeminal ganglion (i.e., intraganglionic convection-enhanced delivery). 13 The patient is treated with a vector genome. In this example, the AAV vector encodes any one of the engineered receptors disclosed herein under the control of the human synapsin-1 (SYN1) promoter for selective neuronal expression. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.1 mg / kg of AZD-0328 or another non-natural ligand as needed (i.e., during pain episodes).

[0301] Example 11. Treatment of Patients Suffering from Obesity In a non-limiting example, a patient suffering from obesity is treated using the compositions and methods disclosed herein. The patient receives 10 doses of AAV on day 1. 13The patient is treated with a vector genome of 0.1 mg / kg AZD-0328 or another non-natural ligand. Ghrelin is operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the gastric branch of the vagus nerve (i.e., intraneurally). In this example, the AAV vector encodes the engineered receptor under the control of the human ghrelin promoter for selective neuronal expression. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.1 mg / kg AZD-0328 or another non-natural ligand daily for excessive weight loss (i.e., appetite suppression).

[0302] Example 12. Treatment of Patients Suffering from Obesity In a non-limiting example, a patient suffering from obesity is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-TRPV1 operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the dorsal root ganglion (i.e., intraganglion) that innervates the pancreas. 13 The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human TRPV1 promoter for selective neuronal expression in nociceptors. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.1 mg / kg of AZD-0328 or another non-natural ligand daily for excessive weight loss.

[0303] Example 13. Treatment of patients suffering from obesity In a non-limiting example, a patient suffering from obesity is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-SIM1 operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the paraventricular nucleus of the hypothalamus (PVH) (i.e., intracranial convection-enhanced delivery). 13The patient is treated with a vector genome. In this example, the AAV vector encodes a channel engineered under the control of the human single mind family BHLH transcription factor 1 (SIM1) promoter for selective neuronal expression in proopiomelanocortin (POMC) neurons and ultimately stimulation of the appetite suppression pathway. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.15 mg / kg of AZD-0328 or another non-natural ligand daily for excessive weight loss (i.e., appetite suppression).

[0304] Example 14. Treatment of patients with PTSD In a non-limiting example, a patient suffering from post-traumatic stress disorder (PTSD) is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-hSYN1 operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the C6 stellate ganglion (i.e., intraganglionally). 13 The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human synapsin-1 (hSYN1) promoter for selective neuronal expression. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.15 mg / kg of AZD-0328 or another non-natural ligand daily for PTSD symptoms (i.e., anxiety).

[0305] Example 15. Treatment of patients suffering from depression In a non-limiting example, a patient suffering from treatment-resistant depression (TRD) is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-hSYN1 operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the vagus nerve (i.e., intraneurally). 13The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human synapsin-1 (hSYN1) promoter for selective neuronal expression. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.1 mg / kg of AZD-0328 or another non-natural ligand daily for depressive symptoms.

[0306] Example 16. Treatment of Patients with GERD In a non-limiting example, a patient suffering from gastroesophageal reflux disease (GERD) is treated using the compositions and methods disclosed herein. On day 1, the patient receives a 1.0 mL volume of AAV-hSYN1 operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein, or a 10 mL volume of AAV-CAG operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein, delivered directly to the lower esophageal sphincter (LES), vagus nerve, and myenteric plexus (i.e., intraneurally) or smooth muscle (intramyomyelinally), respectively. 13 The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human synapsin-1 (hSYN1) promoter for selective neuronal expression or the CAG promoter for expression in LES muscle cells. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328 or another non-natural ligand. The patient orally self-administers 0.15 mg / kg AD-0328 or another non-natural ligand daily for symptoms of GERD (i.e., acid reflux).

[0307] Example 17. Treatment of patients with epilepsy In a non-limiting example, a patient suffering from seizures associated with epilepsy is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-CamKIIα operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to a predefined seizure focus (i.e., intracranially), such as the motor cortex. 13 The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human calcium / calmodulin-dependent protein kinase II alpha (CamKII alpha) promoter for selective neuronal expression in excitatory neurons. Two weeks after injection, the patient returns to the clinic for a prescription for AZD-0328. The patient self-administers 0.1 mg / kg of AZD-0328 orally daily for epilepsy symptoms (i.e., seizures).

[0308] Example 18. Treatment of patients suffering from movement disorders In a non-limiting example, a patient suffering from a movement disorder (e.g., Parkinson's tremor) is treated using the compositions and methods disclosed herein. On day 1, the patient receives 10 mL of AAV-CamKIIα operably linked to a polynucleotide encoding any one of the engineered receptors disclosed herein in a volume of 1.0 mL delivered directly to the subthalamic nucleus (i.e., intracranial STN). 13 The patient is treated with a vector genome. In this example, the AAV vector encodes an engineered receptor under the control of the human calcium / calmodulin-dependent protein kinase IIα (CamKIIα) promoter for selective neuronal expression in excitatory neurons. Two weeks after injection, the patient returns to the clinic for a prescription of AZD-0328. The patient self-administers 0.1 mg / kg of AZD-0328 orally daily for movement disorder symptoms (i.e., tremors).

[0309] Further Numbered Embodiments Further embodiments of the present invention are provided in the following numbered embodiments. EMBODIMENT 1 An engineered receptor, a ligand-binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR) and containing the Cys-loop domain from the human glycine receptor α1 subunit; an ion pore domain derived from the human glycine receptor α1 subunit; the ligand-binding domain comprises (i) 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, or (ii) an amino acid substitution of L131E, wherein the amino acid residues correspond to amino acid residues in α7-nAChR; The engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor. Embodiment 1.1 2. The engineered receptor of embodiment 1, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence further comprises two amino acid substitutions in a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131, or an amino acid substitution of L131E, wherein the amino acid residues correspond to amino acid residues in an α7-nAChR. EMBODIMENT 2 2. The engineered receptor of embodiment 1 or 1.1, wherein 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. EMBODIMENT 3 3. The engineered receptor of any one of embodiments 1, 1.1, or 2, wherein 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. Embodiment 3.1 4. The engineered receptor of any one of embodiments 1-3, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and wherein the amino acid sequence further 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. EMBODIMENT 4 2. The engineered receptor of embodiment 1 or 1.1, wherein the ligand binding domain comprises the amino acid substitution L131E. Embodiment 4.1 5. The engineered receptor of any one of claims 1 to 4, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and wherein the amino acid sequence further comprises the amino acid substitution L131E. EMBODIMENT 5 2. The engineered receptor of any one of embodiments 1-4.1, wherein the Cys-loop domain comprises amino acids 166-172 of SEQ ID NO:2. EMBODIMENT 6 2. The engineered receptor of any one of embodiments 1-4.1, wherein the Cys-loop domain comprises amino acids 166-180 of SEQ ID NO:2. EMBODIMENT 7 7. The engineered receptor of any one of embodiments 1-6, wherein the receptor comprises the β1-2 loop domain from the human glycine receptor α1 subunit. EMBODIMENT 8 8. The engineered receptor of embodiment 7, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:2. Embodiment 8.1 9. The engineered receptor of any one of embodiments 1-8, wherein the engineered receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58-63. EMBODIMENT 9 The engineered receptor of any one of embodiments 1-8, wherein the potency of the engineered receptor for acetylcholine is lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. EMBODIMENT 10 The engineered receptor of embodiment 9, wherein the potency of the engineered receptor for acetylcholine is at least 2-fold lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. EMBODIMENT 11 The engineered receptor of any one of embodiments 1 to 10, wherein the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. EMBODIMENT 12 The engineered receptor of any one of embodiments 1-11, wherein the potency of the engineered receptor against the non-natural ligand is greater than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) against the non-natural ligand. EMBODIMENT 13 The engineered receptor of embodiment 12, wherein the potency of the engineered receptor against the non-natural ligand is at least 2-fold higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) against the non-natural ligand. EMBODIMENT 14 The engineered receptor of any one of embodiments 9-13, wherein determining the potency comprises determining the EC50. EMBODIMENT 15 The engineered receptor of any one of embodiments 1-14, wherein the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. EMBODIMENT 16 The engineered receptor of any one of embodiments 1-15, wherein the efficacy of the engineered receptor in the presence of the non-natural ligand is at least 2-fold higher than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. EMBODIMENT 17 The engineered receptor of any one of embodiments 15-16, wherein determining efficacy comprises determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand. EMBODIMENT 18 18. The engineered receptor of any one of embodiments 11-17, 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. EMBODIMENT 19 19. The engineered receptor of embodiment 18, wherein the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and APN-1125. EMBODIMENT 20 19. The engineered receptor of embodiment 18, wherein the non-natural ligand is TC-5619. EMBODIMENT 21 A polynucleotide comprising a nucleic acid encoding the engineered receptor of any one of embodiments 1 to 20. EMBODIMENT 22 22. The polynucleotide of embodiment 21, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the engineered receptor. EMBODIMENT 23 23. The polynucleotide of embodiment 22, wherein the promoter is a regulatable promoter. EMBODIMENT 24 24. The polynucleotide of embodiment 23, wherein the regulatable promoter is active in excitable cells. EMBODIMENT 25 The polynucleotide of embodiment 24, wherein the excitable cell is a neuron or a muscle cell. EMBODIMENT 26 The polynucleotide of embodiment 25, wherein the excitable cell is a neuron. EMBODIMENT 27 A vector comprising the polynucleotide according to any one of embodiments 21 to 26. EMBODIMENT 28 The vector of embodiment 27, wherein the vector is a plasmid or a viral vector. EMBODIMENT 29 The vector of embodiment 28, wherein the vector is a viral vector selected from the group consisting of an adenoviral vector, a retroviral vector, an adeno-associated viral (AAV) vector, and a herpes simplex viral vector 1 (HSV-1). EMBODIMENT 30 The vector of embodiment 29, wherein the viral vector is an AVV vector and the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof. EMBODIMENT 31 A composition comprising an engineered receptor according to any one of embodiments 1 to 20, a polynucleotide according to any one of embodiments 21 to 26, or a vector according to any one of embodiments 27 to 30. EMBODIMENT 32 A pharmaceutical composition comprising an engineered receptor according to any one of embodiments 1 to 20, a polynucleotide according to any one of embodiments 21 to 26, or a vector according to any one of embodiments 27 to 30, and a pharma- ceutically acceptable carrier. EMBODIMENT 33 A method for producing an engineered receptor in a neuron, the method comprising contacting a neuron with a polynucleotide according to any one of embodiments 21 to 26, a vector according to any one of embodiments 27 to 30, a composition according to embodiment 31, or a pharmaceutical composition according to embodiment 32. EMBODIMENT 34 The method of embodiment 33 or the polynucleotide of embodiment 26, wherein the neuron is a neuron of the peripheral nervous system. EMBODIMENT 35 The method of embodiment 33 or 34 or the polynucleotide of embodiment 26, wherein the neuron is a neuron of the central nervous system. EMBODIMENT 36 The method of any one of embodiments 33 to 35, or the polynucleotide of embodiment 26, wherein the neuron is a nociceptive neuron. EMBODIMENT 37 The method of any one of embodiments 33 to 36, or the polynucleotide of embodiment 26, wherein the neuron is a non-nociceptive neuron. EMBODIMENT 38 The method of any one of embodiments 33 to 37, or the polynucleotide of embodiment 26, 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. EMBODIMENT 39 The method of any one of embodiments 33 to 38, or the polynucleotide of embodiment 26, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers. EMBODIMENT 40 40. The method of embodiment 39, or the polynucleotide of embodiment 26, wherein the neuron is an Aδ afferent fiber. EMBODIMENT 41 The method of embodiment 40, or the polynucleotide of embodiment 26, wherein the Aβ afferent fibers are damaged Aβ afferent fibers. EMBODIMENT 42 The method of embodiment 40, or the polynucleotide of embodiment 26, wherein the Aβ afferent fibers are non-injured Aβ afferent fibers. EMBODIMENT 43 The method of any one of embodiments 33 to 42, or the polynucleotide of embodiment 26, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5. EMBODIMENT 44 The method of any one of embodiments 33 to 43, or the polynucleotide of embodiment 26, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1. EMBODIMENT 45 The method of any one of embodiments 33-44, wherein the contacting step is carried out in vitro, ex vivo, or in vivo. EMBODIMENT 46 46. ​​The method of embodiment 45, wherein the contacting step is performed in vivo in a subject. EMBODIMENT 47 47. The method of embodiment 46, wherein the contacting step comprises administering the polynucleotide, vector, composition, or pharmaceutical composition to the subject. EMBODIMENT 48 46. ​​The method of embodiment 45, wherein the contacting step is carried out in vitro or ex vivo. EMBODIMENT 49 49. The method of embodiment 48, wherein the contacting step comprises lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. EMBODIMENT 50 50. The method of any one of embodiments 33-49, wherein the engineered receptor is capable of localizing to the cell surface of the neuron. EMBODIMENT 51 A method of inhibiting activity of a neuron, comprising: (a) contacting the neuron with an engineered receptor of any one of embodiments 1-20, a polynucleotide of any one of embodiments 21-26, a vector of any one of embodiments 27-30, a composition of embodiment 31, or a pharmaceutical composition of embodiment 32; and (b) contacting the neuron with a non-natural ligand of the engineered receptor. Embodiment 51.1 52. The method of embodiment 51, wherein the neuron is a neuron of the peripheral nervous system. Embodiment 51.2 52. The method of embodiment 51, wherein the neuron is a neuron of the central nervous system. EMBODIMENT 52 51-51, wherein the neuron is a nociceptive neuron 2. A method according to any one of claims 1 to 11. EMBODIMENT 53 51-51, wherein the neuron is a non-nociceptive neuron 2. A method according to any one of claims 1 to 11. EMBODIMENT 54 The method of any one of embodiments 51-53, 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. EMBODIMENT 55 The method of any one of embodiments 51-54, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers. EMBODIMENT 56 56. The method of embodiment 55, wherein the neurons are Aδ afferent fibers. EMBODIMENT 57 57. The method of embodiment 56, wherein the Aβ afferent fibers are damaged Aβ afferent fibers. EMBODIMENT 58 57. The method of embodiment 56, wherein the Aβ afferent fibers are non-injured Aβ afferent fibers. EMBODIMENT 59 59. The method of any one of embodiments 51-58, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5. EMBODIMENT 60 60. The method of any one of embodiments 51-59, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1 1. EMBODIMENT 61 The method of any one of embodiments 51-60, wherein the contacting step (a) is carried out in vitro, ex vivo, or in vivo. EMBODIMENT 62 The method of any one of embodiments 51-61, wherein the contacting step (b) is carried out in vitro, ex vivo, or in vivo. EMBODIMENT 63 The method of any one of embodiments 51-62, wherein the contacting steps (a) and / or (b) are performed in vivo in a subject. EMBODIMENT 64 64. The method of embodiment 63, wherein the contacting step (a) comprises administering an engineered receptor, polynucleotide, vector, or pharmaceutical composition to the subject, and / or the contacting step (b) comprises administering a non-natural ligand to the subject. EMBODIMENT 65 The method of any one of embodiments 51-64, wherein the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. EMBODIMENT 66 66. The method of any one of embodiments 51-65, wherein the engineered receptor is capable of localizing to the cell surface of the neuron. EMBODIMENT 67 1. A method of treating and / or delaying the onset of a neurological disorder in a subject in need thereof, comprising: administering to a subject a therapeutically effective amount of an engineered receptor according to any one of embodiments 1-20, a polynucleotide according to any one of embodiments 21-26, a vector according to any one of embodiments 27-30, a composition according to embodiment 31, or a pharmaceutical composition according to embodiment 32; administering to the subject a non-natural ligand of the engineered receptor. EMBODIMENT 68 68. The method of embodiment 67, wherein the subject is administered the non-natural ligand after step (a). The method of embodiment 67, wherein the subject is administered the non-natural ligand simultaneously with step (a). EMBODIMENT 70 The method of any one of embodiments 67-69, wherein the neurological disorder is a seizure disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spasticity disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation. EMBODIMENT 71 71. The method of embodiment 70, wherein the neurological disorder is allodynia. EMBODIMENT 72 The method of any one of embodiments 67-71, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. EMBODIMENT 73 73. The method of any one of embodiments 67-72, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. EMBODIMENT 74 74. The method of embodiment 73, wherein the non-natural ligand is administered orally. EMBODIMENT 75 The method of any one of embodiments 67-74, wherein the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or into the cisterna magna. EMBODIMENT 76 76. The method of any one of embodiments 67-75, wherein the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally. EMBODIMENT 77 The method of any one of embodiments 67-76, wherein the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. EMBODIMENT 78 The method of any one of embodiments 67-76, wherein the subject suffers from neuropathic pain and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. EMBODIMENT 79 The method according to any one of embodiments 67 to 78, wherein the subject is a human. EMBODIMENT 80 The method of any one of embodiments 67-79, wherein the therapeutically effective amount reduces the severity of signs and / or symptoms of a neurological disorder. Embodiment 81 The method of any one of embodiments 67-80, wherein the therapeutically effective amount delays the onset of signs and / or symptoms of a neurological disorder. EMBODIMENT 82 The method of any one of embodiments 67-81, wherein the therapeutically effective amount eliminates signs and / or symptoms of a neurological disorder. EMBODIMENT 83 The method of any one of embodiments 80-82, wherein the symptom of a neurological disorder is nerve damage, nerve atrophy, and / or seizures. EMBODIMENT 84 84. The method of embodiment 83, wherein the nerve injury is a peripheral nerve injury. EMBODIMENT 85 The method of any one of embodiments 80-84, wherein the symptom of the neurological disorder is pain. EMBODIMENT 86 1. A method of treating and / or delaying the onset of pain in a subject in need thereof, comprising: administering to a subject a therapeutically effective amount of an engineered receptor according to any one of embodiments 1-20, a polynucleotide according to any one of embodiments 21-26, a vector according to any one of embodiments 27-30, a composition according to embodiment 31, or a pharmaceutical composition according to embodiment 32; administering to the subject a non-natural ligand of the engineered receptor. EMBODIMENT 87 The method of embodiment 86, wherein the subject is administered the non-natural ligand after step (a). The method of embodiment 86, wherein the subject is administered the non-natural ligand simultaneously with step (a). EMBODIMENT 89 The method of any one of embodiments 86-88, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. EMBODIMENT 90 The method of any one of embodiments 86-89, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. EMBODIMENT 91 91. The method of embodiment 90, wherein the non-natural ligand is administered orally. EMBODIMENT 92 The method of any one of embodiments 86-91, wherein the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or into the cisterna magna. EMBODIMENT 93 93. The method of any one of embodiments 86-92, wherein the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally. EMBODIMENT 94 The method of any one of embodiments 86-93, wherein the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. EMBODIMENT 95 The method of any one of embodiments 86-94, wherein the subject suffers from neuropathic pain and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. EMBODIMENT 96 The method according to any one of embodiments 86 to 95, wherein the subject is a human. EMBODIMENT 97 The method of any one of embodiments 85-96, wherein the pain is neuropathic pain. EMBODIMENT 98 The method of any one of embodiments 85-97, wherein the pain is associated with, caused by, or results from chemotherapy. EMBODIMENT 99 The method of any one of embodiments 85-98, wherein the pain is associated with, caused by, or results from trauma. EMBODIMENT 100 The method of any one of embodiments 85-99, wherein the subject suffers from allodynia. EMBODIMENT 101 The method according to any one of embodiments 85-100, wherein the pain occurs after a medical procedure. EMBODIMENT 102 The method of any one of embodiments 85-101, wherein the pain is associated with, caused by, or results from childbirth or a Cesarean section. EMBODIMENT 103 The method according to any one of embodiments 85-102, wherein the pain is associated with, caused by, or results from a migraine headache. EMBODIMENT 104 The method of any one of embodiments 85-103, wherein the therapeutically effective amount transiently relieves pain in the subject, permanently relieves pain in the subject, prevents the onset of pain in the subject, and / or eliminates pain in the subject. EMBODIMENT 105 The method according to any one of embodiments 85-104, wherein steps (a) and (b) are performed before the subject experiences pain.

[0310] The foregoing merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the present disclosure and are within its spirit and scope, although not explicitly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present disclosure and the concepts contributed by the inventor to advance the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting the principles, aspects, and embodiments of the present disclosure and specific examples thereof are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Thus, the scope of the present disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure is embodied by the appended claims. In certain embodiments, for example, the following items are provided: (Item 1) An engineered receptor, a. a ligand-binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR) and including the Cys-loop domain from the human glycine receptor α1 subunit; b. an ion pore domain derived from the human glycine receptor α1 subunit; the ligand-binding domain comprises (i) 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, or (ii) an amino acid substitution of L131E, wherein the amino acid residues correspond to those of α7-nAChR; The engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor. (Item 1) 2. The engineered receptor of claim 1, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and further comprises the 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, or the amino acid substitution of L131E, wherein the amino acid residues correspond to the amino acid residues of α7-nAChR. (Item 2) 2. The engineered receptor according to item 1 or 1.1, wherein the ligand-binding domain comprises two amino acid substitutions in a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. (Item 3) 3. The engineered receptor of any one of paragraphs 1, 1.1, or 2, wherein said 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. (Item 3) 4. The engineered receptor of any one of items 1 to 3, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence further 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. (Item 4) 2. The engineered receptor of item 1 or 1.1, wherein the ligand binding domain comprises the amino acid substitution L131E. (Item 4) 5. The engineered receptor of any one of items 1 to 4, wherein the engineered receptor comprises the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence further comprises an amino acid substitution of L131E. (Item 5) 2. The engineered receptor of any one of paragraphs 1 to 4.1, wherein the Cys-loop domain comprises amino acids 166 to 172 of SEQ ID NO:2. (Item 6) 2. The engineered receptor of any one of paragraphs 1 to 4.1, wherein the Cys-loop domain comprises amino acids 166 to 180 of SEQ ID NO:2. (Item 7) 7. The engineered receptor of any one of items 1 to 6, wherein the receptor comprises the β1-2 loop domain from the human glycine receptor α1 subunit. (Item 8) 8. The engineered receptor of item 7, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:2. (Item 8) 9. The engineered receptor according to any one of items 1 to 8, wherein the engineered receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 63. (Item 9) 9. The engineered receptor of any one of items 1 to 8, wherein the potency of the engineered receptor for acetylcholine is lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. (Item 10) 10. The engineered receptor of item 9, wherein the potency of the engineered receptor for acetylcholine is at least 2-fold lower than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. (Item 11) 11. The engineered receptor of any one of items 1 to 10, wherein the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. (Item 12) 12. The engineered receptor of any one of items 1 to 11, wherein the potency of the engineered receptor for the non-natural ligand is greater than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. (Item 13) 13. The engineered receptor of claim 12, wherein the potency of the engineered receptor for the non-natural ligand is at least 2-fold higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. (Item 14) 14. The engineered receptor of any one of paragraphs 9 to 13, wherein determining the potency comprises determining an EC50. (Item 15) 15. The engineered receptor of any one of items 1 to 14, wherein the efficacy of the engineered receptor in the presence of a non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. (Item 16) 16. The engineered receptor of any one of paragraphs 1 to 15, wherein the efficacy of the engineered receptor in the presence of a non-natural ligand is at least 2-fold higher than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. (Item 17) 17. The engineered receptor of any one of items 15-16, wherein determining the efficacy comprises determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand. (Item 18) 18. The engineered receptor of any one of items 11 to 17, wherein the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, APN-1125, TC-5619, and fascinicline / RG3487. (Item 19) 19. The engineered receptor of claim 18, wherein the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and APN-1125. (Item 20) 19. The engineered receptor of item 18, wherein the non-natural ligand is TC-5619. (Item 21) 21. A polynucleotide comprising a nucleic acid encoding the engineered receptor according to any one of items 1 to 20. (Item 22) 22. The polynucleotide of claim 21, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the engineered receptor. (Item 23) 23. The polynucleotide of item 22, wherein the promoter is a regulatable promoter. (Item 24) 24. The polynucleotide of item 23, wherein the regulatable promoter is active in excitable cells. (Item 25) 25. The polynucleotide of item 24, wherein the excitable cell is a neuron or a muscle cell. (Item 26) 26. The polynucleotide of item 25, wherein the excitable cell is a neuron. (Item 27) 27. A vector comprising the polynucleotide according to any one of items 21 to 26. (Item 28) 28. The vector according to item 27, wherein the vector is a plasmid or a viral vector. (Item 29) 29. The vector of item 28, wherein the vector is a viral vector selected from the group consisting of an adenoviral vector, a retroviral vector, an adeno-associated viral (AAV) vector, and a herpes simplex viral vector 1 (HSV-1). (Item 30) 30. The vector of item 29, wherein the viral vector is an AAV vector and the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof. (Item 31) 31. A composition comprising an engineered receptor according to any one of items 1 to 20, a polynucleotide according to any one of items 21 to 26, or a vector according to any one of items 27 to 30. (Item 32) 31. A pharmaceutical composition comprising the engineered receptor according to any one of items 1 to 20, the polynucleotide according to any one of items 21 to 26, or the vector according to any one of items 27 to 30, and a pharma- ceutically acceptable carrier. (Item 33) 32. A method for producing an engineered receptor in a neuron, comprising contacting said neuron with the polynucleotide of any one of items 21 to 26, the vector of any one of items 27 to 30, the composition of item 31, or the pharmaceutical composition of item 32. (Item 34) 37. The method of claim 33 or the polynucleotide of claim 26, wherein the neuron is a neuron of the peripheral nervous system. (Item 35) 35. The method of claim 33 or 34, or the polynucleotide of claim 26, wherein the neuron is a neuron of the central nervous system. (Item 36) 36. The method of any one of items 33 to 35, or the polynucleotide of item 26, wherein the neuron is a nociceptive neuron. (Item 37) 37. The method of any one of items 33 to 36, or the polynucleotide of item 26, wherein the neuron is a non-nociceptive neuron. (Item 38) 38. The method of any one of items 33 to 37, or the polynucleotide of item 26, 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. (Item 39) 39. The method according to any one of items 33 to 38, or the polynucleotide according to item 26, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers. (Item 40) 40. The method of claim 39, or the polynucleotide of claim 26, wherein the neuron is an Aβ afferent fiber. (Item 41) 41. The method of claim 40, or the polynucleotide of claim 26, wherein the Aβ afferent fiber is a damaged Aβ afferent fiber. (Item 42) 41. The method of claim 40, or the polynucleotide of claim 26, wherein the Aβ afferent fibers are non-injured Aβ afferent fibers. (Item 43) 43. The method of any one of items 33 to 42, or the polynucleotide of item 26, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5. (Item 44) 44. The method of any one of items 33 to 43, or the polynucleotide of item 26, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1. (Item 45) 45. The method of any one of items 33 to 44, wherein the contacting step is carried out in vitro, ex vivo, or in vivo. (Item 46) 46. ​​The method of claim 45, wherein the contacting step is performed in vivo in a subject. (Item 47) 47. The method of claim 46, wherein the contacting step comprises administering the polynucleotide, the vector, the composition, or the pharmaceutical composition to the subject. (Item 48) 46. ​​The method of claim 45, wherein the contacting step is carried out in vitro or ex vivo. (Item 49) 50. The method of claim 48, wherein the contacting step comprises lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. (Item 50) 50. The method of any one of items 33 to 49, wherein the engineered receptor is capable of localizing to the cell surface of the neuron. (Item 51) 32. A method of inhibiting activity of a neuron, comprising: (a) contacting the neuron with the engineered receptor of any one of items 1 to 20, the polynucleotide of any one of items 21 to 26, the vector of any one of items 27 to 30, the composition of item 31, or the pharmaceutical composition of item 32; and (b) contacting the neuron with a non-natural ligand of the engineered receptor. (Item 51) 52. The method of claim 51, wherein the neuron is a neuron of the peripheral nervous system. (Item 51) 52. The method of claim 51, wherein the neuron is a neuron of the central nervous system. (Item 52) 3. The method according to any of items 51 to 51.2, wherein the neuron is a nociceptive neuron. (Item 53) 3. The method according to any of items 51 to 51.2, wherein the neuron is a non-nociceptive neuron. (Item 54) 54. The method of any one of items 51 to 53, 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. (Item 55) 55. The method of any one of items 51 to 54, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers. (Item 56) 56. The method of claim 55, wherein the neuron is an Aβ afferent fiber. (Item 57) 57. The method of claim 56, wherein the Aβ afferent fibers are damaged Aβ afferent fibers. (Item 58) 57. The method of claim 56, wherein the Aβ afferent fibers are non-injured Aβ afferent fibers. (Item 59) 59. The method of any one of items 51 to 58, wherein the neuron expresses neurofilament 200 (NF200), Piezo2, and TLR-5. (Item 60) 60. The method of any one of items 51 to 59, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1. (Item 61) 61. The method of any one of items 51 to 60, wherein the contacting step (a) is carried out in vitro, ex vivo, or in vivo. (Item 62) 62. The method of any one of items 51 to 61, wherein the contacting step (b) is carried out in vitro, ex vivo, or in vivo. (Item 63) 63. The method of any one of items 51 to 62, wherein the contacting step (a) and / or (b) is carried out in vivo in a subject. (Item 64) 64. The method of claim 63, 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. (Item 65) 65. The method of any one of items 51 to 64, wherein the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. (Item 66) 66. The method of any one of items 51 to 65, wherein the engineered receptor is capable of localizing to the cell surface of the neuron. (Item 67) 1. A method of 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 according to any one of items 1 to 20, the polynucleotide according to any one of items 21 to 26, the vector according to any one of items 27 to 30, the composition according to item 31, or the pharmaceutical composition according to item 32; b. administering to said subject a non-natural ligand of said engineered receptor. (Item 68) 68. The method of claim 67, wherein the subject is administered the non-natural ligand after step (a). (Item 69) 68. The method of claim 67, wherein the subject is administered the non-natural ligand simultaneously with step (a). (Item 70) 70. The method of any one of items 67-69, wherein the neurological disorder is a seizure disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spasticity disorder, pruritus, Alzheimer's disease, Parkinson's disease, post traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation. (Item 71) 71. The method of claim 70, wherein the neurological disorder is allodynia. (Item 72) 72. The method of any one of items 67 to 71, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. (Item 73) 73. The method of any one of items 67-72, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. (Item 74) 74. The method of claim 73, wherein the non-natural ligand is administered orally. (Item 75) 75. The method of any one of items 67-74, 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 into the cisterna magna. (Item 76) 76. The method of any one of items 67-75, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally. (Item 77) 77. The method of any one of items 67-76, wherein the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. (Item 78) 77. The method of any one of items 67-76, wherein the subject suffers from neuropathic pain and the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. (Item 79) 79. The method according to any one of items 67 to 78, wherein the subject is a human. (Item 80) 80. The method of any one of items 67 to 79, wherein the therapeutically effective amount reduces the severity of signs and / or symptoms of the neurological disorder. (Item 81) 81. The method according to any one of items 67 to 80, wherein the therapeutically effective amount delays the onset of signs and / or symptoms of the neurological disorder. (Item 82) 82. The method according to any one of items 67 to 81, wherein the therapeutically effective amount eliminates signs and / or symptoms of the neurological disorder. (Item 83) 83. The method of any one of items 80 to 82, wherein the symptom of the neurological disorder is nerve damage, nerve atrophy, and / or seizures. (Item 84) 84. The method of claim 83, wherein the nerve injury is a peripheral nerve injury. (Item 85) 85. The method of any one of items 80 to 84, wherein the symptom of the neurological disorder is pain. (Item 86) 1. A method of treating 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 according to any one of items 1 to 20, the polynucleotide according to any one of items 21 to 26, the vector according to any one of items 27 to 30, the composition according to item 31, or the pharmaceutical composition according to item 32; b. administering to said subject a non-natural ligand of said engineered receptor. (Item 87) 87. The method of claim 86, wherein the subject is administered the non-natural ligand after step (a). (Item 88) 87. The method of claim 86, wherein the subject is administered the non-natural ligand simultaneously with step (a). (Item 89) 89. The method of any one of items 86 to 88, wherein the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, APN-1125, TC-5619, and facinicline / RG3487. (Item 90) 90. The method of any one of items 86-89, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. (Item 91) 91. The method of claim 90, wherein the non-natural ligand is administered orally. (Item 92) 92. The method of any one of items 86-91, 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 into the cisterna magna. (Item 93) 93. The method of any one of items 86-92, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally. (Item 94) 94. The method of any one of items 86-93, wherein the subject suffers from trigeminal neuralgia and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. (Item 95) 95. The method of any one of items 86-94, wherein the subject suffers from neuropathic pain and the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. (Item 96) 96. The method according to any one of items 86 to 95, wherein the subject is a human. (Item 97) 97. The method according to any one of items 85 to 96, wherein the pain is neuropathic pain. (Item 98) 98. The method of any one of items 85 to 97, wherein the pain is associated with, caused by, or results from chemotherapy. (Item 99) 99. The method of any one of items 85 to 98, wherein the pain is associated with, caused by, or results from trauma. (Item 100) 99. The method according to any of items 85 to 99, wherein the subject suffers from allodynia. (Item 101) The method according to any one of items 85 to 100, wherein the pain occurs after a medical procedure. (Item 102) 102. The method of any one of items 85 to 101, wherein the pain is associated with, caused by, or results from childbirth or Caesarean section. (Item 103) 103. The method of any one of items 85 to 102, wherein the pain is associated with, caused by, or results from a migraine headache. (Item 104) 104. The method of any one of items 85 to 103, wherein the therapeutically effective amount transiently relieves pain in the subject, permanently relieves pain in the subject, prevents the onset of pain in the subject, and / or eliminates pain in the subject. (Item 105) 105. The method according to any one of items 85 to 104, wherein steps (a) and (b) are carried out before the subject experiences pain.

Claims

[Claim 1] The invention described in the specification.

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