Compositions and methods for neurological diseases

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to US Provisional Application No. 63 / 311,817, filed on February 18, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] Instructions for electronically submitting text documents

[0004] The contents of the electronic sequence listing (SWCH_039_01WO_SeqList_ST26.xml; size: 196,033 bytes; and creation date: February 17, 2023) are incorporated herein by reference in its entirety. Field of the Invention

[0005] The present disclosure relates to engineered receptors and the use of engineered receptors and small molecule ligands in modulating cellular activity and treating disease. Background Art

[0006] Refractory neurological diseases are usually associated with abnormally active neurons. Attempts to develop therapies for these conditions are hampered by the lack of tractable target proteins associated with the disease. For example, unrelieved chronic pain is a serious health problem in the US and around the world. A report from the Institute of Medicine estimates that 116 million Americans suffer from pain that lasts for weeks to years, which results in annual costs of more than $560 billion. There are not enough long-term therapies for chronic pain patients, which leads to huge costs for both society and individuals. Pain usually leads to disability, and even when not disabled, it has a great effect on quality of life. Pain treatment often fails even in the following situations: care provision is optimal, such as careful, well-trained doctors; easy access to opioids; use of adjuvant analgesics; availability of patient-controlled analgesia; and evidence-based use of procedures such as nerve blocks and IT pumps.

[0007] The most commonly used treatments for chronic pain are opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and can cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive failure, hallucinations and other systemic side effects. Despite the widespread use of drugs, their effectiveness in pain relief has a surprisingly low success rate. Large randomized studies using various drugs found that only one in every two or three patients achieved at least 50% pain relief (Finnerup et al., 2005). Follow-up studies using the most developed pharmacological treatments found the same results, indicating no improvement in the efficacy of pain medications (Finnerup et al., Pain, 150(3):573-81, 2010).

[0008] More invasive options for treating pain include nerve blocks and electrical stimulation. A nerve block is an injection of a local anesthetic, usually in the spinal cord, to interrupt pain signals to the brain, with effects that last only weeks to months. In most cases, nerve blocks are not a recommended treatment option (Mailis and Taenzer, Pain Res Manag. 17(3): 150-158, 2012). Electrical stimulation involves delivering an electric current to block pain signals. While this effect can last longer than a nerve block, the electrical leads themselves can present complications: dislocation, infection, breakage, or battery dying. One review found that 40% of patients treated for neuropathy with electrical stimulation experienced one or more device problems (Wolter, 2014).

[0009] The most invasive and least preferred method of managing pain is to completely remove the nerve or part of it that is causing the pain through surgery. This option is recommended only when the patient has exhausted the previous and other less invasive treatments and found them to be ineffective. Radiofrequency nerve ablation uses heat to destroy the problematic nerve and provides longer-lasting pain relief than nerve blocks. However, one study found no difference between the control and treatment groups in partial radiofrequency destruction of the DRG for chronic lumbosacral radicular pain (Geurts et al., 2003). Other surgical methods for surgically removing painful nerves suffer from similar disadvantages and have long-term serious side effects, including sensory or motor deficits, or cause pain elsewhere.

[0010] Methods for treating neurological disorders should be safe, effective, and cost-effective. Gene therapy can provide a non-invasive treatment option for various neurological diseases, including management of pain. However, to date, gene therapy methods have not found widespread use in treating neurological diseases. The present disclosure addresses these needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present disclosure may be best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application document contains at least one drawing in color. It should be emphasized that, in accordance with common practice, the various features of the drawings are not drawn to scale. Instead, the dimensions of the various features are arbitrarily expanded or reduced for clarity. The following figures are included in the drawings:

[0012] Figure 1A-1JHeat maps of the percent quenching of YFP fluorescence in mutants of the engineered chimeric receptor SEQ ID NO: 33 containing the indicated amino acid substitutions following stimulation with different doses of acetylcholine or the indicated non-natural ligands are shown. The ligand dose is written at the top of each graph. The numbers in the boxes indicate the relative amount of quenching observed. Higher levels of quenching indicate higher levels of receptor activation due to the ligand at the indicated dose. The level of quenching is also indicated by a color gradient. Dark = YFP reporter with more than 70% maximal quenching (i.e., most engineered receptors are activated). White = less than 10% quenching (i.e., very few engineered receptors are activated). Negative values ​​represent non-responders with negative quenching due to stimulation artifacts. SEQ ID NO: 29 is a non-responsive chimera that serves as a negative control. CODA283 contains an additional N379K mutation introduced during cloning that is located outside the ligand binding domain and therefore does not affect ligand binding. Figure 1A shows YFP fluorescence quenching using acetylcholine; Figure 1B shows YFP fluorescence quenching using CNL001; Figure 1C Shows YFP fluorescence quenching using TC-6683; Figure 1D Shows YFP fluorescence quenching using TC-5619 / Bradanicline; Figure 1E shows YFP fluorescence quenching using CNL002; Figure 1F shows YFP fluorescence quenching using ABT-126; Figure 1G Shows YFP fluorescence quenching using AZD-0328; Figure 1H Shows YFP fluorescence quenching using Facinicline; Fig. 1I Shows YFP fluorescence quenching using TC-6987; Figure 1J The quenching of YFP fluorescence using varenicline is shown. Abbreviations of the names of the non-natural ligands: abt: ABT-126; ach: acetylcholine; AZD: AZD-0328; brd: TC-5619 (Bradanicline); fac: RG3487 (Facinicline); tc6: TC-6987; var: varenicline.

[0013] Figure 2A-2BConcentration-response curves of CR-11 (Chemogenic receptor-11, an engineered receptor comprising an amino acid sequence having amino acid substitutions of Y115D and L131Q in SEQ ID NO: 33) expressed in HEK 293 cells to acetylcholine and to the non-natural ligand RG-3487 (SA-2, synthetic agonist-2) are shown. Responses were evaluated using manual patch clamp electrophysiology. Currents were normalized to unity of the maximal response. Continuous lines through the data points are the best fits obtained using the Hill equation, with the EC for each ligand being 50 Estimated from concentration-response curves. Figure 2A Concentration-response curves for wild-type and CR-11 receptors to acetylcholine are shown. Figure 2B Concentration-response curves of wild-type and CR-11 receptors to RG-3487 (SA-2) are shown.

[0014] Figure 3 Shown are exemplary chloride currents induced by RG-3487 (SA-2) in adult rat DRG neurons transduced with lentivirus expressing CR-11 (chemogenetic receptor-11, an engineered receptor comprising an amino acid sequence having amino acid substitutions of Y115D and L131Q in SEQ ID NO:33).

[0015] Figure 4A Evoked action potentials of transduced DRG neurons expressing CR-11 (an engineered receptor comprising an amino acid sequence having amino acid substitutions of Y115D and L131Q in SEQ ID NO: 33) or control DRG neurons (no CR-11 expression) are shown under different current injections (50 pA to 700 pA). The upper graph shows the evoked action potentials of control DGF neurons. The lower left graph shows the evoked action potentials of transduced DRG neurons expressing CR-11 in the presence of 3 μM RG-3487 (SA-2). The lower right graph shows the evoked action potentials of transduced DRG neurons expressing CR-11 after washing out RG-3487 (SA-2). Figure 4B Shown are basal intensity values ​​(current required to elicit an action potential) for control DRG neurons and transduced DRG neurons expressing CR-11 in the absence or presence of the indicated ligands.

[0016] Figure 5 Shown are the % of HA tag-positive cells expressing the engineered receptor, normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33 ("Normalized HA %"); and the % 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 5Also shown are the median fluorescence intensity (MFI) of cells expressing the engineered receptors, normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33, as assessed using anti-HA antibodies ("Normalized HAMFI") or fluorescently labeled α-bungarotoxin conjugated to Alexa Fluor 647 ("Normalized AB MFI").

[0017] Fig. 6A Relative surface expression and total expression of the indicated engineered receptors in cultured DRG neurons or in HEK cells are shown. Figure 6B Relative surface and total expression of the indicated engineered receptors in cultured hippocampal neurons are shown. Summary of the invention

[0018] The present disclosure provides engineered receptors comprising a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR), wherein the ligand binding domain comprises an amino acid mutation at an amino acid residue corresponding to R101, Y115, L131, Q139, Y140, S172, Y210, or Y217 of SEQ ID NO: 4. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85% identity with amino acid residues 23-220 of SEQ ID NO: 4. In some embodiments, the ligand binding domain comprises an amino acid mutation at two or more amino acid residues selected from the amino acid residues corresponding to R101, Y115, L131, Q139, Y140, S172, Y210, and Y217 of SEQ ID NO: 4. In some embodiments, the ligand binding domain comprises a mutation at an amino acid residue corresponding to the indicated position of SEQ ID NO: 4:

[0019] a) Y140;

[0020] b) Y115 and L131;

[0021] c) Q139 and S172;

[0022] d) Q139 and Y210;

[0023] e) Q139, S172 and Y217;

[0024] f) Y115, Q139 and S172;

[0025] g) R101, L131, Q139 and Y217;

[0026] h) Y115 and S172;

[0027] i) L141, S172 and Y210; or

[0028] j) R101, L131 and Y217.

[0029] In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand binding domain comprises an amino acid substitution corresponding to the indicated position of SEQ ID NO: 4:

[0030] a)Y140R;

[0031] b) Y115E and L131M;

[0032] c) Q139W and S172D;

[0033] d) Q139W and Y210W;

[0034] e) Q139W, S172D and Y217I;

[0035] f) Y115E, Q139E and S172D;

[0036] g) R101F, L131G, Q139L and Y217F;

[0037] h) Y115Q and S172D;

[0038] i) L141M, S172D and Y210W;

[0039] j) R101F, L131N and Y217F;

[0040] k) L131G;

[0041] l) L131M;

[0042] m)Q139E;

[0043] n)Q139L;

[0044] o) Q139W; or

[0045] p)L141M.

[0046] In some embodiments, the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor, and the LGIC receptor comprises an ion pore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is a human glycine receptor α1, a human glycine receptor α2, or a human glycine receptor α3. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity with amino acids 255-457 of SEQ ID NO: 2, amino acids 260-452 of SEQ ID NO: 83, amino acids 259-464 of SEQ ID NO: 85, or amino acids 259-449 of SEQ ID NO: 87. In some embodiments, the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from a human glycine receptor. 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 ligand binding domain of the engineered receptor comprises the 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 according to any one of SEQ ID NOs: 89-98.

[0047] The present disclosure provides an engineered chimeric ligand-gated ion channel (LGIC) comprising a ligand binding domain derived from a first LGIC and an ion pore domain derived from a second LGIC, wherein the first LGIC is a human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprises an amino acid mutation at an amino acid residue corresponding to R101, Y115, L131, Q139, Y140, S172, Y210 or Y217 of SEQ ID NO:4.

[0048] In some embodiments, the ligand binding domain comprises a mutation at the amino acid residue corresponding to the indicated position of SEQ ID NO: 4:

[0049] a) Y140; b) Y115 and L131;

[0050] c) Q139 and S172;

[0051] d) Q139 and Y210;

[0052] e) Q139, S172 and Y217;

[0053] f) Y115, Q139 and S172;

[0054] g) R101, L131, Q139 and Y217;

[0055] h) Y115 and S172;

[0056] i) L141, S172 and Y210; or

[0057] j) R101, L131 and Y217.

[0058] In some embodiments, the ligand binding domain comprises amino acid substitutions corresponding to the indicated positions of SEQ ID NO: 4:

[0059] a) Y140R;

[0060] b) Y115E and L131M;

[0061] c) Q139W and S172D;

[0062] d) Q139W and Y210W;

[0063] e) Q139W, S172D and Y217I;

[0064] f) Y115E, Q139E and S172D;

[0065] g) R101F, L131G, Q139L and Y217F;

[0066] h) Y115Q and S172D;

[0067] i) L141M, S172D and Y210W;

[0068] j) R101F, L131N and Y217F;

[0069] k) L131G;

[0070] l) L131M;

[0071] m)Q139E;

[0072] n)Q139L;

[0073] o) Q139W; or

[0074] p)L141M.

[0075] In some embodiments, the second LGIC is a human glycine receptor.In some embodiments, the human glycine receptor is human glycine receptor alpha 1.In some embodiments, the engineered chimeric LGIC comprises a polypeptide sequence having at least 85% sequence identity to SEQ ID NO:33.

[0076] In some embodiments, the effectiveness of the engineered receptor to acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) or control receptor to acetylcholine. In some embodiments, the effectiveness of the engineered receptor to acetylcholine is at least 2 times lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) or control receptor to acetylcholine. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is roughly the same as that of human α7 nicotinic acetylcholine receptor (α7-nAChR) or control receptor to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) or control receptor to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is at least 2 times higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) or control receptor to non-natural ligands. In some embodiments, the potency of the engineered receptor for a ligand is determined by the EC50 of the receptor for the ligand according to a YFP fluorescence quenching assay using Lenti-X 293T cells.

[0077] In some embodiments, the efficacy of the engineered receptor in the presence of a non-natural ligand is higher than that of a human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor in the presence of a non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of a non-natural ligand is at least 2 times higher than that of a human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor in the presence of a non-natural ligand. In some embodiments, wherein determining efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of a non-natural ligand.

[0078] In some embodiments, the non-natural ligand is selected from AZD-0328, TC-6987, ABT-126, CNL002, TC-5619, CNL001, TC-6683, Varenicline, and Facinicline / RG3487. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the non-natural ligand is Varenicline.

[0079] The present disclosure provides polynucleotides encoding engineered receptors of the present disclosure. In some embodiments, the polynucleotide encodes an engineered receptor comprising an amino acid sequence of any one of SEQ ID NO: 89-98. 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 excitable cells. In some embodiments, the excitable cells are neurons or muscle cells. In some embodiments, the excitable cells are neurons.

[0080] 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 virus (AAV) vector, and a herpes simplex-1 viral vector (HSV-1). The vector of claim 78, the viral vector is an AVV vector, and wherein the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof. In some embodiments, the capsid protein of the AAV9 vector comprises a T492V mutation according to SEQ ID NO: 81.

[0081] 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 pharmaceutically acceptable carrier.

[0082] The present disclosure provides a method for expressing an engineered receptor in a neuron, comprising contacting the neuron with any of the polynucleotides disclosed herein, any of the vectors disclosed herein, any of the compositions disclosed herein, or any 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 hippocampal neuron, a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is a damaged Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an undamaged Aβ afferent fiber. In some embodiments, wherein the neuron expresses neurofilament 200 (NF200), piezo2, and TLR-5. In some embodiments, the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1.

[0083] In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject. In some embodiments, the contacting step comprises administering a polynucleotide, a vector, a composition, or a pharmaceutical composition to the subject. In some embodiments, the contacting step is performed in vitro or ex vivo. In some embodiments, the contacting step comprises lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor is capable of localizing to the cell surface of a neuron.

[0084] The present disclosure provides a method for inhibiting the activity of a neuron, comprising (a) contacting a neuron with any of the engineered receptors disclosed herein, any of the polynucleotides disclosed herein, any of the vectors disclosed herein, any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein, and (b) contacting a neuron with a non-natural ligand of an engineered receptor. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is a damaged Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an undamaged Aβ afferent fiber. In some embodiments, the neuron expresses neurofilament 200 (NF200), piezo2, and TLR-5. In some embodiments, the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.1.

[0085] 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) is performed in vivo in a subject. In some embodiments, the contacting step (a) includes administering an engineered receptor, a polynucleotide, a carrier or a pharmaceutical composition to the subject; and / or the contacting step (b) includes administering a non-natural ligand to the subject. In some embodiments, the contacting step (a) and / or (b) includes lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication or microinjection. In some embodiments, the engineered receptor is capable of being localized to the cell surface of neurons.

[0086] The present disclosure provides a method for 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 of the engineered receptors disclosed herein, any of the polynucleotides disclosed herein, any of the vectors disclosed herein, any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein, and administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the non-natural ligand is administered to the subject after step (a). In some embodiments, the non-natural ligand is administered to the subject in parallel with step (a).

[0087] In some embodiments, the nervous system disorder is epilepsy, movement disorders, eating disorders, spinal cord injury, neurogenic bladder, allodynia, spastic disorders, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome or atrial fibrillation. In some embodiments, the nervous system disorder is allodynia. In some embodiments, the non-natural ligand is selected from AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, Varenicline and Facinicline / RG3487.

[0088] In some embodiments, the non-natural ligand is administered orally, subcutaneously, superficially or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered subcutaneously, orally, intrathecally, superficially, intravenously, intraganglionically, intraneuronally, intracranially, intraspinally or to the cisterna magna. In some embodiments, the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered by transforaminal injection or intrathecal administration. In some embodiments, the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject. In some embodiments, the subject suffers from neuropathic pain, and wherein the engineered receptor, polynucleotide, carrier, composition or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject. In some embodiments, the subject is a person. In some embodiments, the therapeutically effective amount reduces the severity of the signs and / or symptoms of a nervous system disorder. In some embodiments, the therapeutically effective amount delays the onset of the signs and / or symptoms of a nervous system disorder. In some embodiments, the therapeutically effective amount eliminates signs and / or symptoms of a neurological disorder. In some embodiments, the signs of a neurological disorder are nerve damage, nerve atrophy, and / or epileptic seizures. In some embodiments, the nerve damage is peripheral nerve damage. In some embodiments, the symptom of a neurological disorder is pain.

[0089] The present disclosure provides a method for 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 of the engineered receptors disclosed herein, any of the polynucleotides disclosed herein, any of the vectors disclosed herein, any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein, and administering to the subject a non-natural ligand of the engineered receptor. In some embodiments, the non-natural ligand is administered to the subject after step (a). In some embodiments, the non-natural ligand is administered to the subject in parallel with step (a). In some embodiments, the non-natural ligand is selected from AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, Varenicline, and Facinicline / RG3487.

[0090] In some embodiments, the non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, the non-natural ligand is administered orally. In some embodiments, the engineered receptor, polynucleotide, carrier, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionically, intraneuronally, intracranially, intraspinally, or to the cisterna magna. In some embodiments, the engineered receptor, polynucleotide, carrier, composition, or pharmaceutical composition is administered by transforaminal injection or intrathecally.

[0091] In some embodiments, the subject suffers from trigeminal neuralgia, and wherein 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 wherein the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the dorsal root ganglion (DRG) of the subject.

[0092] 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 caused by chemotherapy. In some embodiments, the pain is associated with, caused by, or caused by 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 caused by childbirth or cesarean section. In some embodiments, the pain is associated with, caused by, or caused by migraine. In some embodiments, the therapeutically effective amount temporarily reduces pain in the subject, permanently reduces pain in the subject, prevents pain attacks in the subject, and / or eliminates pain in the subject. In some embodiments, steps (a) and (b) are performed before pain appears in the subject.

[0093] The present disclosure provides a kit comprising (a) a vector of the present disclosure and (b) a non-natural ligand of an engineered receptor encoded by the vector. The present disclosure provides a kit comprising (a) an engineered receptor of the present disclosure, and (b) a non-natural ligand of an engineered receptor. In some embodiments, the combination of engineered receptors and non-natural ligands is according to any one of the combinations provided in Table 29. In some embodiments, the kit comprises a device suitable for administering the vector.

[0094] The present disclosure provides a method for treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of a polynucleotide of the present disclosure or a vector of the present disclosure. In some embodiments, the polynucleotide is delivered to hippocampal neurons in a subject, or wherein the vector transduces hippocampal neurons in a subject. In some embodiments, the vector is an AAV9 vector comprising a capsid polypeptide, the capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 81. In some embodiments, the focal epilepsy is mesial temporal lobe epilepsy (mTLE). In some embodiments, the polynucleotide or vector is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cistern magna) administration, intracerebral administration, intraventricular administration, or direct injection into an epileptic focus in the hippocampus.

[0095] The present disclosure provides a method for treating neuropathic pain in a subject in need thereof, comprising administering an effective amount of a polynucleotide of the present disclosure or a vector of the present disclosure. In some embodiments, the neuropathic pain is peripheral neuropathy or trigeminal neuralgia. In some embodiments, the polynucleotide is delivered to a dorsal root ganglion neuron or a trigeminal ganglion neuron in a subject, or wherein the vector transduces a dorsal root ganglion neuron or a trigeminal ganglion neuron in a subject. In some embodiments, the vector is an AAV9 vector comprising a capsid polypeptide, the capsid polypeptide comprising or consisting of the following amino acid sequence, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 81, and a non-threonine mutation at a position corresponding to T492 of SEQ ID NO: 81. In some embodiments, the non-threonine mutation is a valine substitution. In some embodiments, the polynucleotide or vector is administered by intrathecal (IT) or intraganglionic (IG) administration.

[0096] In some embodiments, the method further comprises administering a non-natural ligand for the engineered receptor. DETAILED DESCRIPTION

[0097] A. Overview

[0098] Provided are compositions and methods for modulating cell activity using engineered ligand-gated ion channel (LGIC) receptors, polynucleotides encoding engineered LGIC receptors, and gene therapy vectors comprising polynucleotides encoding engineered LGIC receptors. These compositions and methods are particularly useful for modulating neuronal activity, such as for treating disease or for studying neuronal circuits. In addition, reagents, devices, and kits thereof for practicing the subject methods are also provided.

[0099] In particular, the present disclosure provides an engineered receptor that binds to a ligand and sends a signal in response to the ligand. In some embodiments, the ligand is a drug. In some embodiments, the ligand is referred to as a "binding agent". In some embodiments, the engineered receptors described herein show an increased affinity for known agonist ligands. In some embodiments, the engineered receptors described herein show an affinity for antagonist or modulator ligands, and respond to antagonist and / or modulator ligands as if they were agonist ligands. The present disclosure further provides a method for treating a neurological disorder in a subject in need thereof. The present disclosure increases the number of clinical indications for which known drugs can be used by utilizing engineered receptors that respond to known drugs in a manner different from that of wild-type endogenous receptors.

[0100] Before describing the methods and compositions of the present invention, it will be understood that the present disclosure is not limited to the specific methods or compositions described, and therefore may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0101] When providing a numerical range, it is understood that, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed, to one tenth of the lower limit unit. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded from the range, and each range in which any one limit, neither limit, or both limits are included in the smaller range is also included in the present disclosure, subject to the influence of any specifically excluded limits in the specified range. Where the range includes one or both of the limits, the range excluding any one or both of these included limits is also included in the present disclosure.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosure, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials associated with the cited publications. It should be understood that if there is a conflict, the disclosure will supersede the disclosure of any incorporated publication.

[0103] As will be apparent to those skilled in the art reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any of the described methods can be performed in the order of events recited or in any other order that is logically possible.

[0104] Publications throughout this article are provided only because they are disclosed prior to the filing date of this application. Nothing herein should be construed as admitting that the disclosure is not entitled to publish earlier than the disclosure by virtue of prior disclosure. Further, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0105] B. Definition

[0106] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art, and so forth.

[0107] As used in this specification, the term "and / or" means "and" or "or" in this disclosure unless otherwise stated.

[0108] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" means the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In addition, the recitation of numerical ranges throughout this specification specifically includes all integers and decimal points therebetween.

[0109] Throughout this specification, unless the context requires otherwise, the phrase "consisting essentially of" means limiting the scope of the described composition, method, or kit to specified materials or steps that do not materially affect the basic and novel features of the disclosure. For example, a ligand binding domain that "consists essentially of" a disclosed sequence has the amino acid sequence of the disclosed sequence, plus or minus about 5 amino acid residues at the sequence boundaries, such as about 5 residues, 4 residues, 3 residues, 2 residues, or about 1 residue less than the described binding amino acid residues, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the described binding amino acid residues.

[0110] Throughout this specification, unless the context requires otherwise, the phrase "consisting of" means excluding from the composition, method or kit any element, step or ingredient not specified in the claim. For example, a ligand binding domain "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0111] As used in the present application, the terms "about" and "approximately" are used as equivalents. With or without about / approximately, any numeral used in the present application is intended to cover any normal fluctuation understood by those of ordinary skill in the relevant art. In certain embodiments, the terms "about" or "approximately" refer to (greater than or less than) falling into 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a numerical range in a smaller range in the either direction of the reference value, unless otherwise stated or apparent from context (unless such numerals exceed 100% of possible values).

[0112] As used herein, the term "isolated" means that the material is substantially or essentially free from normally accompanying components as found in its native state. In some embodiments, the terms "obtained" or "derived" are used synonymously with isolated.

[0113] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to vertebrates, such as mammals. The mammal can be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee) or human. Tissues, cells or derivatives thereof of the subject (whether obtained in vivo or cultured in vitro) are also contemplated. Human subjects can be adults, adolescents, children (2 to 14 years old), infants (1 to 24 months) or newborns (up to 1 month). In some embodiments, an adult is an elderly person who is about 65 years old or older or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who intends to become pregnant.

[0114] The term "sample" refers to a certain volume and / or mass of biological material analyzed. In some embodiments, the sample includes a tissue sample, a cell sample, a fluid sample, etc. In some embodiments, the sample is taken from a subject (e.g., a human subject) or provided by it. In some embodiments, the sample includes a portion of tissue taken from any internal organ, cancerous, precancerous or non-cancerous tumor, brain, skin, hair (including roots), eyes, muscles, bone marrow, cartilage, white adipose tissue and / or brown adipose tissue. In some embodiments, the fluid sample includes oral swabs, blood, cord blood, saliva, semen, urine, ascites, pleural fluid, spinal fluid, lung lavage fluid, tears, sweat, etc. It will be understood by those of ordinary skill in the art that in some embodiments, "sample" is a "raw sample" because it is obtained directly from a source (e.g., a subject). In some embodiments, "sample" is the result of processing the raw sample, such as removing certain potential contaminating components, separating certain components and / or purifying certain components of interest. In some embodiments, the sample is a cell or cell population (e.g., a neuronal cell). The cell sample can be directly derived from a subject (e.g., a primary sample) or can be a cell line. The cell line can include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0115] As used herein, "treating" or "treatment" refers to delivering a composition (e.g., an engineered receptor and / or ligand) to a subject and / or a cell population to affect a physiological outcome. In specific embodiments, treatment results in an improvement (e.g., reduction, alleviation, or remedy) of one or more disease symptoms. The improvement may be an observable or measurable improvement, or may be an improvement in the subject's overall sense of well-being. The treatment of a disease may refer to reducing the severity of disease symptoms. In some embodiments, treatment may refer to reducing the severity of disease symptoms to a level comparable to the severity before the onset of the disease. In some embodiments, treatment may refer to a short-term (e.g., temporary or acute) and / or long-term (e.g., continuous or chronic) reduction in disease symptoms. In some embodiments, treatment may refer to the alleviation of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of subjects at risk of developing a particular disease to prevent disease development. Preventing disease development may refer to preventing disease symptoms completely, delaying the onset of disease, alleviating the severity of symptoms in a disease that subsequently develops, or reducing the likelihood of disease development.

[0116] 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.

[0117] A "therapeutically effective amount" is the amount of a composition required to achieve the desired therapeutic outcome. A therapeutically effective amount can vary depending on factors such as, but not limited to, the disease state and age, sex, and weight of the subject. Generally, a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes an amount of a composition that is effective for treating a subject.

[0118] "Increase" refers to an increase in value (e.g., increased binding affinity, increased physiological response, increased therapeutic effect, etc.) of at least 5% compared to a reference or control level. For example, an increase can include an increase of 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 500%, 1000% or more. An increase also means an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) higher than a reference or control level.

[0119] "Reduced," "decreased," "attenuated," or their synonyms refer to a decrease in a value of at least 5% compared to a reference or control level (e.g., decreased binding affinity, decreased physiological response, decreased therapeutic reduction, decreased pain in a subject, etc.). For example, a decrease can include a decrease of 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 500%, 1000% or more. A decrease also means 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) lower than a reference or control level.

[0120] The so-called "maintain" or "keep" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to a physiological and / or therapeutic effect comparable to the effect caused by a vehicle or control molecule / composition. A comparable response is a response that is not significantly different or measurably different from a reference response.

[0121] The terms "reference" or "control" levels are used interchangeably throughout this article and refer to the value of a specific physiological and / or therapeutic effect in a subject or sample that has not been treated with a composition described herein, or a subject or sample that has been treated with a vehicle control. In some embodiments, a reference level refers to the value of a specific physiological and / or therapeutic effect measured in a subject or sample prior to administration of a composition described herein (e.g., a baseline level).

[0122] 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.

[0123] "Receptor-ligand binding" and "ligand binding" are used interchangeably herein to 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 signaling intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., a synthetic or non-naturally occurring ligand. For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by a variety of methods known in the art (e.g., detecting association with a radiolabeled ligand).

[0124] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site on a receptor and a ligand. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K d Affinity can be measured by common methods known in the art, including the methods described herein.

[0125] The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims to refer to the binding that occurs between paired species of molecules (e.g., receptors and ligands). The terms "specific binding affinity" or "specific binding" are used interchangeably throughout the specification and claims to refer to the binding that occurs between paired species of molecules (e.g., receptors and ligands). In various embodiments, the specific binding between one or more species is direct. In one embodiment, the affinity of the specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0126] "Signaling" refers to the biochemical or physiological response resulting from the binding of a ligand to a receptor.

[0127] The term "wild type" or "original" is a term of art understood by those skilled in the art and refers to the typical form of an organism, strain, gene, protein or feature as it occurs in nature, as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of the protein that occurs in nature.

[0128] Throughout the specification and claims, the terms "non-naturally occurring," "variant," and "mutant" are used interchangeably to refer to mutants of a native or wild-type composition, such as a variant polypeptide having less than 100% sequence identity with a native or wild-type sequence.

[0129] "Amino acid modification" or "amino acid mutation" can be an amino acid substitution, an amino acid deletion and / or an amino acid insertion. Amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also called conservative mutations, conservative substitutions or conservative variations) are amino acid substitutions in proteins that change a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used herein, "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or substitution of one polar residue for another, such as substitution of arginine for lysine, substitution of glutamic acid for aspartic acid, or substitution of glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, and the like.

[0130] The terms "parent" or "starting material" are used interchangeably throughout the specification and claims to refer to an initial composition, or a protein that is mutated, modified or derivatized to create an engineered composition with new properties. In some embodiments, the parent protein is a chimeric protein.

[0131] The term "engineered" is used throughout the specification and claims to refer to a non-naturally occurring composition, or a protein that has properties that differ from the parent composition, or the protein from which it is derived.

[0132] In general, "sequence identity" or "sequence homology" refers to the correspondence between nucleotides and nucleotides or amino acids and amino acids of two polynucleotides or polypeptide sequences, respectively. Typically, the technique for determining sequence identity includes determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining the "percent identity" of the two or more sequences (polynucleotides or amino acids). The percent identity of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of close matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. The identity percentage can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990), and as described in Altschul, et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); One Foundation Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). In short, the BLAST program defines identity as the number of identical aligned symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The program can be used to determine the percentage identity of the entire length of the compared proteins. Default parameters are provided to optimize searches using short query sequences, for example, using the blastp program. The program also allows the use of SEG filters to mask fragments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The degree of sequence identity is expected to range from about 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0133] As used herein, "substantially identical" refers to sequences having 85% or more, such as 90% or more, such as 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, wherein the activity of the composition is not altered by modifications in the sequence that result in differences in sequence identity.

[0134] As used herein, with respect to the position of an amino acid or nucleotide, the term "corresponds to" or "corresponds to" refers to an amino acid in a first polypeptide sequence that is aligned with a given amino acid in a reference polypeptide sequence when the first polypeptide and the reference polypeptide sequence are aligned, or a nucleotide in a first polynucleotide sequence that is aligned with a given nucleotide in a reference polynucleotide sequence when the first polynucleotide and the reference polynucleotide sequence are aligned. The alignment is performed by a person skilled in the art using software designed for this purpose (e.g., BLAST program version 2.2.9 with default parameters for this version).

[0135] 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 a transcription start site, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional connection between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs the transcription of a nucleic acid corresponding to the second sequence.

[0136] As used herein, the term "viral vector," "viral vector," or "gene delivery vector" refers to a viral particle that acts as a vehicle for nucleic acid delivery and comprises a nucleic acid (e.g., an AAV expression cassette) packaged in a virion. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0137] As used herein, "neuronal activity", "neuronal activity", "neuronal discharge" and variants and synonyms thereof refer to electrical activity generated by stimulation or excitation of neurons. In some embodiments, neuronal activity is measured using an automated or manual patch clamp technique. In some embodiments, determining the activity of a neuron includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the activity level of a neuron depends on, or is affected by, an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential.

[0138] 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 a structural, biochemical, and / or electrical abnormality of the brain, spinal cord, nerves, or any component of the nervous system.

[0139] As used herein, "signs" of disease refer to physical or mental characteristics of a condition that are considered indicative of disease. In some embodiments, a sign is an objective indication of disease. In some embodiments, a sign is objectively evaluated, examined, observed, or measured by someone other than the patient, such as a physician.

[0140] As used herein, "symptoms" of a disease refer to physical or mental characteristics of a condition that are considered indicative of the disease, particularly characteristics that are noticeable to the patient. In some embodiments, the symptom is subjectively assessed by the patient. For example, in some embodiments, the symptom is pain.

[0141] As used herein, "potency" refers to the ability of a receptor as described herein to respond to a specific ligand. Thus, an increase in potency refers to an increase in the responsiveness of a receptor to a specific ligand. Thus, a decrease in potency refers to a decrease in the responsiveness of a receptor to a specific ligand. The potency of a receptor is generally determined herein by the half-maximal effective concentration (EC50) of a specific ligand of a specific receptor. EC50 refers to the ligand concentration that causes a response halfway between the baseline and the maximum value after a specific exposure time. In some embodiments, the response is the opening or closing of an ion channel in a receptor.

[0142] As used herein, "substantially retains potency" of a ligand refers to an engineered receptor whose EC50 for a particular ligand is either unchanged or altered and increased or decreased by less than 2-fold compared to a parent or control receptor.

[0143] As used herein, the "efficacy" of a receptor relative to a ligand refers to a measure of the activity of the receptor in the presence of the ligand. In some embodiments, efficacy refers to the amount of current passing through the receptor under specific conditions (such as in the presence of a specific concentration of the ligand). In some embodiments, determining efficacy includes determining the amount of current passing through the receptor, and / or the base strength of the receptor.

[0144] C. Engineered receptors

[0145] The present disclosure relates to engineered receptors, engineered receptor mutants and methods of using them. As used herein, the term "receptor" refers to any protein located on the surface of a cell and that can mediate signal transduction to and / or from a cell. The term "engineered receptor" is used herein to refer to a receptor that has been experimentally altered to physically and / or functionally differ from a corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. "Wild-type receptor" is used herein to refer to a receptor having a polypeptide sequence identical to that of a protein found in nature. Wild-type receptors include receptors naturally present in humans and orthologs naturally present in other eukaryotic organisms (e.g., protists, fungi, plants or animals, such as yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates). In some embodiments, the parent receptor is a non-natural receptor; that is, it is a receptor that does not exist in nature, for example, a receptor engineered from a wild-type receptor. For example, a parent receptor can be an engineered receptor comprising one or more subunits from a wild-type receptor and one or more subunits from a second wild-type receptor. Thus, the resulting protein comprises subunits from two or more wild-type receptors. Thus, in some embodiments, the parent receptor is a chimeric receptor. Engineered receptors of the present disclosure include, for example, parent receptor mutants and switch receptors.

[0146] In some aspects, the engineered receptor of the present disclosure comprises at least one amino acid mutation relative to the corresponding parent receptor, for example, one or more mutations in one or more domains of the wild-type receptor. In some embodiments, mutation is an amino acid substitution. In some embodiments, the engineered receptor has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or lower sequence identity with the corresponding parent receptor, including all values ​​and sub-ranges therebetween. In some embodiments, the parent receptor variant has 85% or higher sequence identity with the corresponding parent receptor, for example, 90% or higher or 95% or higher with the corresponding parent receptor, for example, about 96%, about 97%, about 98% or about 99% identity, including all values ​​and sub-ranges therebetween. In some embodiments, the engineered receptor (for example, parent receptor mutant) is generated by error-prone PCR.

[0147] In some embodiments, the ligand binding domain (LBD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation relative to the corresponding ligand binding domain of the parent receptor, such as one or more mutations in the ligand binding domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand binding domain of the engineered receptor has 85% or more sequence identity with the ligand binding domain of the corresponding parent receptor, such as 90% or more or 95% or more with the ligand binding domain of the corresponding parent receptor, such as about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity, including all values ​​and sub-ranges therebetween. In some embodiments, the ligand binding domain of the engineered receptor has a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the corresponding ligand binding domain of the parent receptor, including all values ​​and sub-ranges therebetween.

[0148] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation relative to the corresponding ion pore domain of the parent receptor, such as one or more mutations in the ion pore domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ion pore domain of the engineered receptor has 85% or more sequence identity with the ion pore domain of the corresponding parent receptor, such as 90% or more or 95% or more, such as about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity with the ion pore domain of the corresponding parent receptor, including all values ​​and subranges therebetween. In some embodiments, the ion pore domain of the engineered receptor shares a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the corresponding ion pore domain of the parent receptor, including all values ​​and subranges therebetween.

[0149] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation relative to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, substantially reduce, or eliminate the function of the engineered receptor relative to the parent receptor, for example by reducing the binding of an endogenous ligand to the engineered receptor relative to the binding of an endogenous ligand to the parent receptor or by reducing the activity of one or more signal transduction pathways downstream of the engineered receptor that are normally activated in response to the binding of a ligand to the corresponding parent receptor. In some embodiments, the mutation is an amino acid substitution.

[0150] In some embodiments, amino acid mutations are functional gain amino acid mutations relative to the corresponding parent receptor. "Function gain" amino acid mutations refer to, for example, by changing or enhancing the affinity of the engineered receptor to the ligand relative to the binding of the endogenous ligand to the parent receptor or by changing or enhancing the activity of the signal transduction pathway activated in response to the binding of the ligand to the engineered receptor relative to the binding of the endogenous ligand to the corresponding parent receptor, one or more mutations of the function of the engineered receptor modified relative to the parent receptor. In some embodiments, functional gain mutations cause the affinity of the engineered receptor to the ligand to increase. In specific embodiments, functional gain mutations cause the affinity of the engineered receptor to the agonist ligand to increase. In some embodiments, functional gain mutations cause the antagonist ligand to act as an agonist ligand when combined with the engineered receptor (for example, causing an agonist signal transduction pathway rather than an antagonist signal transduction pathway to be activated). In some embodiments, functional gain mutations cause the regulator ligand to act as an agonist ligand when combined with the engineered receptor. In some embodiments, mutations are amino acid substitutions.

[0151] In some embodiments, the subject engineered receptors of the present disclosure, or ligand binding domains and / or ion pore domains thereof, comprise one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations relative to the corresponding parent receptor. In some embodiments, the mutations are amino acid substitutions.

[0152] In some embodiments, loss-of-function mutations and gain-of-function mutations are at the same residue, i.e., they are the same mutations. In other embodiments, loss-of-function mutations and gain-of-function mutations are mutations at different amino acid residues. In some embodiments, mutations are amino acid substitutions. In some embodiments, the subject engineered receptors (or their ligand binding domains and / or ion pore domains) comprising loss-of-function mutations and / or gain-of-function mutations have about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% (including all ranges and subranges therebetween) or less sequence identity with the corresponding parent receptor, such as a wild-type receptor or a non-natural receptor (or its ligand binding domain and / or ion pore domain). In some embodiments, a subject engineered receptor (or a ligand binding domain and / or ion pore domain thereof) shares 85% or more sequence identity with a corresponding parent receptor (or a ligand binding domain and / or ion pore domain thereof), e.g., 85%, 90% or 95% or more sequence identity, and in some cases 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity, including all 4 and subranges therebetween.

[0153] In some aspects, the engineered receptors of the present disclosure comprise a combination of one or more amino acid sequences, such as a subunit derived from a wild-type receptor having one or more amino acid sequences, such as a receptor derived from a subunit of a second wild-type receptor. In other words, the engineered receptors include amino acid sequences that are heterologous to each other, where "heterologous" means not occurring together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, chimeric receptors are used as parent receptors from which the engineered receptors disclosed herein are generated.

[0154] In some embodiments, the parent receptor mutant exhibits increased affinity for an agonist ligand. In some embodiments, a ligand that acts as an antagonist or modulator when bound to a wild-type receptor acts as an agonist when bound to a parent receptor mutant.

[0155] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGIC refers to a large group of transmembrane proteins that allow ions to pass through when activated by a specific ligand. LGICs are composed of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Binding of a ligand to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding causes a dramatic change in the permeability of the channel to one or more specific ions; in fact, no ions can pass through the channel when the channel is inactive or closed, but up to 10 ions can pass through the channel per second when a ligand is bound. 7 ions pass through. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and promote ion influx 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 ion efflux from the cytosol to the extracellular environment. Importantly, LGIC activation results in ion transport across the cell membrane (e.g., Ca 2+ 、Na + , K + , Cl - etc.) without causing the transport of the ligand itself.

[0156] LGIC receptors contain multiple subunits and can be homomeric receptors or heteromeric receptors. Homomeric receptors contain subunits that are all of the same type. Heteromeric receptors contain subunits in which at least one subunit is different from at least one other subunit included in the receptor. For example, the glycine receptor contains 5 subunits, of which there are two types: α subunits, of which there are four isoforms (α1-α4); and β subunits, of which there is a single known isoform. An exemplary homomeric GlyR is a GlyR containing 5 α1-GlyR subunits. Similarly, the homomeric GABA A The receptor may contain β3-GABA ASubunits, and nAchR receptors may include α7-nAchR subunits. Exemplary heteromeric GlyRs may include one or more α-subunits and one or more β-subunits (eg, α1β-GlyR). Subunits of exemplary LGIC receptors are shown in Table 1.

[0157] Table 1: LGIC receptors and subunits

[0158]

[0159]

[0160]

[0161]

[0162] Illustrative examples of LGIC families suitable for use in particular embodiments include, but are not limited to, Cys loop receptors, such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), λ-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchRs); as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaCs), ionotropic glutamate receptors, IP3 receptors, P2X receptors, ryanodine receptors, and zinc-activated channels (ZACs).

[0163] Specific non-limiting examples of LGICs suitable for use in the methods described herein include: HTR3A; HTR3B; HTR3C; HTR3D; HTR3E; ASIC1; ASIC2; ASIC3; SCNN1A; SCNN1B; SCNN1D; SCNN1G; GABRA1; GABRA2; GABRA3; GABRA4; GABRA5; GABRA6; GABRB1; GABRB2; GABRB3; GABRG1; GABRG2; GABRG3; GABRD; GABRE; GABRQ; GABRP; GABRR1; GABRR2; GABRR3; GLRA1; GLRA2; GLRA3; GLRA4; GLRB; GRIA1; GRIA2; GRIA3; GRI A4; GRID1; GRID2; GRIK1; GRIK2; GRIK3; GRIK4; GRIK5; GRIN1; GRIN2A; GRIN2B; GRIN2C; GRIN2D; GRIN3A; GRIN3B; ITPR1; ITPR2; ITPR3; CHRNA1; CHRNA2; CHRNA3; CHRNA4; CHR NA5; CHRNA6; CHRNA7; CHRNA9; CHRNA10; CHRNB1; CHRNB2; CHRNB3; CHRNB4; CHRNG; CHRND; CHRNE; P2RX1; P2RX2; P2RX3; P2RX4; P2RX5; P2RX6; P2RX7; RYR1; RYR2; RYR3; and ZACN.

[0164] TRPV1, TRPM8 and P2X2 are members of a larger LGIC family that share structural features and gating principles. For example, TRPV4, similar to TRPV1, is also triggered by heat rather than capsaicin; and P2X3 is triggered by ATP, but desensitizes faster than P2X2. Therefore, TRPV1, TRPM8 and P2X2 are non-limiting examples of LGICs suitable for use in specific embodiments.

[0165] In one embodiment, the engineered receptor is a TRPV1 or TRPM8 receptor or a mutant protein thereof. TRPV1 and TRPM8 are vanilloid receptors and menthol receptors expressed by nociceptive neurons of the peripheral nervous system. It is believed that both channels are used as non-selective sodium and calcium permeable homotetramers. In addition, these two channels and their main agonists - respectively, capsaicin and cooling compounds such as menthol - are hardly present in the central nervous system. Capsaicin and some cooling compounds (including menthol and icilin) ​​contain potential receptor sites for light-labile blocking groups. The association of light-labile blocking groups with such receptors will produce ligand-gated ion channels in which light acts as an indirect trigger by releasing active ligands.

[0166] In one embodiment, the engineered receptor is a P2X2 receptor or a mutant protein thereof. P2X2 is an ATP-gated non-selective cation channel distinguished by its slower desensitization rate. P2X2 can be used as a selective addressable source of depolarizing current and presents a platform for generating engineered channel-ligand combinations that are completely lacking natural agonists.

[0167] Non-limiting examples of sequences of wild-type LGIC receptors that can be used to generate the engineered receptors of the present disclosure include the following. In the sequence, the signal peptide is italicized, the ligand binding domain is bold, and the ion pore domain is underlined:

[0168] In some embodiments, the wild-type LGIC receptor is the human α1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 2), which is encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1 (SEQ ID NO: 1)):

[0169]

[0170] (SEQ ID NO: 2).

[0171] In some embodiments, the wild-type LGIC receptor is the human α2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO: 83), which is encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1, SEQ ID NO: 82):

[0172]

[0173] (SEQ ID NO: 83).

[0174] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO: 85), which is encoded by the GLRA3 gene (GenBank Accession No. NM_006529.3, SEQ ID NO: 84):

[0175]

[0176] (SEQ ID NO: 85).

[0177] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO: 87), which is encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.3, SEQ ID NO: 86):

[0178]

[0179] (SEQ ID NO: 87)

[0180] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor α7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 4), which is encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5 (SEQ ID NO: 3)):

[0181]

[0182] (SEQ ID NO: 4).

[0183] In some embodiments, the wild-type LGIC receptor is human 5-hydroxytryptamine receptor 3A (5HT3A, GenBank Accession No. NP_998786.2, SEQ ID NO: 6), which is encoded by the HTR3A gene (GenBank Accession No. NM_213621.3, SEQ ID NO: 5):

[0184] (a)

[0185] (SEQ ID NO: 6).

[0186] In some embodiments, the wild-type LGIC receptor is human 5-hydroxytryptamine receptor 3B (5HT3B GenBank Accession No. NP_006019.1, SEQ ID NO: 57), which is encoded by the HTR3B gene (GenBank Accession No. NM_006028.4, SEQ ID NO: 56):

[0187] (a)

[0188] (SEQ ID NO: 57).

[0189] In some embodiments, the wild-type LGIC receptor is human gamma-aminobutyric acid receptor A (GABA-A), subunit beta-3 (GABA-A β3) (GenBank Accession No. NP_000805.1, SEQ ID NO: 8), which is encoded by the GABRB3 gene (GenBank Accession No. NM_000814.5, SEQ ID NO: 7):

[0190] (a) (SEQ ID NO: 8).

[0191] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rhol (ρ1) (GABA-Aρ1) (GenBank Accession No. NP_002033.2, SEQ ID NO: 10), which is encoded by the GABRR1 gene (GenBank Accession No. NM_002042.4, SEQ ID NO: 9):

[0192] (a) (SEQ ID NO: 10).

[0193] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho2 (ρ2) (GABA-Aρ2) (GenBank Accession No. NP_002034.3, SEQ ID NO: 12), which is encoded by the GABRR2 gene (GenBank Accession No. NM_002043.4, SEQ ID NO: 11):

[0194] (a) (SEQ ID NO: 12).

[0195] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit rho3 (ρ3) (GABA-Aρ3) (GenBank Accession No. NP 001099050.1, SEQ ID NO: 14), which is encoded by the GABRR3 gene (GenBank Accession No. NM_001105580.2, SEQ ID NO: 13):

[0196] (a) (SEQ ID NO: 14).

[0197] In some aspects, the engineered receptor is a chimeric LGIC receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence and an ion pore conduction domain sequence derived from at least a first LGIC, or more simply, an "ion pore domain sequence" derived from at least a second LGIC. In some embodiments, the derived amino acid sequence is identical to the corresponding region of the LGIC from which it is derived. In some embodiments, the derived amino acid sequence may contain an alteration in at least one amino acid position compared to the corresponding region of the LGIC from which it is derived. In some embodiments, the amino acid sequence derived from the LGIC sequence differs from the corresponding region of the original amino acid sequence by at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the derived amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% (including all ranges and subranges therebetween) sequence identity with the corresponding region of the LGIC amino acid sequence.

[0198] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequences and ion pore domain sequences of Cys loop receptors are well known in the art and can be easily identified from the literature by using publicly available software such as PubMed, Genbank, Uniprot, etc. In some embodiments, the ligand binding domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with the ligand binding domain of the first LGIC. In some embodiments, the ion pore domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is bold and the ion pore domain is underlined.

[0199] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO: 2). In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to about amino acids 29-235 of GlyRα1, such as amino acids 29-235, amino acids 29-240, amino acids 29-246, amino acids 29-248, amino acids 29-250, or amino acids 29-252 of SEQ ID NO: 2. In certain such embodiments, the ligand binding domain consists essentially of amino acids 29-235 of SEQ ID NO: 2, consists essentially of amino acids 29-240 of SEQ ID NO: 2, consists essentially of amino acids 29-246 of SEQ ID NO: 2, consists essentially of amino acids 29-248 of SEQ ID NO: 2, consists essentially of amino acids 29-250 of SEQ ID NO: 2, consists essentially of amino acids 29-252 of SEQ ID NO: 2. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human GlyRα1.

[0200] In some embodiments, the ligand binding domain of the chimeric receptor includes a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), such as amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ligand binding domain consists essentially of amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human α7-nAChR.

[0201] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), such as amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230 or amino acids 23-231 of SEQ ID NO: 4. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human α7-nAChR.

[0202] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the sequence of the ligand binding domain of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ligand binding domain comprises approximately amino acids 23-247 of 5HT3A (SEQ ID NO: 6), such as amino acids 23-240, amino acids 30-245, amino acids 23-247, amino acids 23-250, and in some cases amino acids 30-255 of SEQ ID NO: 6. In certain embodiments, the ligand binding domain consists essentially of amino acids 23-240 of SEQ ID NO: 6, consists essentially of amino acids 23-245 of SEQ ID NO: 6, consists essentially of amino acids 30-247 of SEQ ID NO: 6, consists essentially of amino acids 23-250 of SEQ ID NO: 6, and consists essentially of amino acids 23-255 of SEQ ID NO: 6. In some such embodiments, the ligand binding domain comprises approximately amino acids 21-239 of 5HT3B (SEQ ID NO:57), e.g., amino acids 21-232, amino acids 21-235, amino acids 21-240, amino acids 21-245, and in some cases amino acids 21-247 of SEQ ID NO:57. In certain embodiments, the ligand binding domain consists essentially of amino acids 21-239 of SEQ ID NO:57, consists essentially of amino acids 21-232 of SEQ ID NO:57, consists essentially of amino acids 21-235 of SEQ ID NO:57, consists essentially of amino acids 21-240 of SEQ ID NO:57, consists essentially of amino acids 21-245 of SEQ ID NO:57. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human 5-hydroxytryptamine receptor 3.

[0203] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-Aβ3. In some such embodiments, the ligand binding domain comprises about amino acids 26-245 of GABA-Aβ3 (SEQ ID NO: 8), such as amino acids 26-240, amino acids 26-245, amino acids 26-248, amino acids 26-250, and in some cases amino acids 26-255 of SEQ ID NO: 8. In certain such embodiments, the ligand binding domain consists essentially of amino acids 26-240 of SEQ ID NO: 8, consists essentially of amino acids 26-245 of SEQ ID NO: 8, consists essentially of amino acids 26-248 of SEQ ID NO: 8, consists essentially of amino acids 26-250 of SEQ ID NO: 8, or consists essentially of amino acids 26-255 of SEQ ID NO: 8. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human GABA-A receptor.

[0204] In some embodiments, the ion pore domain fused to the ligand binding domain conducts anions, for example, it comprises the ion pore domain sequence of a human glycine receptor or a human serotonin receptor. In other embodiments, the ion conducting pore domain fused to the ligand binding domain conducts cations, for example, it comprises the ion pore domain sequence of a human acetylcholine receptor or a human γ-aminobutyric acid receptor A.

[0205] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to about amino acids 245-457 of GlyRα1 (SEQ ID NO: 2), such as amino acids 240-457, amino acids 245-457, amino acids 248-457, amino acids 249-457, amino acids 250-457, amino acids 255-457, or amino acids 260-457 of SEQ ID NO: 2. In some embodiments, the ion pore domain consists essentially of amino acids 245-457 of SEQ ID NO:2, consists essentially of amino acids 248-457 of SEQ ID NO:2, consists essentially of amino acids 249-457 of SEQ ID NO:2, or consists essentially of amino acids 250-457 of SEQ ID NO:2.

[0206] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO: 83. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO: 3883. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 258-452 of SEQ ID NO: 83. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 260-452 of SEQ ID NO: 83.

[0207] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 253-464 of SEQ ID NO: 85. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 257-464 of SEQ ID NO: 85. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 259-464 of SEQ ID NO: 85.

[0208] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 7887). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 253-449 of SEQ ID NO: 87. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 257-449 of SEQ ID NO: 87. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 259-449 of SEQ ID NO: 87.

[0209] In some embodiments, the ion pore domain is derived from the ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with about amino acids 230-502, such as amino acids 227-502, amino acids 230-502, amino acids 231-502, amino acids 232-502 or amino acids 235-502. In certain such embodiments, the ion pore domain consists essentially of amino acids 227-502 of SEQ ID NO:4, consists essentially of amino acids 230-502 of SEQ ID NO:4, consists essentially of amino acids 231-502 of SEQ ID NO:4, consists essentially of amino acids 232-502 of SEQ ID NO:4, or consists essentially of amino acids 235-502 of SEQ ID NO:4.

[0210] In some embodiments, the ion pore domain is derived from the ion pore domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to about amino acids 248-516 of 5HT3A (SEQ ID NO: 6), e.g., amino acids 240-516, amino acids 245-516, amino acids 248-516, amino acids 250-516, or amino acids 255-516 of SEQ ID NO: 6. In certain such embodiments, the ion pore domain consists essentially of amino acids 240-516 of SEQ ID NO: 6, consists essentially of amino acids 245-516 of SEQ ID NO: 6, consists essentially of amino acids 248-516 of SEQ ID NO: 6, consists essentially of amino acids 250-516 of SEQ ID NO: 6, or consists essentially of amino acids 253-516. In some such embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to about amino acids 240-441 of 5HT3B (SEQ ID NO: 57), e.g., amino acids 230-441, amino acids 235-441, amino acids 240-441, amino acids 245-441, or amino acids 250-441 of SEQ ID NO: 57. In certain such embodiments, the ion pore domain consists essentially of amino acids 230-441 of SEQ ID NO:57, consists essentially of amino acids 235-441 of SEQ ID NO:57, consists essentially of amino acids 240-441 of SEQ ID NO:57, consists essentially of amino acids 245-441 of SEQ ID NO:57, or consists essentially of amino acids 250441.

[0211] In some embodiments, the ion pore domain is derived from the ion pore domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-Aβ3. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to about amino acids 246-473 of GABA-Aβ3 (SEQ ID NO: 8), such as amino acids 240-473, amino acids 245-473, amino acids 247-473, amino acids 250-473, or amino acids 253-473 of SEQ ID NO: 8. In certain such embodiments, the ion pore domain consists essentially of amino acids 240-473 of SEQ ID NO:8, amino acids 245-473 of SEQ ID NO:8, amino acids 247-473 of SEQ ID NO:8, amino acids 250-473 of SEQ ID NO:8, or amino acids 253-473 of SEQ ID NO:8.

[0212] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ1 (GABRR1, SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain sequence of human GABA-Aρ1 comprises, consists essentially of, or consists of amino acids 284-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-Aρ1 comprises, consists essentially of, or consists of amino acids 288-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-Aρ1 comprises, consists essentially of, or consists of amino acids 290-479 of SEQ ID NO: 10.

[0213] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ2 (GABRR2, SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ion pore domain sequence of human GABA-Ap2 comprises, consists essentially of, or consists of amino acids 265-466 of SEQ ID NO: 12. In some embodiments, the ion pore domain sequence of human GABA-Ap2 comprises, consists essentially of, or consists of amino acids 269-466 of SEQ ID NO: 12. In some embodiments, the ion pore domain sequence of human GABA-Ap2 comprises, consists essentially of, or consists of amino acids 271-466 of SEQ ID NO: 12.

[0214] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GABA-Aρ3 (GABRR3, SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain sequence of human GABA-A p3 comprises, consists essentially of, or consists of amino acids 271-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-A p3 comprises, consists essentially of, or consists of amino acids 275-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-A p3 comprises, consists essentially of, or consists of amino acids 277-467 of SEQ ID NO: 14.

[0215] In some embodiments, the ion pore domain of the subject chimeric ligand-gated ion channel comprises an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ion pore domain. By "M2-M3 linker domain" or "M2-M3 linker" is meant a sequence within the ion pore domain of the LGIC that is flanked by the C-terminal end of the transmembrane domain 2 (M2) of the receptor at its amino (N) terminal and by the N-terminal end of the transmembrane domain 3 (M3) of the receptor at its carboxyl (C) terminal. The M2-M3 linker of the LGIC can be readily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). In some embodiments, when the ion pore domain of the chimeric receptor comprises a heterologous M2-M3 linker, the M2-M3 linker is derived from the same receptor as the ligand binding domain of the chimeric receptor. For example, when the subject ligand-gated ion channel comprises a ligand binding domain from an AChR and an ion pore domain from a GlyR, its ion pore domain sequence may comprise an M2-M3 linker sequence derived from an AChR. In some embodiments, the ion pore domain is derived from GlyRα1, and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the native M2-M3 linker sequence removed from the ion pore domain corresponds to about amino acids 293-313 of GlyRα1 (SEQ ID NO: 2), e.g., amino acids 304-310, 293-306, 298-310, 305-311, 302-313, etc. In some such embodiments, the inserted M2-M3 linker is derived from approximately amino acids 281-295 of α7-nAChR (SEQ ID NO: 4), e.g., amino acids 290-295, 281-290, 281-295, 283-295, 287-292, etc., or the inserted M2-M3 linker is derived from a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to amino acids 281-295 or 283-295 of α7-nAChR.

[0216] In some embodiments, the ligand binding domain of the subject chimeric ligand-gated ion channel comprises a Cys loop domain sequence that is heterologous to the Cys loop sequence of the ligand binding domain. By "Cys loop domain sequence" or "Cys loop sequence" is meant a domain within the ligand binding domain of the Cys loop LGIC that forms a ring structure flanked by cysteines at the N- and C-termini. Without wishing to be bound by theory, it is believed that upon ligand binding to the ligand binding domain, the Cys loop structurally moves to be adjacent to the M2-M3 loop, and this movement mediates the biophysical transition of ligand binding in the extracellular domain to signal transduction in the ion pore domain (as reviewed in Miller and Smart, Trends in Pharmacological Sci 2009: 31(4)). Replacing the endogenous Cys loop sequence with a heterologous Cys loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases at least 5 times or 6 times, and at least 7 times, 8 times, 9 times or 10 times at certain doses. The Cys loop domain of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the chimeric receptor comprises a heterologous Cys loop sequence, the Cys loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the subject chimeric ligand-gated ion channel comprises a ligand binding domain from an AChR and an ion pore domain from a GlyR, the subject ligand-gated ion channel can comprise a ligand binding domain sequence from an AChR (except for the sequence of the Cys loop domain, which is instead derived from a GlyR). In some embodiments, the ligand binding domain is derived from α7-nAChR and the Cys loop sequence is derived from GLyR. In some embodiments, the Cys loop sequence removed from the ligand binding domain corresponds to about amino acids 150-164 of α7-nAChR SEQ ID NO: 4), such as amino acids 150-157 of α7-nAChR. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 166-180 of GlyRα1 (SEQ ID NO: 2), such as amino acids 166-172 of GlyRα1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 166-180 of GlyRα1.

[0217] In some embodiments, the inserted Cys loop sequence is derived from about amino acids 172-186 of GlyRα2 (SEQ ID NO: 83), such as amino acids 172-178 of GlyRα2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 172-186 of GlyRα2. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 171-185 of GlyRα3 (SEQ ID NO: 85 or 87), such as amino acids 171-177 of GlyRα3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 171-185 of GlyRα3. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 198-212 of GABA-ρ1 (SEQ ID NO: 10), such as amino acids 198-204 of GABA-ρ1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 198-212 of GABA-ρ1. In some embodiments, the inserted Cys loop sequence is derived from about amino acids 178-192 of GABA-ρ2 (SEQ ID NO: 12), such as amino acids 178-184 of GABA-ρ2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 178-192 of GABA-ρ2. In some embodiments, the inserted Cys loop sequence is derived from approximately amino acids 184-198 of GABA-Aρ3 (SEQ ID NO: 14), such as amino acids 184-190 of GABA-Aρ3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 184-198 of GABA-Aρ3.

[0218] In some embodiments, the ligand binding domain of the subject chimeric ligand-gated ion channel comprises a β1-2 loop domain sequence that is heterologous to the β1-2 loop domain sequence of the ligand binding domain. By "β1-2 loop domain sequence" or "β1-2 loop or β1-β2 loop" is meant a domain within the ligand binding domain of the Cys loop LGIC, which is flanked by the C-terminus of the β1 sheet at its N-terminus and by the N-terminus of the β2 sheet at its C-terminus. Without wishing to be bound by theory, it is believed that the β1-2 loop contributes to the biophysical transition of mediating ligand binding in the extracellular domain to the ion pore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). It is believed that, after ligand binding, the β1-2 loop, together with the Cys loop, comes to the immediate vicinity of the M2-M3 loop to mediate the biophysical transition of signal transduction in the ion pore domain where the ligand in the extracellular domain resides (as reviewed by Miller and Smart above). Replacing the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases at least 5 times or 6 times, and at least 7 times, 8 times, 9 times or 10 times at certain doses. The β1-2 loop of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the chimeric receptor comprises a heterologous β1-2 loop sequence, the β1-2 loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the subject chimeric ligand-gated ion channel comprises a ligand binding domain derived from an AChR and an ion pore domain derived from a GlyR, the sequence of the β1-2 loop domain of the ligand binding domain can be derived from GlyR. In some embodiments, the ligand binding domain is derived from α7-nAChR. In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to about amino acids 64-72 or 67-70 of α7-nAChR (SEQ ID NO: 4), such as amino acids 67-70, amino acids 66-71, or amino acids 64-72 of α7-nAChR. In some embodiments, the inserted β1-2 loop sequence is about amino acids 79-85 of GlyRα1 (SEQ ID NO: 2), such as amino acids 80-85, 81-84, 79-85, or 81-84 of GlyRα1, with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα2, and the inserted β1-2 loop corresponds to about amino acids 86-91 of GlyRα2 (SEQ ID NO: 83), with up to 3, up to 2, up to 1, or no amino acid mutations.In some embodiments, the ion pore domain is derived from GlyRα3, and the inserted β1-2 loop corresponds to about amino acids 85-90 of GlyRα3 (SEQ ID NO: 85 or 87), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Aρ1, and the inserted β1-2 loop corresponds to about amino acids 112-117 of GABA-Aρ1 (SEQ ID NO: 10), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Aρ2, and the inserted β1-2 loop corresponds to about amino acids 92-97 of GABA-Aρ2 (SEQ ID NO: 12), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GABA-Aρ3 and the inserted β1-2 loop corresponds to approximately amino acids 98-103 of GABA-Aρ3 (SEQ ID NO: 14) with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the mutations are amino acid substitutions.

[0219] In some embodiments, the present disclosure provides a chimeric LGIC receptor comprising a ligand binding domain derived from a human α7-nAChR and an ion pore domain derived from a human glycine receptor, wherein the ligand binding domain comprises one or more amino acid substitutions of the present disclosure. In some embodiments, the human glycine receptor is a human glycine receptor α1, a human glycine receptor α2, or a human glycine receptor α3. In some embodiments, the ligand binding domain comprises a Cys loop domain derived from a human glycine receptor. In some embodiments, the ligand binding domain comprises a β1-2 loop domain derived from a human glycine receptor.

[0220] In some embodiments, the present disclosure provides a chimeric LGIC receptor comprising a ligand binding domain derived from a human α7-nAChR and an ion pore domain derived from a human GABA receptor, wherein the ligand binding domain comprises one or more amino acid substitutions of the present disclosure. In some embodiments, the human GABA receptor is human GABA-Aρ1, human GABA-Aρ2, or human GABA-Aρ3. In some embodiments, the ligand binding domain comprises a Cys ring domain derived from a human GABA receptor. In some embodiments, the ligand binding domain comprises a β1-2 ring domain derived from a human GABA receptor.

[0221] Non-limiting examples of chimeric LGIC receptor sequences of the present disclosure include sequences disclosed herein as SEQ ID NO: 15-SEQ ID NO: 52. In some embodiments, the chimeric LGIC receptor or polynucleotide encoding the same has 85% or more sequence identity to the sequences provided herein as SEQ ID NO: 15-SEQ ID NO: 52, such as 90% or more, 93% or more, or 95% or more, i.e., about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequences provided herein as SEQ ID NO: 15-SEQ ID NO: 52. In the sequences, the signal peptide is italicized, the ligand binding domain is bold, and the ion pore domain is underlined.

[0222] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 linked) comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0223] (SEQ ID NO: 16, encoded by SEQ ID NO: 15).

[0224] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (R228 linked) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0225] (SEQ ID NO: 17).

[0226] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (V224 junction) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0227] (SEQ ID NO: 18).

[0228] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (Y233 linked) chimera comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0229] (SEQ ID NO: 19).

[0230] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 linked) comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) fused to a human GlyRα1 ion pore domain (underlined) comprising an α7-nAChR M2-M3 linker (lowercase):

[0231] (a) (SEQ ID NO: 21, encoded by SEQ ID NO: 20):

[0232] (b) (SEQ ID NO: 23, encoded by SEQ ID NO: 22):

[0233] (c) (SEQ ID NO: 25, encoded by SEQ ID NO: 24);

[0234] (d) (SEQ ID NO: 27, encoded by SEQ ID NO: 26);

[0235] (e) (SEQ ID NO: 29, encoded by SEQ ID NO: 28); or

[0236] (f) (SEQ ID NO: 31, encoded by SEQ ID NO: 30).

[0237] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1 Cys loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the following SEQ ID No: 33:

[0238]

[0239] (SEQ ID NO: 33, encoded by SEQ ID NO: 32).

[0240] (a)

[0241] (SEQ ID NO: 35. Encoded by SEQ ID NO: 34).

[0242] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising the human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising the GlyRα1 β1-2 loop sequence (lowercase); fused to the human GlyRα1 ion pore domain (underlined):

[0243] (a) (SEQ ID NO: 37, encoded by SEQ ID NO: 36).

[0244] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1β1-2 loop sequence (lowercase) and a Cys loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined):

[0245] (a) (SEQ ID NO: 39, encoded by SEQ ID NO: 38).

[0246] (b)

[0247]

[0248] (SEQ ID NO:41, encoded by SEQ ID NO:40).

[0249] (c)

[0250] (SEQ ID NO: 43. Encoded by SEQ ID NO: 42).

[0251] (d) (SEQ ID NO: 45, encoded by SEQ ID NO: 44).

[0252] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1β1-2 loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined) comprising a human α7-nAChR M2-M3 linker (lowercase):

[0253] (a) (SEQ ID NO: 47, encoded by SEQ ID NO: 46).

[0254] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) comprising a GlyRα1 Cys loop sequence (lowercase); fused to a human GlyRα1 ion pore domain (underlined) comprising a human α7-nAChR M2-M3 linker (lowercase):

[0255] (a) (SEQ ID NO: 49, encoded by SEQ ID NO: 48).

[0256] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (R241 linked), comprising the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold), fused to the human GlyRα1 ion pore domain (underlined):

[0257] (a) (SEQ ID NO: 50).

[0258] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (V236 junction) comprising the human 5HT3A serotonin receptor signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0259] (a) (SEQ ID NO: 51).

[0260] In some embodiments, the chimeric LGIC receptor is a GABRB3 / GLRA1 chimera (Y245 linked) comprising the human GABA-Aβ3 signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0261] (a) (SEQ ID NO: 52).

[0262] C1. Amino Acid Mutation

[0263] As described above, in some aspects, the subject engineered receptor comprises at least one amino acid mutation that changes the potency of the ligand on the engineered receptor relative to the potency of the ligand on the unmutated parent receptor. In other words, one or more amino acid mutations, such as loss-of-function mutations or gain-of-function mutations, change the potency of the engineered receptor for the ligand relative to the potency of the unmutated parent receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, one or more mutations are in the ligand binding domain of the engineered receptor. In some embodiments, such as when the ligand binding domain of the engineered receptor is a Cys loop receptor protein, the one or more amino acid mutations are substitutions at residues corresponding to the following residues of α7-nAChR (SEQ ID NO: 4), the residues selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213, and Y217. In some embodiments, one residue is substituted. In some embodiments, 2, 3, 4, or 5 or more residues are substituted, for example, 6, 7, 8, 9, or 10 residues are substituted. In certain embodiments, the residue corresponds to a residue of α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188. In certain embodiments, one or more substitutions are within the α7-nAChR sequence.

[0264] In some embodiments, one or more substitutions reduce the potency of the engineered receptor for acetylcholine and non-natural ligands by, e.g., 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In certain embodiments, one or more substitutions are substitutions corresponding to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of α7-nAChR. In other cases, one or more substitutions selectively reduce the potency of acetylcholine to the engineered receptor. In other words, one or more substitutions reduce the effectiveness of engineered receptors to acetylcholine while substantially maintaining the effectiveness of non-natural ligands, or, compared to its reduction of the responsiveness of engineered receptors to non-natural ligands, otherwise reduce the effectiveness of engineered receptors to acetylcholine by 2 times or more, such as 3 times, 4 times, 5 times or more, in some cases 10 times, 20 times, 50 times, 100 times or more. In some embodiments, the substitution corresponds to L131E, L131S, L131T, L131D or S172D of α7-nAChR. In other embodiments, one or more substitutions selectively reduce the effectiveness of non-natural ligands to engineered receptors. In other words, one or more substitutions reduce the effectiveness of engineered receptors to non-natural ligands while substantially maintaining the effectiveness of acetylcholine, or, compared to its reduction of the effectiveness of engineered receptors to acetylcholine, otherwise reduce the effectiveness of engineered receptors to non-natural ligands by 2 times or more, such as 3 times, 5 times or more, in some cases 10 times, 20 times or 50 times or more. In some embodiments, the substitution corresponds to W77M, Y115W, S172T or S172C of α7-nAChR. In certain embodiments, one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126 and Facinicline / RG3487.

[0265] In other embodiments, one or more substitutions increase, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more or 100 times the effectiveness of engineered receptors to acetylcholine and / or non-natural ligands. In some embodiments, substitutions correspond 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 embodiments, one or more substitutions increase the effectiveness of acetylcholine and non-natural ligands. In some embodiments, the substitution corresponds to L131N, S170G, S170A, S170L, S170U, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q or S188T of α7-nAChR. In other cases, one or more substitutions selectively increase the potency of acetylcholine to the engineered receptor. In other words, it increases the potency of the engineered receptor to acetylcholine by 2 times or more, such as 3 times, 4 times or 5 times or more, and in some cases 10 times, 20 times, 50 times or 100 times, compared to the potency of the engineered receptor to the non-natural ligand increased by one or more substitutions. In some embodiments, the substitution corresponds to the substitution of L141W, S172T, S172C, S188P and S188W of α7-nAChR. In certain embodiments, one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126 and Facinicline / RG3487. In some other cases, one or more substitutions selectively increase the potency of the non-natural ligand to the engineered receptor. In other words, compared with one or more substitutions increasing the potency of the engineered receptor to acetylcholine, one or more substitutions increase the potency of the engineered receptor to the non-natural ligand by 2 times or more, for example 3 times, 5 times or more, in some cases 10 times, 20 times or 50 times or more.

[0266] In some embodiments, the amino acid residue mutated in the subject engineered receptor is not the amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7nAChR (SEQ ID NO: 4). In some embodiments, the mutation is an amino acid substitution. In some embodiments, the amino acid residue mutated in the subject engineered receptor is the amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7nAChR (SEQ ID NO: 4). In some embodiments, the substitution is not a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution is a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, when such a substitution is present in an engineered receptor, it is present in combination with one or more amino acid mutations described herein.

[0267] In some embodiments, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 4) mediate the binding of the natural ligand acetylcholine. In some embodiments, mutations at these residues can reduce the binding of acetylcholine and are therefore considered loss-of-function mutations. In some embodiments, residues W77, Y115, N129, V130, L131, Q139, L141, S170, Y210, C212, C213, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand AZD0328 to the receptor, and mutations of these residues can increase the affinity of AZD0328 and / or other ligands for the receptor and are therefore considered gain-of-function mutations. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues of the ligand binding domain of a α7-nAChR (SEQ ID NO: 4) or a chimeric receptor comprising the ligand binding domain of a α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of: W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213, and Y217. In some embodiments, the mutation is an amino acid substitution. In certain embodiments, the mutation in one or more amino acid residues of the ligand binding domain of the α7-nAChR (SEQ ID NO: 4) or a chimeric receptor comprising the ligand binding domain of the α7-nAChR is a substitution at one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217.

[0268] In some embodiments, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 4) can mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues can reduce the binding of acetylcholine and / or nicotine. In some embodiments, R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand ABT126, and mutations at one or more of these residues can increase the affinity of ABT126 and / or other ligands for α7-nAChR. In some embodiments, the mutation is an amino acid substitution. In some embodiments, R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of α7-nAChR can mediate the 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 α7-nAChR. In some embodiments, R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand Facinicline / RG3487, and mutation of one or more of these residues can increase the affinity of Facinicline / RG3487 and / or other ligands for α7-nAChR. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues of the ligand binding domain of the α7-nAChR or a chimeric receptor comprising the ligand binding domain of the α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of: R101, Y115, T128, N120, N129, L131, L141, S170, W171, S172, S188, Y210, C212, C213, and Y217. In some embodiments, the one or more amino acid residues alter the binding of acetylcholine and / or nicotine to the α7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212, and Y217 of the α7-nAChR. In certain such embodiments, the amino acid is selected from C212 and S170. In some embodiments, mutation of one or more amino acid residues alters the binding of ABT126 to α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, L131, L141, W171, S172, S188, Y210 and Y217 of α7-nAChR.In certain such embodiments, the amino acid is selected from R101, S188 and Y210. In some embodiments, mutations in one or more amino acid residues alter the binding of TC6987 to α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210 and C213. In some embodiments, mutation of one or more amino acid residues alters the binding of Facinicline / RG3487 to α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from Y210, R101 and N129.

[0269] In some embodiments, residues W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3 (SEQ ID NO: 6) can mediate the binding of serotonin, and mutations at one or more of these residues can reduce the binding of serotonin to 5HT3. D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3 mediate the binding of the non-natural ligand cilansetron, and mutations at one or more of these residues can increase the affinity of cilansetron and / or other ligands for 5HT3. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain of a chimeric receptor comprising the ligand binding domain of 5HT3A, wherein the one or more amino acid residues are selected from the group consisting of: D64, I66, W85, R87, Y89, N123, Y136, Y138, G146, N147, Y148, K149, T176, S177, S178, L179, W190, R191, F221, E224, Y228, Y229, and E2311. In some embodiments, mutations in one or more amino acid residues alter the binding of serotonin to 5HT3, wherein the one or more amino acids are selected from the group consisting of W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3A. In certain such embodiments, the amino acids are selected from Y136, Y138, N147, K149, and L179. In some embodiments, mutations in one or more amino acid residues alter the binding of cilansetron to 5HT3, wherein the one or more amino acid residues are selected from the group consisting of D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3A. In certain such embodiments, the amino acid is selected from D64, 166, Y89, N123, T176, W190, R191, F221, E224, and E231.

[0270] In some embodiments, one or more mutations that affect the ability of a ligand to modulate LGIC activity are in the ion pore domain of the LGIC. In some embodiments, the mutation is an amino acid substitution. For example, residue T279 of the serotonin receptor 5HT3A mediates the manner in which ligands modulate channel activity, such that mutation of this residue to, for example, serine (T279S) switches the effect from antagonism (i.e., reducing the activity of the LGIC) to agonism (i.e., promoting the activity of the channel). In some embodiments, the subject ligand-gated ion channel comprises a mutation in one or more amino acid residues of the ion pore domain of human 5HT3A (SEQ ID NO: 6) or the ion pore domain of a chimeric LGIC receptor comprising the 5HT3A ion pore domain, wherein the substitution is in the amino acid corresponding to 279 of SEQ ID NO: 6. In certain embodiments, the substitution is a T279S substitution relative to SEQ ID NO: 6.

[0271] The present disclosure provides engineered receptors having two or more mutations (such as amino acid substitutions) compared to a parent receptor. In some embodiments, the parent receptor comprises a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR). In some embodiments, the parent receptor is a chimeric receptor. In some embodiments, the parent receptor comprises an ion pore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is a human glycine receptor α1, a human glycine receptor α2, or a human glycine receptor α3. In some embodiments, the ligand binding domain of the engineered receptor comprises a Cys ring domain derived from a human glycine receptor. In some embodiments, the parent receptor comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the engineered receptor comprises two amino acid substitutions compared to a parent receptor comprising an amino acid sequence of SEQ ID NO: 33. In some embodiments, the ligand binding domain of the engineered receptor comprises a β1-2 ring domain from a human glycine receptor α1 subunit.

[0272] In some embodiments, the ligand binding domain of the engineered receptor comprises amino acid substitutions at two or more amino acid residues selected from amino acid residues corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172, Y210 and Y217 of human α7-nAChR (SEQ ID NO: 4).

[0273] 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, Y115 and S172, 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, Y115 and S172, and Y115 and L131. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution of S172D. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution of Y115D. 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, Y115Q and S172D, Y115E and L131M, and Y115D and L131E. In some embodiments, the ligand binding domain comprises an amino acid substitution of L131E.

[0274] In some embodiments, the ligand binding domain comprises one or more amino acid substitutions at an amino acid residue selected from the group consisting of Y140, R101, L131, Y115, and Y210, wherein the amino acid residue corresponds to an amino acid residue of an α7-nAChR.

[0275] In some embodiments, the ligand binding domain comprises an amino acid substitution of R101W and / or Y210V. In some embodiments, the ligand binding domain comprises two or more amino acid substitutions at an amino acid residue selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at an amino acid residue selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of: R101 and L131, Y115 and Y210, R101 and Y210. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of R101F and L131G, R101F and L131D, Y115E and Y210W, R101W and Y210V, R101F and Y210V, R101F and Y210F, R101M and L131A, and R101M and L131F. In some embodiments, the ligand binding domain comprises three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand binding domain comprises amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0276] In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution of R101F or R101M. In some embodiments, the amino acid substitution at residue L131 is L131G, L131D, L131A, L131F, or L131N.

[0277] In some embodiments, the ligand binding domain comprises a hydrophobic amino acid substitution at residue Y210 and an amino acid substitution of R101W or R101F. In some embodiments, the amino acid substitution at residue Y210 is Y210V, Y210F or Y210W.

[0278] In some embodiments, the ligand binding domain comprises an amino acid substitution of Q139W and an additional amino acid substitution at a residue selected from S172, Y210, and Y217.

[0279] Those skilled in the art will readily recognize appropriate control receptors for comparison with the engineered receptors of the present disclosure. In some embodiments, the control receptor is identical in sequence to the engineered receptor except for one or more distinguished amino acid mutations (e.g., substitutions). In all cases, reference to a control receptor is intended to indicate that the stated property change (e.g., efficacy to a ligand) is the result of an amino acid mutation of an engineered receptor of the present disclosure.

[0280] The present invention provides an engineered receptor, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor and comprises: (a) a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprising a Cys ring domain derived from a human glycine receptor α1 subunit; and (b) an ion pore domain derived from a human glycine receptor α1 subunit. In some embodiments, the engineered receptor is derived from a parent engineered receptor comprising or consisting of an amino acid sequence of SEQ ID NO: 33, and further comprises one or more amino acid substitutions based on the parent engineered receptor.

[0281] In some embodiments, the potency of engineered receptors to acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine. In some embodiments, the potency of engineered receptors to acetylcholine is at least about 1.5 times lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine (e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, the potency of engineered receptors to acetylcholine is assessed by the EC50 of acetylcholine of the engineered receptor determined by the cell reporter using YFP fluorescence quenching as described in Example 2 of the present disclosure. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 100uM, at least 200uM, at least 300uM, at least 500uM, at least 700uM, at least 1mM, at least 2mM, at least 3mM, at least 4mM, at least 5mM, at least 6mM, at least 7mM, at least 8mM, at least 9mM or at least 10mM. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 1mM. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 3mM. In some embodiments, having a higher EC50 for acetylcholine allows the higher expression level of the engineered receptor in cells or on cell surfaces without having a significant amount of electricity entering the cell in the presence of acetylcholine at physiological concentrations.

[0282] In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is roughly the same as that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands by at least about 1.5 times (e.g., low about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, determining potency comprises determining an EC50 based on a cell reporter assay using YFP fluorescence quenching as described in Example 2 of the present disclosure. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 15 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 150 ... In some embodiments, the EC50 of the engineered receptor to non-natural ligands is less than 10nM. In some embodiments, the EC50 of the engineered receptor to non-natural ligands is less than 100nM. In some embodiments, the EC50 of the engineered receptor to non-natural ligands is less than 100nM. In some embodiments, the EC50 of the engineered receptor to non-natural ligands is less than 100nM. In some embodiments, the EC50 of the engineered receptor to non-natural ligands is less than 1uM.

[0283] In some embodiments, the efficacy of engineered receptors in the presence of non-natural ligands is higher than the efficacy of human α7 nicotinic acetylcholine receptors (α7-nAChR) in the presence of non-natural ligands. In some embodiments, the efficacy of engineered receptors in the presence of non-natural ligands is higher than the efficacy of human α7 nicotinic acetylcholine receptors (α7-nAChR) in the presence of non-natural ligands by at least about 1.5 times (for example, low about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, wherein determining the efficacy includes determining in vitro the amount of the current passing through the engineered receptor in the presence of the non-natural ligand.

[0284] In some aspects, the subject ligand-gated ion channel comprises one or more non-desensitizing mutations. In some embodiments, the mutation is an amino acid substitution. When used for ligand-gated ion channels, "desensitization" refers to the gradual reduction of ion fluxes under the long-term presence of agonists. This causes the neuron to gradually lose the effectiveness of the ligand. The so-called non-desensitizing mutation means an amino acid mutation that prevents LGIC from becoming desensitized to the ligand, thereby preventing the neuron from becoming less responsive or unresponsive to the ligand. By introducing the LGIC carrying the mutation into the neuron and analyzing the current flux during long-term exposure to the ligand, non-desensitizing mutations can be easily identified. If the LGIC does not include a non-desensitizing mutation, the current will recover from the peak value to the steady state during the long-term exposure, and if the LGIC includes a non-desensitizing mutation, the current will maintain the peak flux during exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (V294L processes the preprotein to remove the signal peptide) and the L321V mutation in human GABA-A receptor GABRB3 (L296V processes the preprotein to remove the signal peptide). In some embodiments, the desensitizing mutation is to replace the amino acid residues at or near the C-terminus of LGIC with a desensitizing sequence (e.g., a sequence having 90% or greater identity to IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53) derived from the C-terminus of a protein encoded by GABAR1), for example, replacing 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, for example, 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.

[0285] In some aspects, the subject ligand-gated ion channel comprises one or more conversion mutations. In some embodiments, the mutation is an amino acid substitution. The so-called conversion mutation refers to a mutation that changes the permeability of the LGIC ion pore domain so that it becomes permeable to the conduction of non-natural ions (i.e., ions that are not naturally allowed to pass). In some cases, the mutation converts the permeability from cations to anions, such as replacing amino acid residues 260-281 (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 54) in human α7-nAChR (CHRNA7) with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 55), or the corresponding amino acids in another LGIC that is permeable to cations. In some cases, the mutation converts the permeability from anion to cation, for example, amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with glutamic acid (E) (such as A293E substitution in GLRA1, which converts the LGIC from anion permeable to calcium permeable), or amino acid residue 278 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is deleted, amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with glutamic acid (E), and amino acid residue 293 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC is substituted with valine (V) (such as P278Δ, A279E, T293V in GLRA1, which converts the LGIC from anion permeable to cation permeable).

[0286] In some embodiments, the parent receptor mutant library is produced from a limited number of parent receptors. Methods known in the art (including fallibility PCR) can be used to mutate the parent receptor. In some embodiments, the parent receptor mutant library is then transfected into yeast or mammalian cells, and screened with high throughput to identify functional receptors (for example, to identify the parent receptor mutant that can be carried out in response to the part for signal transduction). In some embodiments, the functional parent receptor mutant identified in the primary screening is then expressed in mammalian cells, and the effect of the part is screened, for example, by plate reader and / or electrophysiological assay as described herein. Then it is possible to select the binding affinity that shows the increase of the agonist ligand in the secondary screening or to enable the use of antagonists or regulator ligands as the parent receptor mutants of agonists, and further in vitro and / or in vivo verification and characterization are measured. Such screening assays are known in the art, e.g., Armbruster, BN et al. (2007) PNAS, 104, 5163-5168; Nichols, CD and Roth, BL (2009) Front. Mol. Neurosci. 2, 16; Dong, S. et al. (2010) Nat. Protoc. 5, 561-573; Alexander, GM et al. (2009) Neuron 63, 27-39; Guettier, JM et al. (2009) PNAS 106, 19197-19202; Ellefson JW et al. (2014) Nat Biotechnol . 32(1):97-101; Maranhao AC and Ellington AD. (2017) ACS Synth Biol. 20;6(1):108-119; Talwar S et al. (2013) PLoS One;8(3):e58479; Gilbert DF et al. (2009) Front Mol Neurosci. 30;2:17; Lynagh and Lynch, (2010), Biol Chem. 14:285(20), 14890-14897; Islam R. et al. (2016) ACS Chem Neurosci. 21;7(12):1647-1657; and Myers et al. (2008) Neuron. 8:58(3):362-373.

[0287] C2. Exemplary Chimeric LGICs

[0288] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to L131S and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor having no substitution or only one of such substitutions. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor having no such substitution or only one of such substitutions. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA534 (SEQ ID NO: 59), and the control receptor is CODA71, CODA333, or CODA377.

[0289] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to L131T and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor having no substitution or only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of acetylcholine>3mM. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor having no such substitution or only one of such substitutions. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA535 (SEQ ID NO: 60) and the control receptor is CODA71, CODA335, or CODA377.

[0290] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to L131D and S172D in the LBD of human α7-nAChR. In some embodiments, compared with a control receptor without such substitution or only having one of such substitutions, such engineered receptors have a lower efficacy (e.g., as determined by a higher EC50) for acetylcholine. In some embodiments, compared with a control receptor without such substitution or only having one of such substitutions, such an engineered receptor substantially retains the efficacy (or has a higher efficacy) for non-natural ligands. In some embodiments, compared with a control receptor without such substitution and / or only having one of such substitutions, such an engineered receptor has a higher efficacy (lower EC50) for CNL002. In some embodiments, the non-natural ligand of such an engineered receptor is CNL002, AZD-0328 or Facinicline. In some embodiments, the non-natural ligand is Facinicline. In some embodiments, the non-natural ligand is AZD-0328. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA536 (SEQ ID NO: 58) and the control receptor is CODA71, CODA339 or CODA377.

[0291] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to Y115D and S170T in the LBD of human α7-nAChR. In some embodiments, compared with a control receptor without such substitution and / or only having one of such substitutions (e.g., only S170T), such engineered receptors have a lower efficacy (e.g., as determined by a higher EC50) for acetylcholine. In some embodiments, such engineered receptors have an EC50 of acetylcholine>3mM. In some embodiments, compared with a control receptor without such substitution or only having one of such substitutions (e.g., only S170T), such engineered receptors substantially retain the efficacy (or have a higher efficacy) to non-natural ligands. In some embodiments, the non-natural ligand of such engineered receptors is Facinicline or TC-6987. In some embodiments, the non-natural ligand is Facinicline. In some embodiments, the non-natural ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA805 (SEQ ID NO: 63) and the control receptor is CODA71, CODA282, or CODA109.

[0292] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to Y115D and L131Q in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor without such substitution or with only one of such substitutions (e.g., only L131Q). In some embodiments, such engineered receptors have an EC50 of acetylcholine>3mM. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, the non-natural ligand of such engineered receptors is AZD-0328, Facinicline, or TC-6987. In some embodiments, the non-natural ligand is Facinicline. In some embodiments, the non-natural ligand is AZD-0328. In some embodiments, the non-natural ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA806 (SEQ ID NO: 62) and the control receptor is CODA71, CODA282, or CODA334.

[0293] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to Y115D and L131E in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower efficacy (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, such engineered receptors substantially retain the efficacy (or have a higher efficacy) for non-natural ligands compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, the non-natural ligand ligand of such engineered receptors is TC-5619, AZD-0328, Facinicline or TC-6987. In some embodiments, the non-natural ligand is TC-5619. In some embodiments, the non-natural ligand is Facinicline. In some embodiments, the non-natural ligand is AZD-0328. In some embodiments, the non-natural ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA807 (SEQ ID NO: 61) and the control receptor is CODA71, CODA282, or CODA340.

[0294] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to R101F and L131G in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower efficacy (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, such engineered receptors substantially retain the efficacy (or have a higher efficacy) for non-natural ligands compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, the non-natural ligands of such engineered receptors are CNL001, TC-5619, CNL002, AZD-0328, TC-6987 or varenicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC-5619. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is AZD-0328. In some embodiments, the non-natural ligand is TC-6987. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1025 (SEQ ID NO: 65) and the control receptor is CODA71, CODA236 or CODA325.

[0295] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to R101F and L131D in the LBD of human α7-nAChR. In some embodiments, compared with a control receptor without such substitution or only having one of such substitutions, such engineered receptors have a lower efficacy for acetylcholine (e.g., as determined by a higher EC50). In some embodiments, compared with a control receptor without such substitution or only having one of such substitutions, such an engineered receptor substantially retains the efficacy (or has a higher efficacy) to a non-natural ligand. In some embodiments, the non-natural ligand of such an engineered receptor is CNL001, TC-5619, CNL002, or TC-6987. In some embodiments, a non-natural ligand is CNL001. In some embodiments, a non-natural ligand is TC-5619. In some embodiments, a non-natural ligand is CNL002. In some embodiments, a non-natural ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1027 (SEQ ID NO: 66) and the control receptor is CODA71, CODA236, or CODA339.

[0296] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to Y115E and Y210W in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower efficacy (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, such engineered receptors substantially retain the efficacy (or have a higher efficacy) for non-natural ligands compared to a control receptor without such substitution or with only one of such substitutions. In some embodiments, the non-natural ligands of such engineered receptors are TC-5619, ABT-0126, or CNL002. In some embodiments, the non-natural ligands are TC5619 / Bradanicline. In some embodiments, the non-natural ligands are ABT-0126. In some embodiments, the non-natural ligands are CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1039 (SEQ ID NO: 67) and the control receptor is CODA71, CODA283, or CODA409.

[0297] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101W and Y210V in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor having no such substitution or only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of acetylcholine>3mM. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor having no such substitution or only one of such substitutions. In some embodiments, the non-natural ligand is TC-5619 / bradanicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1045 (SEQ ID NO:68) and the control receptor is CODA71, CODA238, or CODA405.

[0298] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101F and Y210V in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor having no such substitution or only one of such substitutions (e.g., R101F). In some embodiments, such engineered receptors have an EC50 of acetylcholine of >3mM. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors having no such substitution or only one of such substitutions (e.g., Y210V). In some embodiments, the non-natural ligand is TC-5619 / Bradanicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1047 (SEQ ID NO:69) and the control receptor is CODA71, CODA236, or CODA405.

[0299] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101F and Y210F in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor without such substitutions or with only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of >1mM acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor without such substitutions or with only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of <= about 10nM of CNL001 and / or an EC50 of <= about 30nM of TC5619 / Bradanicline. In some embodiments, the non-natural ligand of such engineered receptors is CNL001 or TC5619 / Bradanicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / Bradanicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1048 (SEQ ID NO: 70) and the control receptor is CODA71, CODA236 or CODA407.

[0300] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101M and L131A in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor without such substitutions or with only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of >1mM acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor without such substitutions or with only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of <= about 10nM of CNL001, an EC50 of <= about 3nM of TC5619 / Bradanicline, and / or an EC50 of <= about 3nM of varenicline. In some embodiments, the non-natural ligand of such engineered receptors is CNL001, TC5619 / Bradanicline or varenicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / Bradanicline. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1053 (SEQ ID NO: 71), and the control receptor is CODA71, CODA237 or CODA326.

[0301] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101M and L131F in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of >1mM or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one of such substitutions. In some embodiments, such engineered receptors have an EC50 of <= about 1nM of CNL001 and / or an EC50 of <= about 3nM of varenicline. In some embodiments, the non-natural ligand is CNL001, TC5619 / Bradanicline or varenicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / Bradanicline. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1054 (SEQ ID NO: 72) and the control receptor is CODA71, CODA237 or CODA330.

[0302] In some embodiments, the engineered receptor comprises a ligand binding domain derived from a human α7-nAChR and amino acid substitutions corresponding to R101W, Y115E, and Y210W in the LBD of a human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >3mM or >10mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, the non-natural ligand is TC-5619 / Bradanicline. In some embodiments, such engineered receptors have an EC50 of <= about 1nM for TC-5619 / Bradanicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1055 (SEQ ID NO: 73) and the control receptor is CODA71, CODA238, CODA283, or CODA409.

[0303] In some embodiments, the engineered receptor comprises a ligand binding domain derived from a human α7-nAChR and amino acid substitutions corresponding to R101F, Y115E, and Y210W in the LBD of a human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >3mM or >10mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, the non-natural ligand is TC-5619 / Bradanicline. In some embodiments, such engineered receptors have an EC50 of <= about 10nM for TC-5619 / Bradanicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1056 (SEQ ID NO: 74) and the control receptor is CODA71, CODA236, CODA283, or CODA409.

[0304] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to W77F, R101F, and L131D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >1mM or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of <= about 10nM of CNL002. In some embodiments, the non-natural ligand is CNL001, CNL002, or ABT-126. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1138 (SEQ ID NO: 75) and the control receptor is CODA71, CODA217, CODA236 or CODA339.

[0305] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101F, L131N, and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an ECs0 of >1mM or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of <= about 1nM of CNL001, or an EC50 of <= about 10nM of CNL002. In some embodiments, the non-natural ligand is CNL001 or CNL002. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1140 (SEQ ID NO: 76) and the control receptor is CODA71, CODA236, CODA337 or CODA377.

[0306] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to Q139E and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one of such substitutions (e.g., S172D). In some embodiments, such an engineered receptor substantially retains potency (or has higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one of such substitutions. In some embodiments, the non-natural ligand is CNL001 or CNL002. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1157 (SEQ ID NO: 77) and the control receptor is CODA71, CODA945, or CODA377.

[0307] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to S172D and Y210W in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor without such a substitution or with only one of such a substitution. In some embodiments, such engineered receptors have an EC50 of >3mM or >10mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have a higher potency) for non-natural ligands compared to a control receptor without such a substitution or with only one of such a substitution. In some embodiments, such engineered receptors have an EC50 of <= about 10nM of CNL001. In some embodiments, the non-natural ligand of such engineered receptors is CNL001, CNL002, or ABT-126. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1173 (SEQ ID NO: 78) and the control receptor is CODA71, CODA377 or CODA409.

[0308] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution at an amino acid residue corresponding to Y140 of human α7-nAChR. In some embodiments, the amino acid substitution is Y140I. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor without such a substitution. In some embodiments, the engineered receptor is a chimeric LGIC (e.g., CODA952, SEQ ID NO: 64), which comprises a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1, and the control receptor is CODA71 (SEQ ID NO: 33). In some embodiments, such an engineered receptor substantially retains potency (or has higher potency) for non-natural ligands compared to a control receptor without such a substitution. In some embodiments, the non-natural ligand of such engineered receptors is CNL001, TC-5619 / Bradanicline, CNL002, or Facinicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC-5619 / Bradanicline. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is Facinicline.

[0309] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution at an amino acid residue corresponding to Y140 of human α7-nAChR. In some embodiments, the amino acid substitution is Y140C. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor without such a substitution. In some embodiments, the engineered receptor is a chimeric LGIC (e.g., CODA965, SEQ ID NO: 88), which comprises a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1, and the control receptor is CODA71 (SEQ ID NO: 33). In some embodiments, such an engineered receptor substantially retains potency (or has higher potency) for non-natural ligands compared to a control receptor without such a substitution. In some embodiments, the non-natural ligand of such engineered receptors is CNL001, TC-5619 / Bradanicline, CNL002, ABT-126, or TC-6987. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC-5619 / Bradanicline. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the non-natural ligand is TC-6987.

[0310] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution at an amino acid residue corresponding to Y140 of human α7-nAChR. In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and an amino acid substitution corresponding to Y140R in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency (e.g., as determined by a higher EC50) for acetylcholine compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such a substitution or have only one or two of such substitutions. In some embodiments, the non-natural ligand is TC-5619. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA967 (SEQ ID NO: 89) and the control receptor is CODA71.

[0311] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to Y115E and L131M in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such substitutions or have only one or two of such substitutions. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1101 (SEQ ID NO:90) and the control receptor is CODA71.

[0312] In some embodiments, the engineered receptor comprises a ligand binding domain derived from a human α7-nAChR and amino acid substitutions corresponding to Q139W and S172D in the LBD of a human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such a substitution or have only one or two of such substitutions. In some embodiments, the non-natural ligand is ABT-126 or CNL002. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1167 (SEQ ID NO: 91) and the control receptor is CODA71.

[0313] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to Q139W and Y210W in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such substitutions or have only one or two of such substitutions. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1168 (SEQ ID NO:92) and the control receptor is CODA71.

[0314] In some embodiments, the engineered receptor comprises a ligand binding domain derived from a human α7-nAChR and amino acid substitutions corresponding to Q139W, S172D, and Y217I in the LBD of a human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such a substitution or have only one or two of such substitutions. In some embodiments, the non-natural ligand is ABT-126 or CNL001. In some embodiments, the non-natural ligand is ABT-126. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1245 (SEQ ID NO: 93) and the control receptor is CODA71.

[0315] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to Y115E, Q139E, and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such substitutions or have only one or two of such substitutions. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1261 (SEQ ID NO:94) and the control receptor is CODA71.

[0316] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101F, L131G, Q139L, and Y217F in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such substitutions or have only one or two of such substitutions. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1458 (SEQ ID NO:95) and the control receptor is CODA71.

[0317] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to Y115Q and S172D in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such substitutions or have only one or two of such substitutions. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1471 (SEQ ID NO: 96) and the control receptor is CODA71.

[0318] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to L141M, S172D, and Y210W in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, the non-natural ligand is CNL001 or CNL002. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1500 (SEQ ID NO: 97) and the control receptor is CODA71.

[0319] In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR and amino acid substitutions corresponding to R101F, L131N, and Y217F in the LBD of human α7-nAChR. In some embodiments, such engineered receptors have a lower potency for acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such a substitution or has only one or two of such substitutions. In some embodiments, such engineered receptors have an EC50 of >0.4mM, >1mM, or >3mM for acetylcholine. In some embodiments, such engineered receptors substantially retain potency (or have higher potency) for non-natural ligands compared to control receptors that do not have such a substitution or have only one or two of such substitutions. In some embodiments, the non-natural ligand is varenicline or CNL002. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human GlyRα1. In some embodiments, the engineered receptor is CODA1507 (SEQ ID NO: 98) and the control receptor is CODA71.

[0320] Table 10 below provides a summary of these exemplary engineered receptors.

[0321] Table 10: Exemplary Engineered Receptors

[0322]

[0323]

[0324]

[0325] D. Ligand

[0326] In some embodiments, the ligands of the present disclosure refer to having a known effect on mammalian cells. The terms "binding agent" or "agent" are used interchangeably herein and refer to exogenous drugs or compounds (e.g., agonists, antagonists, or modulators known to act as receptors) with a known mechanism of action on mammalian cells. Such ligands may also be referred to as "binding agents". The ligands of the present disclosure may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the ligands include drugs or compounds that have been approved by the US Food and Drug Administration (FDA) for clinical use in the treatment of specific diseases (e.g., neurological diseases). In some embodiments, the ligands include drugs or compounds that have not been approved by the FDA for clinical use, but have been tested in one or more clinical trials, are currently being tested in one or more clinical trials, and / or are expected to be tested in one or more clinical trials. In some embodiments, the ligands include drugs or compounds that are not approved by the FDA for clinical use but are routinely used in laboratory research. In some embodiments, the ligand is an analog of one of the above-mentioned ligands. In a specific embodiment, the ligand is selected from any one of the ligands in Tables 2-9 below. In some embodiments, the ligand is selected from the group consisting of AZD0328, ABT-126, AQW-051, cannabidiol, cilansetron, PH-399733, FACINICLINE / RG3487 / MEM-3454, TC-6987, CNL002, and TC-5619 / AT-101. In some embodiments, the ligand is selected from the group consisting of ABT-126, AZD-0328, CNL0025, RG3487, TC-6987, CNL001, TC-6683, varenicline, and TC-5619.

[0327] In certain embodiments, the ligand is an analog of cilansetron, for example as described by one of the following compound formulas 2-7 in its R or S enantiomer:

[0328]

[0329] In some embodiments, the ligand acts as an agonist. As used herein, the term "agonist" refers to a ligand that induces a signaling response. In some embodiments, the ligand acts as an antagonist. As used herein, the term antagonist refers to a ligand that inhibits a signaling response.

[0330] In some embodiments, the ligand is AZD-0328 according to the formula:

[0331]

[0332] In some embodiments, the ligand is TC-6987 according to the formula:

[0333]

[0334] In some embodiments, the ligand is ABT-126 according to the formula:

[0335]

[0336] In some embodiments, the ligand is TC-5619 / Bradanicline according to the formula:

[0337]

[0338] In some embodiments, the ligand is TC-6683 according to the formula:

[0339]

[0340] In some embodiments, the ligand is varenicline according to the formula:

[0341]

[0342] In some embodiments, the ligand is Facinicline / RG3487 according to the formula:

[0343]

[0344] In some embodiments, the ligand is CNL001.

[0345] In some embodiments, the ligand is CNL002.

[0346] In some embodiments, the ligand is an anxiolytic, anticonvulsant, antidepressant, antipsychotic, antiemetic, nootropic, antibiotic, antifungal, antiviral, or antiparasitic.

[0347] Table 2: Ligands of glycine receptor (GlvR)

[0348]

[0349]

[0350] Table 3: Ligands of the lambda-aminobutyric acid A receptor (GABA-A)

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Table 4: Ligands of 5-HT3 receptors

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Table 5: Ligands of Nicotinic Acetylcholine Receptor (nAchR)

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] Table 6: Ligands of ATP-gated P2X receptor cation channels (P2X)

[0373]

[0374]

[0375]

[0376] Table 7: Ligands of inward rectifier potassium channels (Kir)

[0377]

[0378] Table 8: Ligands of voltage-dependent potassium channels (KCNO / Kv7)

[0379] Agonists Modulators / Binders Antagonists Diazoxide Azimilide Flupirtine Amiodarone Minoxidil Bretylium Nicorandil Clofilium Pinacidil Dalfampridine Retigabine Dofetilide E-4031 Ibutilide Nifekalant Sematilide Sotalol Sulfonylurea Tedisamil

[0380] Table 9: Ligands of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

[0381] Agonists Modulators / Binders Antagonists 8-Cyclopentyl-1,3-dipropylxanthine Bumetanide 8-Methoxypsoralen Crofelemer Apigenin Glibenclamide CTP-656 Ibuprofen Genistein IBMX Ivacaftor Lumacaftor

[0382] E. Polynucleotide

[0383] In various illustrative embodiments, the present disclosure contemplates, in part, polynucleotides, polynucleotides encoding engineered receptor polypeptides and fusion polypeptides comprising LGIC and its subunits and mutant proteins, viral vector polynucleotides, and compositions comprising the same.

[0384] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence" or "nucleic acid" are used interchangeably. The terms refer to a polymeric form of nucleotides (deoxynucleotides or ribonucleotides or their analogs) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined according to connection analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the modification of the nucleotide structure can be performed before or after assembling the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The polynucleotide may be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a DNA / RNA hybrid. The polynucleotide may be single-stranded or double-stranded. The polynucleotide includes, but is not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozyme, synthetic RNA, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA (-)), synthetic RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more (including all ranges and subranges therebetween) nucleotides (ribonucleotides or deoxynucleotides or modified forms of either type of nucleotides) in length, as well as all intermediate lengths. It will be readily understood that in this context, "intermediate lengths" refers to any length between the cited values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.In specific embodiments, the polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all ranges and subranges therebetween, to a reference sequence described herein or known in the art, wherein, unless otherwise specified, the variants typically retain at least one biological activity of the reference sequence.

[0385] As used herein, the term "gene" may refer to a polynucleotide sequence including enhancers, promoters, introns, exons, etc. In a specific embodiment, the term "gene" refers to a polynucleotide sequence encoding a polypeptide, regardless of whether the polynucleotide sequence is the same as a genomic sequence encoding the polypeptide.

[0386] As used herein, "cis-acting sequence", "cis-acting regulatory sequence" or "cis-acting nucleotide sequence" or equivalents refer to polynucleotide sequences associated with the expression, e.g., transcription and / or translation, of a gene. In one embodiment, a cis-acting sequence regulates transcription because the cis-acting sequence is a binding site for a polypeptide that inhibits or reduces transcription or a polynucleotide sequence associated with a transcription factor binding site that contributes to transcriptional inhibition. Examples of cis-acting sequences that regulate the expression of a polynucleotide sequence and that can be operably linked to the polynucleotides of the present disclosure to regulate the expression of a subject engineered receptor are well known in the art and include elements such as promoter sequences (e.g., CAG, CMV, SYN, CamKII, TRPV1), Kozak sequences, enhancers, post-transcriptional regulatory elements, miRNA binding elements, and polyadenylation sequences.

[0387] As a non-limiting example, a promoter sequence is a DNA regulatory region that can bind to RNA polymerase in a cell and initiate transcription of a downstream (3' direction) coding sequence. For the purpose of defining the present invention, a promoter sequence is defined by a transcription initiation site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. A transcription initiation site and a protein binding domain responsible for binding RNA polymerase will be found within the promoter sequence. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Each promoter can be used to drive each vector of the present invention. For example, a promoter can be a constitutively active promoter, i.e., a promoter that is active in the absence of an externally applied ligand, for example, CMV IE1 promoter, SV40 promoter, GAPDH promoter, actin promoter. A promoter can be an inducible promoter, i.e., a promoter whose activity is regulated when a ligand is applied to a cell, for example, doxycycline, tet-on or tet-off promoter, estrogen receptor promoter, etc. The promoter may be a tissue-specific promoter, ie, a promoter that is active in certain types of cells.

[0388] In some embodiments, the promoter is active in excitable cells. "Excitable cells" means cells that are activated by changing membrane potential, such as neurons or muscle cells, such as dorsal root ganglion neurons, motor neurons, excitatory neurons, inhibitory neurons, or sensory neurons. Promoters active in excitable cells that would be useful in the polynucleotide compositions of the present invention would include neuronal promoters, such as synaptobrevin (SYN), TRPV1, Na v 1.7 Na v 1.8 Na v 1.9, CamKII, NSE and Advillin promoters; myocyte promoters, such as desmin (Des), α-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC) promoters; and ubiquitous promoters, such as CAG, CBA, E1Fa, Ubc, CMV and SV40 promoters.

[0389] As used herein, "regulatory elements for inducible expression" refers to a polynucleotide sequence that is operably linked to a polynucleotide to be expressed and responds to the presence or absence of a molecule that binds to the element to increase (turn on) or decrease (turn off) the expression of the polynucleotide operably linked thereto, an enhancer, or a functional fragment thereof. Illustrative regulatory elements for inducible expression include, but are not limited to, tetracycline responsive promoters, ecdysone responsive promoters, cumate responsive promoters, glucocorticoid responsive promoters, estrogen responsive promoters, RU-486 responsive promoters, PPAR-γ promoters, and peroxide inducible promoters.

[0390] "Regulatory elements for transient expression" refers to polynucleotide sequences that can be used to briefly or temporarily express a polynucleotide sequence. In specific embodiments, one or more regulatory elements for transient expression can be used to limit the duration of the polynucleotide. In certain embodiments, the preferred duration of polynucleotide expression is about minutes, hours, or days. Illustrative regulatory elements for transient expression include, but are not limited to, nuclease target sites, recombinase recognition sites, and inhibitory RNA target sites. In addition, to some extent, in specific embodiments, regulatory elements for inducible expression can also help control the duration of polynucleotide expression.

[0391] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or to a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" encompass polynucleotides in which one or more nucleotides have been added or deleted or modified or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. In specific embodiments, the polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all ranges and subranges therebetween, to a reference sequence described herein or known in the art, wherein, unless otherwise specified, the variants typically retain at least one biological activity of the reference sequence.

[0392] In one embodiment, polynucleotides include nucleotide sequences that hybridize with target nucleic acid sequences under stringent conditions. Hybridization under "stringent conditions" describes a hybridization scheme in which nucleotide sequences at least 60% identical to each other maintain hybridization. Typically, stringent conditions are selected to be approximately 5 ° C lower than the thermal melting point (Tm) of a specific sequence under a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize with the target sequence when balanced. Since the target sequence is usually present in excess, 50% of the probes are occupied when balanced under Tm.

[0393] As used herein, the statement "sequence identity" or, for example, including "a sequence that is 50% identical to ..." refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size) and multiplying the result by 100 to produce a percentage of sequence identity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12, but typically 15 to 18 and often at least 25 monomer units in length, comprising nucleotides and amino acid residues. Because two polynucleotides can each include (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) a sequence that differs between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides within a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, typically about 50 to about 100, more typically about 100 to about 150, wherein a sequence is compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. The comparison window can include about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (the reference sequence does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for the comparison window can be performed by computerized implementations of the algorithm (GAP, BESTFIT, FASTA, and TFASTA of the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., producing the highest percentage of homology over the comparison window) generated by any of the various methods selected.Reference may also be made to the BLAST family of programs as disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis may be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0394] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that occur on either side of it in its naturally occurring state, for example, a fragment that has been removed from sequences that are normally adjacent to a DNA fragment. In specific embodiments, "isolated polynucleotide" refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but have been made by the hand of man.

[0395] Terms describing the orientation of a polynucleotide include: 5′ (usually the end of the polynucleotide with a free phosphate group) and 3′ (usually the end of the polynucleotide with a free hydroxyl group (OH)). Polynucleotide sequences can be annotated in a 5′-3′ orientation or a 3′-5′ orientation. For DNA and mRNA, the 5′-3′ strand is named the “sense,” “positive,” or “coding” strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, rather than thymine (T) in DNA]. For DNA and mRNA, the 3′-5′ complementary strand, which is the strand transcribed by RNA polymerase, is named the “template,” “antisense,” “negative,” or “non-coding” strand. As used herein, the term “reverse orientation” refers to a 5′ to 3′ sequence written in a 3′ to 5′ orientation or a 3′ to 5′ sequence written in a 5′ to 3′ orientation.

[0396] The term "flanking" refers to a polynucleotide sequence between an upstream polynucleotide sequence and / or a downstream polynucleotide sequence, i.e., 5' and / or 3', relative to the sequence. For example, a sequence "flanking" two other elements (e.g., ITRs) indicates that one element is located 5' of the sequence and the other element is located 3' of the sequence; however, there may be an intervening sequence between them.

[0397] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTAC 5'. The latter sequence is often written as the reverse complement 5'CATGACT 3' with the 5' end on the left and the 3' end on the right. A sequence that is equal to its reverse complement is called a palindrome. Complementarity can be "partial", in which only some of the bases in the bases of the nucleic acid are matched according to the base pairing rules. Alternatively, there can be "complete" or "full" complementarity between nucleic acids.

[0398] As used herein, the term "nucleic acid box" or "expression cassette" refers to a polynucleotide sequence within a larger polynucleotide such as a vector, which is sufficient to express one or more RNAs from the polynucleotide. The expressed RNA can be translated into a protein, can be used as a guide RNA or an inhibitory RNA to target other polynucleotide sequences for cleavage and / or degradation. In one embodiment, the nucleic acid box contains one or more polynucleotides of interest. In another embodiment, the nucleic acid box contains one or more expression control sequences operably linked to one or more polynucleotides of interest. The polynucleotide comprises one or more polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide used as a template for a transcriptional inhibitory polynucleotide, such as LGIC and its subunits and mutant proteins, as contemplated herein. In a specific embodiment, the polynucleotide of interest encodes a polypeptide or fusion polypeptide having one or more enzymatic activities (such as nuclease activity) and / or chromatin remodeling or epigenetic modification activity.

[0399] The vector may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. In a preferred embodiment of the present disclosure, the nucleic acid cassette includes one or more expression control sequences (e.g., a promoter or enhancer operable in a neuronal cell) operably linked to a polynucleotide encoding an engineered receptor, such as a LGIC or a subunit thereof or a mutant protein. The cassette may be removed from or inserted into other polynucleotide sequences, such as a plasmid or a viral vector, as a single unit.

[0400] In one embodiment, the polynucleotides contemplated herein include 1, 2, 3, 4, 5, 6, 7, 8, 9 or more nucleic acid cassettes, any number or combination of which may be in the same or opposite orientation.

[0401] In addition, it will be appreciated by those of ordinary skill in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences that can encode fragments of polypeptides or their variants, as contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon selection, are specifically contemplated by the present disclosure. In one embodiment, polynucleotides comprising specific allele sequences are provided. Alleles are endogenous polynucleotide sequences that are altered due to one or more mutations such as deletions, additions and / or substitutions of nucleotides.

[0402] In some embodiments, the present disclosure provides polynucleotides encoding the engineered receptors described herein. In some embodiments, the present disclosure provides polynucleotides encoding the chimeric engineered LGIC receptors described herein.

[0403] In some embodiments, the disclosure provides polynucleotides encoding engineered receptors, the engineered receptors comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of one of SEQ ID NOs 89-98. In some embodiments, the disclosure provides polynucleotides encoding engineered receptors, the engineered receptors comprising an amino acid sequence of one of SEQ ID NOs 89-98. In some embodiments, the disclosure provides polynucleotides encoding engineered receptors, the engineered receptors consisting of an amino acid sequence of one of SEQ ID NOs 89-98.

[0404] F. Vector

[0405] In some aspects of the present disclosure, nucleic acid molecules, i.e., polynucleotides encoding engineered receptors, are delivered to a subject. In some cases, nucleic acid molecules encoding engineered receptors are delivered to a subject via a vector. In various embodiments, a vector includes one or more polynucleotide sequences considered herein. The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred polynucleotides are usually connected to a carrier nucleic acid molecule, such as inserted into a carrier nucleic acid molecule. The vector may contain a sequence that guides autonomous replication in a cell or may contain a sequence sufficient to allow integration into host cell DNA. The vector may deliver a target polynucleotide to an organism, a cell, or a cell component. In some cases, the vector is an expression vector. As used herein, "expression vector" refers to a vector, such as a plasmid, that can promote expression and replication of a polynucleotide incorporated therein. Typically, the nucleic acid sequence to be expressed is operably connected to a cis-acting regulatory sequence, such as a promoter and / or enhancer sequence and is controlled by transcriptional regulation of a promoter and / or enhancer. In certain cases, a vector is used to deliver a nucleic acid molecule encoding an engineered receptor of the present disclosure to a subject.

[0406] In a specific embodiment, any carrier suitable for expression cassette or polynucleotides encoding engineered receptors can be introduced into neuronal cells.Illustrative examples of suitable carriers include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes and viral vectors.In some cases, carriers are circular nucleic acids, such as plasmids, BACs, PACs, YACs, cosmids, Fox plasmids, etc.In some cases, circular nucleic acid molecules can be used to deliver nucleic acid molecules encoding engineered receptors to a subject.For example, the plasmid DNA molecules encoding engineered receptors can be introduced into the cells of the subject, thus the DNA sequence encoding the engineered receptors is transcribed into mRNA and mRNA "message" is translated into a protein product.The circular nucleic acid vector will generally include a regulatory element for regulating the expression of the target protein.For example, the circular nucleic acid vector can include any number of promoters, enhancers, terminators, splicing signals, replication origins, start signals, etc.

[0407] In some cases, the vector may include a replicon. A replicon may be any nucleic acid molecule capable of self-replication. In some cases, a replicon is an RNA replicon derived from a virus. Various suitable viruses (e.g., RNA viruses) may be obtained, including but not limited to alphaviruses, picornaviruses, flaviviruses, coronaviruses, pestiviruses, rubella viruses, caliciviruses, and hepatitis viruses.

[0408] In some embodiments, the vector is a non-viral vector. "Non-viral vector" means any delivery vehicle that does not include a viral capsid or envelope, such as lipid nanoparticles (anionic (negatively charged), neutral or cationic (positively charged)), heavy metal nanoparticles, polymer-based particles, plasmid DNA, minicircle DNA, microcarrier DNA, ccDNA, synthetic RNA, exosomes, etc. Non-viral vectors can be delivered by any suitable method as will be fully understood in the art, including, for example, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection. See, for example, Chen et al. Mol. Therapy, Methods and Clinical Development. 2016 Jan; Vol 3, issue 1; and Hardy, CE et al. Genes (Basel). 2017 Feb; 8 (2): 6.5.

[0409] In other embodiments, the vector is a viral vector. "Viral vector" means a delivery vehicle including a viral capsid or envelope around a polynucleotide encoding a RNA or polypeptide of interest. In some cases, the viral vector is derived from a replication-defective virus. Non-limiting examples of viral vectors suitable for delivering nucleic acid molecules of the present invention to a subject include those derived from adenovirus, retrovirus (e.g., lentivirus), adeno-associated virus (AAV) and herpes simplex-1 (HSV-1). Illustrative examples of suitable viral vectors include, but are not limited to, retroviral vectors (e.g., lentiviral vectors), vectors based on herpes virus and vectors based on parvovirus (e.g., vectors based on adeno-associated virus (AAV), AAV-adenovirus chimeric vectors and vectors based on adenovirus).

[0410] As used herein, the term "parvovirus" encompasses all parvoviruses, including autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include members of the genus Parvovirus, the genus Erythrovirus, the genus Densovirus, the genus Iteravirus, and the genus Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute 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).

[0411] 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 rh10, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV.

[0412] In a preferred embodiment, the vector is an AAV vector. In a specific case, the viral vector is an AAV5, AAV6 or AAV-9 vector.

[0413] The genome organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule with a length of less than about 5,000 nucleotides (nt). Reverse terminal repeats (ITRs) are flanked by unique coding nucleotide sequences of non-structural replication (Rep) proteins and structural (VP) proteins. VP proteins (VP1, VP2 and VP3) form capsids and contribute to the tropism of the virus. The terminal 145nt ITR is self-complementary and is organized so that the energetically stable intramolecular duplex formed by forming a T-shaped hairpin can be formed. These hairpin structures are used as the starting point for viral DNA replication, thereby being used as primers for cellular DNA polymerase complexes. After wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and plays a role in the replication of the viral genome.

[0414] In some cases, the outer protein "capsid" of the viral vector is present in nature, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In certain cases, the capsid is synthetically engineered (e.g., by directed evolution or rational design) to have certain unique properties that do not exist in nature, such as altered tropism, increased transduction efficiency, or immune evasion. An example of a rationally designed capsid is a mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid protein. Non-limiting examples of viral vectors whose VP3 capsid proteins have been synthetically engineered and suitable for use with the compositions and methods provided herein include: AAV1 (Y705+731F+T492V), AAV2 (Y444+500+730F+I491V), AAV3 (Y705+731F), AAV5 (Y436+693+719F), AAV6 (Y705+731F+T492V), AAV8 (Y733F), AAV9 (Y731F) and AAV10 (Y733F). Non-limiting examples of viral vectors that have been engineered by directed evolution and suitable for use with the compositions and methods provided herein include AAV-7m8 and AAV-ShH10. In some embodiments, the viral vector comprises an AAV capsid protein comprising an amino acid mutation at one or more positions corresponding to T492, Y705, Y731, or any combination thereof of an AAV6 capsid protein, wherein the AAV capsid protein is serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or another AAV serotype. In some embodiments, the one or more positions are two or more positions, two positions, or three positions. In some embodiments, the viral vector comprises an AAV capsid protein comprising one or more amino acid substitutions corresponding to T492V, Y705F, or Y731F of an AAV6 capsid protein, or any combination thereof, wherein the AAV capsid protein is serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or another AAV serotype. In some embodiments, the one or more substitutions are two or more substitutions, two substitutions, or three substitutions.

[0415] In some embodiments, the viral vector comprises an AAV5 capsid protein comprising a mutation at one or more amino acid positions selected from Y693 and Y719. In some embodiments, the viral vector comprises an AAV5 capsid protein comprising one or more mutations selected from Y693F and Y719F. In some embodiments, the viral vector comprises an AAV5 capsid protein comprising an amino acid mutation Y693F+Y719F. In some embodiments, the AAV5 capsid protein comprises, or consists of, an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.

[0416] In some embodiments, the viral vector comprises an AAV6 capsid protein comprising a mutation at one or more amino acid positions selected from T492, Y705, and Y731. In some embodiments, the viral vector comprises an AAV6 capsid protein comprising one or more mutations selected from T492V, Y705F, and Y731F. In some embodiments, the viral vector comprises an AAV6 capsid protein comprising the amino acid mutations T492V+Y705F+Y731F. In some embodiments, the AAV6 capsid protein comprises, or consists of, an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

[0417] In some embodiments, the viral vector comprises an AAV9 capsid protein comprising a mutation at one or more amino acid positions selected from T492, Y705, and Y731. In some embodiments, the viral vector comprises an AAV9 capsid protein comprising one or more mutations selected from T492V, Y705F, and Y731F. In some embodiments, the viral vector comprises an AAV9 capsid protein comprising an amino acid mutation T492V+Y705F+Y731F. In some embodiments, the AAV9 capsid protein comprises, or consists of, an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0418] In some embodiments, the viral vector is an AAV9 vector or a variant thereof. In some embodiments, the capsid polypeptide of the AAV9 vector comprises a sequence according to SEQ ID NO 81, is essentially composed of it, or is composed of it. In some embodiments, the capsid polypeptide of the present disclosure comprises the following amino acid sequence, is essentially composed of the following amino acid sequence, or is composed of the following amino acid sequence, and the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with SEQ ID NO 81. In some embodiments, the capsid polypeptide of the present disclosure comprises the following amino acid sequence, is essentially composed of the following amino acid sequence, or is composed of the following amino acid sequence, and the amino acid sequence has a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated (substituted, deleted and / or added) amino acids compared to the sequence of SEQ ID NO 81. In some embodiments, the capsid polypeptide of the AAV9 vector comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 99.7% sequence identity to SEQ ID NO 81, wherein the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 of SEQ ID NO 81. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) amino acids compared to the sequence of SEQ ID NO 81, wherein the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 of SEQ ID NO 81. In some embodiments, the substitution is T492V. In some embodiments, the substitution is T492I. In some embodiments, the substitution is T492L. In some embodiments, the T492 residue is substituted by a hydrophobic amino acid selected from valine (Val), leucine (Leu) and isoleucine (Ile). In some embodiments, the T492 residue is substituted by a hydrophobic amino acid selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met) and tryptophan (Trp).

[0419] In some embodiments, a viral vector comprising an AAV capsid protein (or a mutant of the disclosure) facilitates targeting expression of an engineered receptor to a subpopulation of cells or neurons in a subject. In some embodiments, the neurons are nociceptors.

[0420] "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 referred to as "heterologous" to ITRs because such combinations are not usually found in nature. When present in insect host cells expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors can replicate and be packaged into infectious viral particles. When rAAV vectors are incorporated into larger nucleic acid constructs (e.g., in chromosomes or in another vector such as a plasmid or baculovirus for cloning or transfection), rAAV vectors are often referred to as pro-vectors that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary auxiliary functions.

[0421] In certain embodiments, any AAV ITR can be used in an AAV vector, including ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16. In a preferred embodiment, the AAV vectors contemplated herein include one or more AAV2 ITRs.

[0422] The payload capacity of a rAAV vector comprising two ITRs is about 4.4 kB. A self-complementary rAAV vector contains a third ITR and packages both strands of the recombinant portion of the vector, leaving only about 2.1 kB for the polynucleotides contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0423] The use of a dual rAAV vector strategy has achieved an expanded packaging capacity that is approximately twice the packaging capacity of rAAV (about 9kB). The dual vector strategy useful in generating the rAAV considered herein includes, but is not limited to, splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybridization). In the dual AAV trans-splicing strategy, the splicing donor (SD) signal is placed at the 3' end of the 5' half vector, and the splicing acceptor (SA) signal is placed at the 5' end of the 3' half vector. After co-infection of the same cell by a dual AAV vector and head-to-tail concatenation of the two halves mediated by inverted terminal repeats (ITRs), trans-splicing results in the production of mature mRNA and full-size proteins (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the large transgene expression cassettes contained in the two halves of the dual AAV vector can contain homologous overlapping sequences (the dual AAV overlaps at the 3' end of the 5' half vector and at the 5' end of the 3' half vector) that will mediate the reconstruction of a single large genome by homologous recombination (Duan et al., 2001). This strategy depends on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on adding highly recombinogenic regions from exogenous genes (i.e., alkaline phosphatase; Ghosh et al., 2008; Ghosh et al., 2011) to the trans-splicing vector. To increase recombination between the dual AAVs, the added region is placed downstream of the SD signal in the 5' half vector and upstream of the SA signal in the 3' half vector.

[0424] "Hybrid AAV" or "hybrid rAAV" refers to a rAAV genome packaged with a capsid of a different AAV serotype (and preferably, a different serotype than the one or more AAV ITRs) and may otherwise be referred to as a pseudotyped rAAV. For example, the rAAV type 1, rAAV type 2, rAAV type 3, rAAV type 4, rAAV type 5, rAAV type 6, rAAV type 7, rAAV type 8, rAAV type 9, rAAV type 10, rAAV type 11, rAAV type 12, rAAV type 13, rAAV type 14, rAAV type 15, or rAAV type 16 genome can be in AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type 14, AAV type 15, or AAV type 16. 16 capsid or a variant thereof, provided that the AAV capsid and genome (and preferably, the one or more AAV ITRs) are of different serotypes. In certain embodiments, the pseudotyped rAAV particles can be referred to as having an "x / y" type, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid, e.g., a 2 / 5 rAAV particle has ITRs from AAV2 and a capsid from AAV6.

[0425] "Host cells" include cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the present disclosure. Host cells may include cells that produce viruses and cells infected with viral vectors. In specific embodiments, host cells are infected in vivo with the viral vectors contemplated herein. In certain embodiments, the term "target cell" is used interchangeably with host cell and refers to an infected cell of a desired cell type.

[0426] High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. Nos. 5,658,776, 6,566,118, 6,989,264, and 6,995,006; U.S. 2006 / 0188484, WO 98 / 22607, WO 2005 / 072364, and WO / 1999 / 011764; and Viral Vectors for Gene Therapy: Methods and Protocols, Machida, ed., Humana Press) 2003; Samulski et al., (1989) Journal of Virology (J. Virology) 63, 3822; Xiao et al., (1998) Journal of Virology 72, 2224; Inoue et al., (1998) Journal of Virology 72, 7024. Methods for producing pseudotyped AAV vectors have also been reported (e.g., WO 00 / 28004), as well as various modifications or formulations of AAV vectors to reduce their immunogenicity when administered in vivo (see, e.g., WO 01 / 23001; WO 00 / 73316; WO 04 / 112727; WO 05 / 005610; WO 99 / 06562).

[0427] G. Pharmaceutical Compositions

[0428] In some embodiments, the disclosure provides a composition comprising a polynucleotide or a vector, the polynucleotide encoding the engineered receptor described herein, the vector comprising a polynucleotide encoding the engineered receptor described herein. In some embodiments, the composition further comprises a ligand as described herein. The pharmaceutical preparation comprises a subject polynucleotide (RNA or DNA) encoding the engineered receptor, a vector carrying a polynucleotide (RNA or DNA) encoding the subject engineered receptor, or a ligand present in a pharmaceutically acceptable vehicle. In some embodiments, the disclosure provides a first composition and a second composition, the first composition comprising a polynucleotide or a vector, the polynucleotide encoding the engineered receptor described herein, the vector comprising a polynucleotide encoding the engineered receptor described herein, and the second composition comprising a ligand as described herein.

[0429] "Pharmaceutically acceptable vehicle" can be a vehicle used in mammals such as humans, which is approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other recognized pharmacopoeias. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier for use in mammals with which the compound of the present disclosure is formulated. Such pharmaceutical vehicles can be liquids, such as water and oil, including those oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can be saline, gum arabic, gelatin, starch paste, talc, keratin, silicone, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to mammals, the compounds and compositions of the present disclosure and pharmaceutically acceptable vehicles, excipients or diluents can be sterile. In some cases, when the compound of the present disclosure is administered intravenously, an aqueous medium can be used as a vehicle, such as water, saline solution, and aqueous glucose solution and aqueous glycerol solution.

[0430] The pharmaceutical composition can take the form of capsules, tablets, pills, spheres, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or any other form suitable for administration to mammals. In some cases, the pharmaceutical composition is formulated according to conventional procedures for use as a pharmaceutical composition suitable for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulating them are described below: Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, incorporated herein by reference.

[0431] The choice of excipient will be determined in part by the particular carrier, as well as by the particular method used to administer the composition.Thus, there is a wide variety of formulations suitable for the pharmaceutical compositions of the present disclosure.

[0432] In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for intradermal administration. In some embodiments, the pharmaceutical composition is formulated for intraperitoneal administration. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for infusion. In some embodiments, the pharmaceutical composition is formulated for intracranial administration. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for intranasal administration. In some embodiments, the pharmaceutical composition is formulated for intraganglionic administration. In some embodiments, the pharmaceutical composition is formulated for intraspinal administration. In some embodiments, the pharmaceutical composition is formulated for intraventricular administration. In some embodiments, the pharmaceutical composition is formulated for cisterna magna administration. In some embodiments, the pharmaceutical composition is formulated for intraneural administration. In some embodiments, the pharmaceutical composition is formulated for delivery to neuronal cells.

[0433] For example, the carrier can be formulated into a preparation for injection by dissolving, suspending or emulsifying the carrier in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and if desired, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0434] As another example, the carrier can be formulated into a preparation suitable for oral administration, including (a) a liquid solution, such as an effective amount of the compound dissolved in a diluent such as water or saline; (b) a capsule, sachet or tablet, each containing a predetermined amount of the active ingredient such as a solid or granules; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. The tablet form can contain one or more of the following: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, cross-linked sodium carboxymethyl cellulose, talc, magnesium stearate, stearic acid and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents and pharmacologically compatible excipients. Lozenge forms may contain the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, as well as pastilles containing the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia, emulsions, gels, and similar dosage forms containing in addition to the active ingredient such excipients as described herein.

[0435] As another example, the subject formulations of the present disclosure can be formulated into aerosol formulations for administration by inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. Aerosol formulations can also be formulated into medicaments for non-pressurized preparations, such as for use in a nebulizer or atomizer.

[0436] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulation may be presented in unit dose or multi-dose sealed containers such as ampoules and vials and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid excipients, such as water, for injection immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0437] The formulation suitable for topical application can be presented in creams, gels, pastes or foams, which, in addition to the active ingredient, also contain a suitable carrier. In some embodiments, the topical formulation contains one or more components selected from structurants, thickeners or gelling agents and emollients or lubricants. The structurants often used include long-chain alcohols such as stearyl alcohol and glycerol ethers or esters and oligo(ethylene oxide) ethers or their esters. Thickeners and gelling agents include polymers and esters thereof of, for example, acrylic acid or methacrylic acid, polyacrylamide and naturally occurring thickeners, such as agar, carrageenan, gelatin and guar gum. The example of an emollient includes triglycerides, fatty acid esters and amides, waxes (such as beeswax, spermaceti or carnauba wax), phospholipids (such as lecithin) and sterols and fatty acid esters thereof. The topical formulation may further include other components, such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (i.e., sunscreens), etc.

[0438] The compounds of the present disclosure can be formulated for topical application. The vehicle for topical application can be in one of various forms, such as lotion, cream, gel, ointment, stick, spray or paste. The vehicle can include various types of carriers, including but not limited to solution, aerosol, emulsion, gel and liposome. The carrier can be formulated into an emulsion such as having an oil-in-water or water-in-oil matrix. The suitable hydrophobic (oily) component used in the emulsion includes, for example, vegetable oil, animal fat and oil, synthetic hydrocarbon and its ester and alcohol (including polyester) and organopolysiloxane oil. Such emulsions also include emulsifiers and / or surfactants, such as nonionic surfactants, to disperse and suspend the discontinuous phase in the continuous phase.

[0439] Suppository formulations are also provided by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.

[0440] Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be provided, wherein each dosage unit (e.g., teaspoonful, tablespoonful, tablet or suppository) contains a predetermined amount of a composition comprising one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may include one or more inhibitors in a composition as a solution in sterile water, saline or another pharmaceutically acceptable carrier.

[0441] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present disclosure and a pharmaceutically acceptable diluent, carrier or vehicle, the predetermined amount of which is calculated to be sufficient to produce the desired effect. The specifications of the novel unit dosage forms of the present disclosure depend on the specific compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0442] Dosage levels may vary depending on the specific compound, the nature of the delivery vehicle, etc. The desired dosage for a given compound can be readily determined by a variety of means.

[0443] The dosage administered to animals, particularly humans, in the context of the present disclosure should be sufficient to affect a prophylactic or therapeutic response in the animal within a reasonable time frame, for example, as described in more detail below. The dosage will depend on a variety of factors, including the strength of the specific compound employed, the condition of the animal and the weight of the animal, as well as the severity of the illness and the stage of the disease. The size of the dosage will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a specific compound.

[0444] In pharmaceutical dosage forms, the compound may be administered in the form of a free base, a pharmaceutically acceptable salt of the compound or the compound may also be used alone or in suitable combination with other pharmaceutically active compounds and in combination therewith.

[0445] H. Clinical Application and Treatment Methods

[0446] The compositions and methods disclosed herein can be used to treat nervous system diseases or conditions. In some aspects of the present disclosure, a method for treating a nervous system disease or condition in a subject is provided, the method comprising introducing an engineered receptor into a neuronal cell and providing an effective amount of a ligand for activating the engineered receptor to control the activity of the cell, thereby alleviating the pain in the subject. In some aspects, the carrier or composition disclosed herein is used to manufacture a drug for treating a nervous system disease or condition.

[0447] In some aspects of the present disclosure, there is provided a method for treating a neurological disease or condition of a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding an engineered receptor of the present disclosure. In some embodiments, the polynucleotide is delivered to hippocampal neurons. In some embodiments, the polynucleotide is delivered to dorsal root ganglion neurons or trigeminal ganglion neurons. In some embodiments, the method further comprises administering a small molecule ligand / drug / agonist of an activated engineered receptor, the engineered receptor sequentially regulates the activity of target neurons, and thereby treats a neurological disease or condition (e.g., pain or epilepsy).

[0448] In some aspects of the present disclosure, a method for treating focal epilepsy in a subject is provided, the method comprising administering to the subject an effective amount of a polynucleotide encoding an engineered receptor of the present disclosure. In some embodiments, the polynucleotide is delivered to hippocampal neurons. In some embodiments, the method further comprises administering a small molecule ligand that activates the engineered receptor, the engineered LGIC sequentially modulating the activity of the target neuron, and thereby treating focal epilepsy in the subject.

[0449] In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to prepare a drug for treating a neurological disease or condition. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to prepare a drug for treating a neurological disease or condition associated with hippocampal neuron disorders, such as focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome and fragile X syndrome. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat focal epilepsy. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat schizophrenia. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat autism spectrum disorder. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat Alzheimer's disease. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat Rett syndrome. In some aspects, the polynucleotides, vectors or compositions disclosed herein are used to treat fragile X syndrome.

[0450] This disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating epilepsy in a subject. In some embodiments, the epilepsy is focal epilepsy. In some embodiments, the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0451] In some embodiments, the compositions and methods herein can be used to improve the level of epileptic seizures in a subject. In some embodiments, the compositions and methods herein can be used to prevent or control the level of epileptic seizures in a subject. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonic, absence, or atonic seizures.

[0452] In some embodiments, the compositions and methods herein may reduce the number or frequency of seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%, including all ranges and subranges therebetween. In some embodiments, the compositions and methods herein may reduce the number or frequency of seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, including all ranges and subranges therebetween.

[0453] In some embodiments, the compositions and methods herein may reduce the level and / or duration of seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%, including all ranges and subranges therebetween. In some embodiments, the compositions and methods herein may reduce the level and / or duration of seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, including all ranges and subranges therebetween.

[0454] In various embodiments, a method for controlling, managing, preventing or treating epilepsy (e.g., focal epilepsy) in a subject comprises administering to the subject an effective amount of a polynucleotide encoding an engineered receptor of the disclosure. Without wishing to be bound by any particular theory, the disclosure contemplates the use of engineered receptors to modulate neuronal activity to alleviate epilepsy in a subject.

[0455] In various embodiments, vectors and / or polynucleotides encoding engineered receptors that activate or depolarize neuronal cells are administered to (or introduced into) one or more neuronal cells that control epilepsy. In the presence of a ligand, neuronal cells expressing the engineered receptors are activated and reduce sensitivity to epilepsy.

[0456] In some embodiments, the epilepsy is focal epilepsy (focal seizures). Focal epilepsy is a neurological disorder in which the main symptom is recurrent seizures that affect one hemisphere (half) of the brain.

[0457] Focal seizures are divided into four categories: (a) focal conscious seizures, in which the subject is aware of what is happening during the seizure; (b) focal impaired consciousness seizures; in which the subject is confused or unaware of what is happening during the seizure or cannot remember it; (c) focal motor seizures, in which the subject moves to some degree - anything from twitching to spasms to rubbing hands to walking around; and (d) focal non-motor seizures, in which the subject does not twitch or make other movements during the seizure. Instead, it causes changes in how the subject feels or thinks (for example, feeling strong emotions, strange sensations, or symptoms like a racing heartbeat, goose bumps, or heat or cold waves).

[0458] Focal epilepsy is characterized by seizures originating from a specific part (lobe) of the brain. Focal epilepsy includes idiopathic location-related epilepsy (ILRE), frontal lobe epilepsy, temporal lobe epilepsy, parietal lobe epilepsy, and occipital lobe epilepsy.

[0459] Idiopathic localization-related epilepsy (ILRE) is caused by unknown factors.

[0460] Frontal lobe epilepsy is the term for recurring seizures that begin in the frontal lobe (the area of ​​the brain behind the forehead).

[0461] Temporal lobe epilepsy is a term for recurrent seizures that begin in the temporal lobes (parts of the brain located on either side of the head behind the temples and cheekbones). The temporal lobes are the area of ​​the brain that most often causes seizures. The medial (middle) portion of the two temporal lobes is very important in epilepsy—it is often the source of seizures and can be easily damaged or scarred. Medial temporal lobe epilepsy (mTLE) is the most common form of epilepsy in people. Many times, its pathophysiological basis is hippocampal sclerosis. Therefore, one strategy for treating focal epilepsy (such as mTLE) is to target hippocampal neurons.

[0462] Parietal lobe epilepsy. The parietal lobe is the part of the brain located on the top and sides of the head. The parietal lobe, called the "associative cortex," is responsible for connecting meaning to brain function. It is here that the brain creates visual images, recognizes sounds as words, and associates touch sensations with specific objects. In some ways, the parietal lobe is where perception is integrated with physical reality.

[0463] Occipital lobe epilepsy. Occipital lobe epilepsy is the term for recurring seizures that begin in the occipital lobe, the part at the back of the brain that is primarily responsible for vision.

[0464] In some aspects of the present disclosure, there is provided a method for treating neuropathic pain such as peripheral neuropathy and trigeminal neuralgia in a subject, the method comprising administering to the subject an effective amount of a polynucleotide, wherein the polynucleotide encodes an engineered receptor of the present disclosure. In some embodiments, the polynucleotide is delivered to DRG or TGG neurons. In some embodiments, the method further comprises administering a small molecule ligand that activates the engineered receptor, the engineered LGIC sequentially modulates the activity of the target neuron, and thereby treats neuropathic pain in the subject, such as peripheral neuropathy and trigeminal neuralgia.

[0465] In some aspects, polynucleotides disclosed herein, vectors or compositions are used to prepare medicines for treating nervous system diseases or conditions. In some aspects, polynucleotides disclosed herein, vectors or compositions are used to prepare medicines for treating nervous system diseases or conditions (e.g., neuropathic pain) associated with DRG / TGG neuron disorders. In some aspects, polynucleotides disclosed herein, vectors or compositions are used to prepare medicines for treating spinal cord-related diseases such as spasms, spinal cord injuries and avulsions. In some aspects, polynucleotides disclosed herein, vectors or compositions are used to treat spasms. In some embodiments, treating spasms includes transducing neurons in the spine. In some aspects, polynucleotides disclosed herein, vectors or compositions are used to treat spinal cord injuries. In some aspects, polynucleotides disclosed herein, vectors or compositions are used to treat avulsions. In some embodiments, treating spinal cord injuries or avulsions includes transducing neurons in the dorsal horn.

[0466] In some embodiments, neuropathic pain is peripheral neuropathy. Peripheral neuropathy refers to the condition caused when the nerves that carry information from and to the rest of the body to and from the brain and spinal cord are damaged or sick. The range of various peripheral neuropathy ranges from carpal tunnel syndrome (traumatic injury common after long-term repeated use of hands and wrists, such as using computers) to nerve damage connected to diabetes. In general, peripheral neuropathy can be divided into mononeuropathy and polyneuropathy. Mononeuropathy includes carpal tunnel syndrome, ulnar nerve palsy, radial nerve palsy and peroneal nerve palsy. When multiple peripheral nerves in the whole body fail at the same time, polyneuropathy occurs. Polyneuropathy can have many reasons, including exposure to certain toxins, such as complications caused by alcohol abuse, malnutrition (especially vitamin B deficiency) and diseases (such as cancer or renal failure). One of the most common forms of chronic polyneuropathy is diabetic neuropathy, which is a disease that occurs in people with diabetes. More serious in people with poorly controlled blood sugar levels. Although less common, diabetes also causes mononeuropathy. One of the most serious polyneuropathies is Guillain-Barré syndrome, a rare disease that strikes when the body's immune system attacks nerves in the body just as they leave the spinal cord. Symptoms tend to appear quickly and worsen rapidly, sometimes leading to paralysis. Early symptoms include weakness and tingling, which can eventually spread up the arms. More severe cases can cause blood pressure problems, heart rhythm problems, and breathing difficulties. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic form of Guillain-Barré disease, with symptoms that last for months or even years. Early diagnosis and treatment are crucial for people with CIDP, 30% of whom are at risk of eventually becoming confined to a wheelchair.

[0467] In some embodiments, the neuropathic pain is trigeminal neuralgia. Trigeminal neuralgia (TN), also known as tic douloureux, is a chronic pain condition that affects the trigeminal nerve or the 5th cranial nerve, one of the most widespread nerves in the head. The trigeminal nerve is a group of cranial nerves in the head. It is the nerve responsible for providing sensation to the face. One trigeminal nerve goes to the right side of the head and the other goes to the left side. Each of these nerves has three different branches. After the trigeminal nerve leaves the brain and travels inside the skull, it divides into three smaller branches that control sensation throughout the face: ophthalmic nerve (V1): The first branch controls sensation in a person's eyes, upper eyelids, and forehead. Maxillary nerve (V2): The second branch controls sensation in the lower eyelids, cheeks, nostrils, upper lip, and upper gums. Mandibular nerve (V3): The third branch controls sensation in the lower jaw, lower lip, lower gums, and some muscles used for chewing.

[0468] TN is a form of neuropathic pain. The typical or "classic" form of the condition (called "type 1" or TN1) causes extreme, sporadic, sudden burning or shock-like facial pain, with each episode lasting from a few seconds to up to two minutes. These attacks can occur in rapid succession, with a salvo lasting up to two hours. The "atypical" form of the condition (called "type 2" or TN2) is characterized by persistent pain, burning, tingling, and pain that is slightly less intense than type 1. Both forms of pain can occur in the same person, sometimes at the same time. The intensity of the pain can lead to physical and mental incapacity. TN is associated with a variety of conditions. TN can be caused by blood vessels pressing on the trigeminal nerve as it leaves the brainstem. This compression causes the protective layer (myelin sheath) around the nerve to wear or become damaged. People with multiple sclerosis can also experience symptoms of TN, a disease that causes the myelin sheath of the trigeminal nerve to degenerate. In rare cases, symptoms of TN can be caused by tumors pressing on the nerve or tangles of arteries and veins called arteriovenous malformations. Injury to the trigeminal nerve (which may be the result of sinus surgery, oral surgery, stroke, or facial trauma) can also produce neuropathic facial pain.

[0469] In some cases, the methods and compositions of the present disclosure are used to treat epilepsy. Compositions described herein can be used to prevent or control epileptic seizures. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonic, absence or atonic seizures. In some cases, the compositions and methods herein can prevent epileptic seizures or reduce the number of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100%, including all ranges and sub-ranges therebetween.

[0470] In some cases, the methods and compositions of the present disclosure are used to treat eating disorders. Eating disorders may be mental disorders limited by abnormal eating behaviors that have a negative impact on the physical or mental health of the experimenter. In some cases, eating disorders are anorexia nervosa. In other cases, eating disorders are bulimia nervosa. In some cases, eating disorders are pica, rumination, avoidance / restrictive food intake disorders, binge eating disorder (BED), other specified feeding and diet disorders (OSFED), compulsive overeating, diabetic eating disorders, health food anorexia, selective eating disorders, alcohol anorexia, hyperphagia during pregnancy (pregorexia) or gourmand syndrome (Gourmand syndrome). In some cases, the composition comprises a G protein-coupled receptor that increases or decreases the production of one or more molecules related to the eating disorder. In some cases, the composition comprises a ligand-gated ion channel that changes the production of one or more molecules related to the eating disorder. The one or more molecules associated with eating disorders may include, but are not limited to, molecules of the hypothalamus-pituitary-adrenal (HPA) axis, including vasopressin, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), Cortisol, epinephrine or norepinephrine; and serotonin, dopamine, neuropeptide Y, leptin or ghrelin.

[0471] In some cases, the compositions and methods are used to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumor), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that can be treated by the compositions and methods herein include: aspirin, agraphia, alcoholism, alexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger syndrome, ataxia, ataxia telangiectasia, attention deficit hyperactivity disorder, speech and auditory processing disorder, autism spectrum, bipolar disorder, Bell's palsy, brachial plexus injury, brain injury, brain trauma, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, cephalic vein disorder (Cephalic disorder), cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cerebral spinal stenosis, Charcot-Marie-Tooth disease, cerebellar tonsillar herniation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, Coffin-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital bilateral facial palsy, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative injury disorder, Cushing's syndrome, cyclothymic inclusion disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson disease, DeMorsier's syndrome syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas diseasedisease), delayed sleep phase syndrome, dementia, dermatomyositis, developmental coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, articulation disorder, autonomic neuropathy, calculation disorder, writing disorder, movement disorder, reading disorder, muscle tone disorder, empty sella syndrome, encephalitis, encephalocele, cerebral trigeminal angiomatosis, fecal incontinence, enuresis, epilepsy, intellectual disorder of female epilepsy, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry's disease, Fahr's syndrome, syncope, familial spastic paralysis, febrile seizures, Fisher syndrome, Friedreich's ataxia. ataxia, fibromyalgia, Fovego's syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher's disease, generalized epilepsy with febrile seizures plus, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, spherocytoid leukoencephalopathy, gray matter heterotopia, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, globulin pigment syndrome, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary ataxia of the polyneuritic type, herpes zoster oticus, herpes zoster, Hirayama syndrome, Hirschsprung's disease, Holmes-Adie syndrome, holoprosencephaly, Huntington's disease), hydrocephalus, anencephaly, hydrocephalus, hypercortisolism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmenti, infantile Refsum disease, infantile spasms, inflammatory myopathy, intracranial cyst, increased intracranial pressure, homomorphic dicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kingsborne syndrome, Kleine-Levine syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndromesyndrome, Landau-Kleffner syndrome, lateral medullary (Wallenberg) syndrome, learning disorders, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, ablative leukoencephalopathy, dementia with Lewy bodies, lissencephaly, locked-in syndrome, intervertebral disk disease, lumbar spinal stenosis, Lyme disease-neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, macromegaly, Malde debarquement, megalencephalic leukoencephalopathy with subcortical cysts, megalencephaly, Melkersson-Rosenthal syndrome, Menieres disease, meningitis, Menkes disease disease), metachromatic leukodystrophy, microcephaly, microsomia, migraine, Miller Fisher syndrome, mini-stroke (transient ischemic attack), misophonia, mitochondrial myopathy, Mobius syndrome, monomelic muscular atrophy, motor skill disorders, moyamoya disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinoclastic diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neuro-Behcet's disease disease), neurofibromatosis, neuroleptic malignant syndrome, neurologic manifestations of AIDS, neurologic sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal translocation disease, neuropathy, neurasthenia, Niemann-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, Ohtawara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, persistent afterimages, paresthesias, Parkinson's disease, paramyotonia congenita, paraneoplastic disorders, paroxysmal seizures, Parry-Romberg syndrome syndrome), PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, generalized developmental disorder, optically active sneeze reflex, phytanic acid storage disease, Pick's disease, nerve compression, pituitary tumor, PMG, polyneuropathy, poliomyelitis, polymicrogyria, polymyositis, trepanencephaly, postpoliomyelitis, postherpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemiatrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrantanopia, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen encephalitis encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retroviral myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg syndrome, Saint Vitus dance, Sandhoff disease, Schilder's disease, schizencephaly, sensory processing disorder, developmental failure, shaken baby syndrome, herpes zoster, hypotensive syndrome, Sjögren's syndrome syndrome), sleep apnea, narcolepsy, Snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinobulbar muscular atrophy, spinocerebellar ataxia, schizencephaly, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, stroke, Stitch-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, Sydenham's chorea, syncope, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, advanced psychosis, Tarlov cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered cord syndrome, myotonic cataract, thoracic outlet syndrome, trigeminal neuralgia Douloureux, Todd's palsy, Tourette's syndrome, toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, Von Hippel-Lindau disease (VHL), Viliuisk's encephalomyelitis (VE), Wallenberg's syndrome, West's syndrome, whiplash, Williams syndrome, Wilson's disease, or Zellweger's syndrome.

[0472] In some cases, the compositions and methods disclosed herein can be used to treat brain cancer or brain tumors. Non-limiting examples of brain cancers or tumors that may be amenable to treatment with the vectors and compositions described herein include: gliomas, including anaplastic astrocytoma (grade III glioma), astrocytoma (grade II glioma), brain stem glioma, ependymoma, ganglioglioma, ganglioneuroma, malignant glioma (grade IV glioma), glioma, juvenile pilocytic astrocytoma (JPA), low grade astrocytoma (LGA), medullablastoma, mixed glioma, oligodendroglioma, optic nerve glioma, fibrous astrocytoma (grade I glioma), and primitive neuroectodermal (PNET); skull base tumors, including acoustic neuroma (vestibular schwannoma), acromegaly, adenoma, chondrosarcoma, chordoma, craniopharyngioma, epidermoid tumor, glomus jugulare tumor, infratentorial meningioma, meningioma, pituitary adenoma, pituitary tumor, Rathke's cleft cyst (Rathke's cleftcyst); metastatic cancer, including intracranial metastases and metastatic brain tumors; other brain tumors, including brain cysts, choroid plexus papilloma, CNS lymphoma, glial cysts, cystic tumors, dermoid tumors, germ cell tumors, lymphomas, nasal cancer, nasopharyngeal tumors, pineal tumors, pineoblastomas, pineocytomas, supratentorial meningiomas, and hemangiomas; spinal cord tumors, including astrocytomas, ependymomas, meningiomas, and schwannomas.

[0473] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing or treating pain in a subject. "Pain" refers to the uncomfortable feelings and / or unpleasant sensations of a subject's body. The perception of pain can range from mild and occasional to severe and continuous. Pain can be classified as acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain or psychogenic pain. In some cases, pain is caused by or associated with a disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include: neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, pain associated with Parkinson's disease, epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and nociceptive pain, including pain associated with central nervous system trauma, strains / sprains, and burns; myocardial infarction, acute pancreatitis, postoperative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0474] The compositions and methods herein can be used to alleviate the pain level of a subject. In some cases, the pain level of a subject alleviates at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 99% or about 100%, including all ranges and sub-ranges therebetween. The pain level of a subject can be assessed by a variety of methods. In some cases, pain level (that is, a verbal report of the pain level that a human subject expresses himself experiencing) is assessed by self-report. In some cases, pain level is assessed by behavioral indicators of pain, for example, facial expression, limb movement, vocalization, restlessness and defense. For example, these types of assessments can be useful when the subject (e.g., infants, unconscious subjects, non-human subjects) is unable to self-report. Pain levels can be assessed after treatment with a composition of the present disclosure compared to the pain level experienced by the subject prior to treatment with the composition.

[0475] In various embodiments, a method for controlling, managing, preventing or treating pain in a subject comprises administering to the subject an effective amount of an engineered receptor contemplated herein. Without wishing to be bound by any particular theory, the present disclosure contemplates the use of the vectors disclosed herein to modulate neuronal activity to alleviate pain in a subject.

[0476] In various embodiments, a vector encoding an engineered receptor that activates or depolarizes a neuron is applied to (or introduced into) one or more neuron cells that reduce pain sensation, such as inhibitory interneurons. In the presence of a ligand, the neuron cells expressing the engineered receptor are activated and reduce sensitivity to pain, thereby enhancing the analgesic effect of stimulating these neuron cells.

[0477] In various embodiments, a vector encoding an engineered receptor that inactivates or hyperpolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells that increase pain sensation or sensitivity to pain, e.g., nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc. In the presence of a ligand, the neuronal cell expressing the engineered receptor is inactivated and has reduced sensitivity to pain, and analgesia is enhanced.

[0478] Targeting the expression of engineered receptors to a subset of nociceptors can be achieved by one or more of the following: selecting a vector (e.g., AAV1, AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV5, AAV5(Y436+693+719F), AAV6, AAV6(VP3 variant Y705F / Y731F / T492V), AAV-7m8, AAV8, AAV8(Y733F), AAV9, AAV9(VP3 variant Y731F), AAV10(Y733F), and AAV-ShH10); selecting a promoter; and a means of delivery.

[0479] In certain embodiments, the compositions and methods contemplated herein are effective in reducing pain. Illustrative examples of pain suitable for treatment with the vectors, compositions and methods contemplated herein include, but are not limited to, acute pain, chronic pain, neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-associated neuropathy, nerve injury, rheumatoid arthritis pain, osteoarthritis pain, burns, back pain, eye pain, visceral pain, cancer pain (e.g., bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, postherpetic neuralgia, postoperative pain, poststroke pain, and menstrual pain.

[0480] Pain can be classified as acute or chronic. "Acute pain" refers to pain that starts suddenly and is usually sharp in quality. Acute pain may be mild and last only a few moments, or it may be severe and last for weeks or months. In most cases, acute pain does not last more than three months, and the pain disappears when the underlying cause of the pain has been treated or has been cured. However, unrelieved acute pain may lead to chronic pain. "Chronic pain" refers to pain that persists or recurs, persists beyond the normal course of an acute illness or injury, or persists for more than three to six months, and it adversely affects the health of an individual. In a specific embodiment, the term "chronic pain" refers to pain that continues when it should not continue. Chronic pain can be nociceptive pain or neuropathic pain.

[0481] In some embodiments, the pain is associated with or developed as a result of an injury, infection, or medical intervention. In some embodiments, the infection causes nerve damage. In some embodiments, the medical intervention is a surgical procedure, such as a surgical procedure to the central core of the body. In some embodiments, the medical intervention is a surgical procedure to remove part or all of one or more tissues, tumors, or organs in the body. In some embodiments, the medical intervention is an amputation. In specific embodiments, the compositions and methods contemplated herein are effective in reducing acute pain. In specific embodiments, the compositions and methods contemplated herein are effective in reducing chronic pain.

[0482] Clinical pain is present when discomfort and abnormal sensitivity are characteristic of the patient's symptoms. Individuals may experience a variety of pain symptoms. Such symptoms include: 1) spontaneous pain that may be dull, burning, or tingling; 2) excessive pain responses to noxious stimuli (hyperalgesia); and 3) pain produced by normally harmless stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Although patients with various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and therefore may require different treatment strategies. Therefore, pain can also be divided into a variety of different subtypes based on different pathophysiologies, including nociceptive pain, inflammatory pain, and neuropathic pain.

[0483] 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.

[0484] Nociceptive pain is induced by tissue damage or by strong stimulation that may cause damage. Moderate to severe acute nociceptive pain is the prominent feature of pain from central nervous system trauma, strain / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical operation), post-traumatic pain, renal colic, cancer pain and back pain. Cancer pain can be chronic pain, such as tumor-related pain (for example, bone pain, headache, facial pain or visceral pain) or pain related to cancer therapy (for example, post-chemotherapy syndrome, chronic postoperative pain syndrome or post-radiation syndrome). Cancer pain can also occur in response to chemotherapy, immunotherapy, hormone therapy or radiotherapy. Back pain may be due to disc herniation or rupture or lumbar intervertebral facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament abnormalities. Back pain can naturally subside, but back pain continues in some patients more than 12 weeks, and back pain becomes a chronic condition that may make people particularly weak.

[0485] Neuropathic pain can be defined as pain caused or initiated by a primary lesion or functional disorder of the nervous system. Causes of neuropathic pain include, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0486] Neuropathic pain may be associated with a pain disorder, the term pain disorder refers to a disease, disorder or condition associated with or caused by pain. Illustrative examples of pain disorders include arthritis, allodynia, classic trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia of pain, causalgia, sciatica disorders, degenerative joint disorders, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis, or pain associated with cancer.

[0487] The inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue damage or the presence of foreign substances, which leads to swelling and pain. Arthritic pain is a common inflammatory pain.

[0488] Other types of pain suitable for treatment with the vectors, compositions and methods contemplated herein include, but are not limited to, pain caused by musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis and pyomyositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredema and skeletal muscle ischemia; headaches, such as migraine (including migraine with aura and migraine without aura), cluster headaches, tension-type headaches, mixed headaches and headaches associated with vascular disorders; and maxillofacial pain, including dental pain, ear pain, burning mouth syndrome and temporomandibular myofascial pain.

[0489] A variety of pain scales can be used to determine the effective amount of the compositions and methods contemplated herein for reducing the amount of pain experienced by a human subject. Patient self-report can be used to assess whether pain is reduced; see, e.g., Katz and Melzack (1999) Surg. Clin. North Am. 79: 231. Alternatively, an observational pain scale can be used. The LANSS pain scale can be used to assess whether pain is reduced; see, e.g., Bennett (2001) Pain 92: 147. A visual analog pain scale can be used; see, e.g., Schmader (2002) Clin. J. Pain 18: 350. A Likert pain scale can be used; e.g., where 0 is no pain, 5 is moderate pain, and 10 is the worst possible pain. Self-report pain scales for children include, e.g., the Face Pain Scale; the Wong-Baker FACES pain rating scale; and a color analog scale. Self-report pain scales for adults include, for example, the visual analogue scale; the verbal numeric rating scale; the verbal descriptive scale; and the Brief Pain Inventory.Pain measurement scales include, for example, the Alder Hey Triage Pain Score (Stewart et al. (2004) Arch. Dis. Child. 89:625); Behavioral Pain Scale (Payen et al. (2001) Critical Care Medicine 29:2258); Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129); Nonverbal Pain Indicator Checklist (Feldt (2000) Pain Manager. Nurs. 1:13); Critical Illness Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); COMFORT Scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); Dallas Pain Questionnaire (Ozguler et al. (2002) Spine Pain Questionnaire). =2783); Algometer pain index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Face Pain Scale-Revised (Hicks et al. (2001) Pain 93:173); Face Leg Movement Crying Soothing Scale; McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptor Difference Scale (Gracely and Kwilosz (1988) Pain 35:279); Numerical 11 point Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numerical Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310); Wong-Baker FACES pain rating scale; and visual analog scale (Huskisson (1982) J. Rheumatol. 9:768).

[0490] In a specific embodiment, there is provided a method for alleviating the pain of a subject, the method comprising introducing an engineered receptor into a neuronal cell and controlling the activity of the cell by providing an effective amount of a ligand for activating the engineered receptor, thereby alleviating the pain of the subject. The method provides significant analgesia for pain without off-target effects, such as general central nervous system inhibition. In certain embodiments, compared with an untreated subject, the method reduces the neuropathic pain of the subject by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including all ranges and subranges therebetween). In some embodiments, the method includes the step of measuring the subject's pain before and after administration of the ligand, wherein the subject's pain is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, including all ranges and subranges therebetween. In such cases, the measurement can occur 4 hours or more after administration of the ligand, for example 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days or 4 days or more after administration of the ligand.

[0491] In a specific embodiment, the vector contemplated herein is administered or introduced into one or more neuronal cells. The neuronal cells can be a mixed population of neuronal cells of the same type or different types of neuronal cells. In one embodiment, the neuronal cells are nociceptors or peripheral sensory neurons. Illustrative examples of sensory neurons include but are not limited to dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In one embodiment, the neuronal cells are inhibitory interneurons involved in neuronal pain circuits.

[0492] In some cases, the vector encoding the engineered receptor is administered to a subject in need. Non-limiting examples of methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cisterna magna administration, and intraneural administration. In some cases, administration may involve a liquid formulation of an injection vector. In some cases, administration may involve a solid formulation of an oral delivery vector. In some cases, oral formulations may be administered with food. In specific embodiments, the vector is administered to a subject parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneurally, intraganglionically, intraspinally, or intraventricularly to introduce the vector into one or more neuronal cells. In various embodiments, the vector is rAAV.

[0493] In one embodiment, AAV is administered to sensory neurons or nociceptors, e.g., DRG neurons, TGG neurons, etc., by intrathecal (IT) or intraganglionic (IG) administration. The IT route delivers AAV to the cerebrospinal fluid (CSF). This route of administration may be suitable for treating, for example, chronic pain or other peripheral nervous system (PNS) or central nervous system (CNS) indications. In animals, IT administration has been achieved by inserting an IT catheter through the cisterna magna and advancing its caudal end to the lumbar level. In humans, IT delivery can be readily performed by lumbar puncture (LP), a conventional invasive procedure with an excellent safety profile.

[0494] In certain cases, the vector can be administered to the subject by intraganglionic administration. Intraganglionic administration can involve direct injection into one or more ganglia. The IG approach can deliver AAV directly to the DRG or TGG parenchyma. In animals, IG administration to DRG is performed by open neurosurgery, which is undesirable in humans because it will require complex and invasive procedures. In humans, minimally invasive CT imaging guidance technology can be used to safely target DRG. Customized needle assemblies for convection enhanced delivery (CED) can be used to deliver AAV to the DRG parenchyma. In a non-limiting example, the vector of the present disclosure can be delivered to one or more dorsal root ganglia and / or trigeminal ganglia for the treatment of chronic pain. In another non-limiting example, the vector of the present disclosure can be delivered to the nodose ganglion (vagus nerve) to treat epilepsy.

[0495] In yet another specific case, the vector can be administered to the subject by intracranial administration (i.e., directly administered to the brain). In a non-limiting example of intracranial administration, the vector of the present disclosure can be delivered to the cerebral cortex to treat, for example, epileptic seizure foci, to the paraventricular hypothalamus to treat, for example, satiety disorders, or to the central nucleus of the amygdala to treat, for example, satiety disorders. In another specific case, the vector can be administered to the subject by intraneural injection (i.e., directly injected into the nerve). The nerve can be selected based on the indication to be treated, for example, injected into the sciatic nerve to treat chronic pain or injected into the vagus nerve to treat epilepsy or satiety disorders. In yet another specific case, the vector can be administered to the subject by subcutaneous injection, for example, injected into the sensory nerve terminal to treat chronic pain.

[0496] The vector dose can be expressed as the number of vector genome units delivered to the subject. As used herein, "vector genome unit" refers to the number of individual vector genomes administered in a dose. The size of a single vector genome generally depends on the type of viral vector used. The vector genome of the present disclosure can be about 1.0 kilobases, 1.5 kilobases, 2.0 kilobases, 2.5 kilobases, 3.0 kilobases, 3.5 kilobases, 4.0 kilobases, 4.5 kilobases, 5.0 kilobases, 5.5 kilobases, 6.0 kilobases, 6.5 kilobases, 7.0 kilobases, 7.5 kilobases, 8.0 kilobases, 8.5 kilobases, 9.0 kilobases, 9.5 kilobases, 10.0 kilobases to greater than 10.0 kilobases. Therefore, a single vector genome can contain up to or greater than 10,000 nucleotide base pairs. In some cases, the vector dose can be about 1×10 6 pcs, 2×10 6 pcs, 3×10 6 pcs, 4×10 6 pcs, 5×10 6 pcs, 6×10 6 pcs, 7×10 6 8×10 6 pcs, 9×10 6 pcs, 1×10 7 pcs, 2×10 7 pcs, 3×10 7 pcs, 4×10 7 pcs, 5×10 7 pcs, 6×10 7 pcs, 7×10 7 8×10 7 pcs, 9×10 7 pcs, 1×10 8 pcs, 2×10 8 pcs, 3×10 8 pcs, 4×10 8pcs, 5×10 8 pcs, 6×10 8 pcs, 7×10 8 8×10 8 pcs, 9×10 8 pcs, 1×10 9 pcs, 2×10 9 pcs, 3×10 9 pcs, 4×10 9 pcs, 5×10 9 pcs, 6×10 9 pcs, 7×10 9 8×10 9 pcs, 9×10 9 pcs, 1×10 10 pcs, 2×10 10 pcs, 3×10 10 pcs, 4×10 10 pcs, 5×10 10 pcs, 6×10 10 pcs, 7×10 10 8×10 10 pcs, 9×10 10 pcs, 1×10 11 pcs, 2×10 11 pcs, 3×10 11 pcs, 4×10 11 pcs, 5×10 11 pcs, 6×10 11 pcs, 7×10 11 8×10 11 pcs, 9×10 11 pcs, 1×10 12 pcs, 2×10 12 pcs, 3×10 12 pcs, 4×10 12 pcs, 5×10 12 pcs, 6×10 12 pcs, 7×10 12 8×10 12 pcs, 9×10 12 pcs, 1×10 13 pcs, 2×10 13 pcs, 3×10 13 pcs, 4×10 13 pcs, 5×10 13 pcs, 6×10 13 pcs, 7×10 13 8×10 13 pcs, 9×10 13 pcs, 1×10 14 pcs, 2×10 14 pcs, 3×1014 pcs, 4×10 14 pcs, 5×10 14 pcs, 6×10 14 pcs, 7×10 14 8×10 14 pcs, 9×10 14 pcs, 1×10 15 pcs, 2×10 15 c3×10 15 pcs, 4×10 15 pcs, 5×10 15 pcs, 6×10 15 pcs, 7×10 15 8×10 15 pcs, 9×10 15 pcs, 1×10 16 pcs, 2×10 16 pcs, 3×10 16 pcs, 4×10 16 pcs, 5×10 16 pcs, 6×10 16 pcs, 7×10 16 8×10 16 ,9×10 16 pcs, 1×10 17 pcs, 2×10 17 pcs, 3×10 17 pcs, 4×10 17 pcs, 5×10 17 pcs, 6×10 17 pcs, 7×10 17 8×10 17 pcs, 9×10 17 pcs, 1×10 18 pcs, 2×10 18 pcs, 3×10 18 pcs, 4×10 18 pcs, 5×10 18 pcs, 6×10 18 pcs, 7×10 18 8×10 18 pcs, 9×10 18 pcs, 1×10 19 pcs, 2×10 19 pcs, 3×10 19 pcs, 4×10 19 pcs, 5×10 19 pcs, 6×10 19 pcs, 7×10 19 8×10 19 pcs, 9×10 19 pcs, 1×10 20pcs, 2×10 20 pcs, 3×10 20 pcs, 4×10 20 pcs, 5×10 20 pcs, 6×10 20 pcs, 7×10 20 8×10 20 pcs, 9×10 20 or more vector genome units.

[0497] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 1×10 9 genome particles / mL, at least about 1×10 10 genome particles / mL, at least about 5×10 10 genome particles / mL, at least about 1×10 11 genome particles / mL, at least about 5×10 11 genome particles / mL, at least about 1×10 12 genome particles / mL, at least about 5×10 12 genome particles / mL, at least about 6×10 12 genome particles / mL, at least about 7×10 12 genome particles / mL, at least about 8×10 12 genome particles / mL, at least about 9×10 12 genome particles / mL, at least about 10×10 12 genome particles / mL, at least about 15×10 12 genome particles / mL, at least about 20×10 12 genome particles / mL, at least about 25×10 12 genome particles / mL, at least about 50×10 12 genome particles / mL or at least about 100×10 12 As used with reference to viral titer, the term "genomic particle (gp)" or "genomic equivalent" or "genomic copy" (gc) refers to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by methods well understood in the art, such as genomic DNA quantitative PCR or, for example, Clark et al. (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: 272-278.

[0498] The carrier of the present disclosure can be applied with a certain volume of fluid. In some cases, the carrier can be applied with the following volume: about 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, 6.0mL, 7.0mL, 8.0mL, 9.0mL, 10.0mL, 11.0mL, 12.0mL, 13.0mL, 14.0mL, 15.0mL, 16.0mL, 17.0mL, 18.0mL, 19.0mL, 20.0mL or more than 20.0mL. In some cases, the carrier dosage can be expressed as the concentration or titer of the carrier applied to the subject. In this case, the carrier dosage can be expressed as the number of carrier genome units in each volume (i.e., genome units / volume).

[0499] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 5×10 9 infectious units / mL, at least about 6×10 9 infectious units / mL, at least about 7×10 9 infectious units / mL, at least about 8×10 9 infectious units / mL, at least about 9×10 9 infectious units / mL, at least about 1×10 10 infectious units / mL, at least about 1.5×10 10 infectious units / mL, at least about 2×10 10 infectious units / mL, at least about 2.5×10 10 infectious units / mL, at least about 5×10 10 infectious units / mL, at least about 1×10 11 infectious units / mL, at least about 2.5×10 11 infectious units / mL, at least about 5×10 11 infectious units / mL, at least about 1×10 12 infectious units / mL, at least about 2.5×10 12 infectious units / mL, at least about 5×10 12 infectious units / mL, at least about 1×10 13 infectious units / mL, at least about 5×10 13 infectious units / mL, at least about 1×10 14Infectious units / mL. As used with reference to viral titer, the terms "infectious unit (iu)", "infectious particles" or "replication units" refer to the number of recombinant AAV vector particles that have the ability to infect and replicate, as measured by the infectious center assay (also called replication center assay) as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0500] In certain embodiments, the vectors contemplated herein are administered to a subject at a titer of at least about 5×10 10 transduction units / mL, at least about 1×10 11 transduction units / mL, at least about 2.5×10 11 transduction units / mL, at least about 5×10 11 transduction units / mL, at least about 1×10 12 transduction units / mL, at least about 2.5×10 12 transduction units / mL, at least about 5×10 12 transduction units / mL, at least about 1×10 13 transduction units / mL, at least about 5×10 13 transduction units / mL, at least about 1×10 14 As used with reference to viral titers, the term "transduction unit (tu)" refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay described, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0501] The dosage of the vector will generally be determined by the route of administration. In a specific example, an intraganglionic injection may contain about 1×10 9 To about 1×10 13 In another specific...

Claims

1. An engineered receptor comprising a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR), wherein the ligand binding domain comprises an amino acid mutation at an amino acid residue corresponding to R101, Y115, L131, Q139, Y140, S172, Y210 or Y217 of SEQ ID NO:

4.

2. The engineered receptor of claim 1, wherein the ligand binding domain comprises an amino acid sequence that is at least 85% identical to amino acid residues 23-220 of SEQ ID NO:

4.

3. The engineered receptor of claim 1 or 2, wherein the ligand binding domain comprises amino acid mutations at two or more amino acid residues selected from the group consisting of amino acid residues corresponding to R101, Y115, L131, Q139, Y140, S172, Y210, and Y217 of SEQ ID NO:

4.

4. The engineered receptor of any one of claims 1-3, wherein the ligand binding domain comprises a mutation at an amino acid residue corresponding to the indicated position of SEQ ID NO: 4: a) Y140; b) Y115 and L131; c) Q139 and S172; d) Q139 and Y210; e) Q139, S172 and Y217; f) Y115, Q139 and S172; g) R101, L131, Q139 and Y217; h) Y115 and S172; i) L141, S172 and Y210; or j) R101, L131 and Y217.

5. The engineered receptor of any one of claims 1-4, wherein the mutation is an amino acid substitution.

6. The engineered receptor of claim 4, wherein the ligand binding domain comprises an amino acid substitution corresponding to the indicated position of SEQ ID NO: 4: a)Y140R; b) Y115E and L131M; c) Q139W and S172D; d) Q139W and Y210W; e) Q139W, S172D and Y217I; f) Y115E, Q139E and S172D; g) R101F, L131G, Q139L and Y217F; h) Y115Q and S172D; i) L141M, S172D and Y210W; j) R101F, L131N and Y217F; k) L131G; l) L131M; m)Q139E; n)Q139L; o) Q139W; or p)L141M.

7. The engineered receptor of any one of claims 1-6, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor comprising an ion pore domain derived from a human glycine receptor.

8. The engineered receptor of claim 7, wherein the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3.

9. The engineered receptor of claim 7, wherein the ion pore domain comprises an amino acid sequence that is at least 85% identical to amino acids 255-457 of SEQ ID NO:2, amino acids 260-452 of SEQ ID NO:83, amino acids 259-464 of SEQ ID NO:85, or amino acids 259-449 of SEQ ID NO:

87.

10. The engineered receptor of any one of claims 7-9, wherein the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from a human glycine receptor.

11. The engineered receptor of claim 10, wherein the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:

2.

12. The engineered receptor of claim 10, wherein the Cys loop domain comprises amino acids 166-180 of SEQ ID NO:

2.

13. The engineered receptor of any one of claims 1-12, wherein the ligand binding domain of the engineered receptor comprises the β1-2 loop domain from the human glycine receptor α1 subunit.

14. The engineered receptor of claim 13, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:

2.

15. The engineered receptor of any one of claims 1-14, wherein the engineered receptor comprises an amino acid sequence according to any one of SEQ ID NOs: 89-98.

16. An engineered chimeric ligand-gated ion channel (LGIC) comprising a ligand binding domain derived from a first LGIC and an ion pore domain derived from a second LGIC, wherein the first LGIC is a human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprises an amino acid mutation at an amino acid residue corresponding to R101, Y115, L131, Q139, Y140, S172, Y210 or Y217 of SEQ ID NO:

4.

17. The engineered chimeric LGIC of claim 16, wherein the ligand binding domain comprises a mutation at an amino acid residue corresponding to the indicated position of SEQ ID NO:4: a) Y140; b) Y115 and L131; c) Q139 and S172; d) Q139 and Y210; e) Q139, S172 and Y217; f) Y115, Q139 and S172; g) R101, L131, Q139 and Y217; h) Y115 and S172; i) L141, S172 and Y210; or j) R101, L131 and Y217.

18. The engineered chimeric LGIC of claim 16, wherein the ligand binding domain comprises amino acid substitutions corresponding to the indicated positions of SEQ ID NO: 4: a)Y140R; b) Y115E and L131M; c) Q139W and S172D; d) Q139W and Y210W; e) Q139W, S172D and Y217I; f) Y115E, Q139E and S172D; g) R101F, L131G, Q139L and Y217F; h) Y115Q and S172D; i) L141M, S172D and Y210W; j) R101F, L131N and Y217F; k) L131G; l) L131M; m)Q139E; n)Q139L; o) Q139W; or p)L141M.

19. The engineered chimeric LGIC of any one of claims 16-18, wherein the second LGIC is a human glycine receptor.

20. The engineered chimeric LGIC of claim 19, wherein the human glycine receptor is human glycine receptor alpha 1.

21. The engineered chimeric LGIC of claim 20, comprising a polypeptide sequence having at least 85% sequence identity to SEQ ID NO:

33.

22. The engineered receptor of any one of claims 1-21, wherein the engineered receptor is less potent for acetylcholine than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor.

23. The engineered receptor of claim 22, wherein the engineered receptor is at least 2-fold less potent for acetylcholine than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor.

24. The engineered receptor of any one of claims 1-23, wherein the engineered receptor has about the same potency for a non-natural ligand as the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor for the non-natural ligand.

25. The engineered receptor of any one of claims 1-24, wherein the engineered receptor has a higher potency for a non-natural ligand than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor for the non-natural ligand.

26. The engineered receptor of claim 25, wherein the engineered receptor has at least 2-fold greater potency for a non-natural ligand than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor for the non-natural ligand.

27. The engineered receptor of any one of claims 22-26, wherein the potency of the engineered receptor for a ligand is determined by the EC50 of the receptor for the ligand according to a YFP fluorescence quenching assay using Lenti-X 293T cells.

28. The engineered receptor of any one of claims 1-27, wherein the engineered receptor has a greater potency in the presence of a non-natural ligand than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor in the presence of the non-natural ligand.

29. The engineered receptor of any one of claims 1-28, wherein the engineered receptor has at least 2-fold greater potency in the presence of a non-natural ligand than the human α7 nicotinic acetylcholine receptor (α7-nAChR) or a control receptor in the presence of the non-natural ligand.

30. The engineered receptor of any one of claims 28-29, wherein determining the efficacy comprises determining the amount of current through the engineered receptor in vitro in the presence of the non-natural ligand.

31. The engineered receptor of any one of claims 24-30, wherein the non-natural ligand is selected from AZD-0328, TC-6987, ABT-126, CNL002, TC-5619, CNL001, TC-6683, varenicline, and Facinicline / RG3487.

32. The engineered receptor of claim 31, wherein the non-natural ligand is ABT-126.

33. The engineered receptor of claim 31, wherein the non-natural ligand is varenicline.

34. A polynucleotide encoding the engineered receptor of any one of claims 1-33.

35. A polynucleotide encoding an engineered receptor comprising the amino acid sequence of any one of SEQ ID NOs: 89-98.

36. The polynucleotide of claim 34 or 35, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the engineered receptor.

37. The polynucleotide of claim 36, wherein the promoter is a regulatable promoter.

38. The polynucleotide of claim 37, wherein the regulatable promoter is active in excitable cells.

39. The polynucleotide of claim 38, wherein the excitable cell is a neuron or a muscle cell.

40. The polynucleotide of claim 39, wherein the excitable cell is a neuron.

41. A vector comprising the polynucleotide of any one of claims 34-40.

42. The vector of claim 41, wherein the vector is a plasmid or a viral vector.

43. The vector of claim 42, wherein the vector is a viral vector selected from the group consisting of an adenoviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, and a herpes simplex-1 viral vector (HSV-1).

44. The vector of claim 43, wherein the viral vector is an AAV vector, and wherein the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof; optionally, the capsid protein of the AAV9 vector comprises a T492V mutation according to SEQ ID NO:

81.

45. A composition comprising the engineered receptor of any one of claims 1-33, the polynucleotide of any one of claims 34-40, or the vector of any one of claims 41-44.

46. ​​A pharmaceutical composition comprising the engineered receptor of any one of claims 1-33, the polynucleotide of any one of claims 34-40, or the vector of any one of claims 41-44; and a pharmaceutically acceptable carrier.

47. A method of expressing an engineered receptor in a neuron, comprising contacting the neuron with the polynucleotide of any one of claims 34-40, the vector of any one of claims 41-44, the composition of claim 45, or the pharmaceutical composition of claim 46.

48. The method of claim 47 or the polynucleotide of claim 40, wherein the neuron is a neuron of the peripheral nervous system.

49. The method of claim 47 or 48, or the polynucleotide of claim 40, wherein the neuron is a neuron of the central nervous system.

50. The method of any one of claims 47-49 or the polynucleotide of claim 40, wherein the neuron is a nociceptive neuron.

51. The method of any one of claims 47-50 or the polynucleotide of claim 40, wherein the neuron is a non-nociceptive neuron.

52. The method of any one of claims 47-51 or the polynucleotide of claim 40, wherein the neuron is a hippocampal neuron, a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron.

53. The method of any one of claims 47-52 or the polynucleotide of claim 40, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers.

54. The method of claim 53 or the polynucleotide of claim 40, wherein the neurons are A[beta] afferent fibers.

55. The method of claim 54 or the polynucleotide of claim 40, wherein the A[beta] afferent fibers are damaged A[beta] afferent fibers.

56. The method of claim 54 or the polynucleotide of claim 40, wherein the A[beta] afferent fibers are undamaged A[beta] afferent fibers.

57. The method of any one of claims 47-56 or the polynucleotide of claim 40, wherein the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5.

58. The method of any one of claims 47-57 or the polynucleotide of claim 40, wherein the neuron does not express TrpV1, prostatic acid phosphatase, NaV1.

1.

59. The method of any one of claims 47-58, wherein the contacting step is performed in vitro, ex vivo, or in vivo.

60. The method of claim 59, wherein the contacting step is performed in vivo in a subject.

61. The method of claim 60, wherein the contacting step comprises administering the polynucleotide, the vector, the composition, or the pharmaceutical composition to the subject.

62. The method of claim 61, wherein the contacting step is performed in vitro or ex vivo.

63. The method of claim 62, wherein the contacting step comprises lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection.

64. The method of any one of claims 47-63, wherein the engineered receptor is capable of localizing to the cell surface of a neuron.

65. A method for inhibiting neuronal activity, comprising (a) contacting the neuron with an engineered receptor as described in any one of claims 1-33, a polynucleotide as described in any one of claims 34-40, or a vector as described in any one of claims 41-44, a composition as described in claim 45, or a pharmaceutical composition as described in claim 46, and (b) contacting the neuron with a non-natural ligand for the engineered receptor.

66. The method of claim 65, wherein the neuron is a neuron of the peripheral nervous system.

67. The method of claim 65, wherein the neuron is a neuron of the central nervous system.

68. The method of any one of claims 65-67, wherein the neuron is a nociceptive neuron.

69. The method of any one of claims 65-67, wherein the neuron is a non-nociceptive neuron.

70. The method of any one of claims 65-69, 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.

71. The method of any one of claims 65-70, wherein the neurons are Aδ afferent fibers, C fibers, or Aβ afferent fibers.

72. The method of claim 71, wherein the neurons are A[beta] afferent fibers.

73. The method of claim 72, wherein the A[beta] afferent fibers are damaged A[beta] afferent fibers.

74. The method of claim 72, wherein the A[beta] afferent fibers are undamaged A[beta] afferent fibers.

75. The method of any one of claims 65-74, wherein the neuron expresses neurofilament 200 (NF200), piezo 2, and TLR-5.

76. The method of any one of claims 65-75, wherein the neurons do not express TrpV1, prostatic acid phosphatase, NaV1.

1.

77. The method of any one of claims 65-76, wherein the contacting step (a) is performed in vitro, ex vivo, or in vivo.

78. The method of any one of claims 65-77, wherein the contacting step (b) is performed in vitro, ex vivo, or in vivo.

79. The method of any one of claims 65-78, wherein the contacting steps (a) and / or (b) are performed in vivo in a subject.

80. The method of claim 79, 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.

81. The method of any one of claims 65-80, wherein the contacting steps (a) and / or (b) comprise lipofection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection.

82. The method of any one of claims 65-81, wherein the engineered receptor is capable of localizing to the cell surface of a neuron.

83. A method for treating and / or delaying the onset of a neurological disorder in a subject in need thereof, comprising: a administering to the subject a therapeutically effective amount of the engineered receptor of any one of claims 1-33, the polynucleotide of any one of claims 34-40, or the vector of any one of claims 41-44, the composition of claim 45, or the pharmaceutical composition of claim 46, and b. Administering a non-natural ligand for the engineered receptor to the subject.

84. The method of claim 83, wherein the non-natural ligand is administered to the subject after step (a).

85. The method of claim 83, wherein the non-natural ligand is administered to the subject concurrently with step (a).

86. The method of any one of claims 83-85, wherein the nervous system disorder is an epileptic disorder, a movement disorder, an eating disorder, a spinal cord injury, a neurogenic bladder, allodynia, a spastic disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, memory loss, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation.

87. The method of claim 86, wherein the neurological disorder is allodynia.

88. The method of any one of claims 83-87, wherein the non-natural ligand is selected from AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, varenicline, and Facinicline / RG3487.

89. The method of any one of claims 83-88, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously.

90. The method of claim 89, wherein the non-natural ligand is administered orally.

91. The method of any of claims 83-90, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or to the cisterna magna.

92. The method of any one of claims 83-91, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally. E 93. The method of any one of claims 83-92, wherein the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

94. The method of any one of claims 83-92, wherein the subject suffers from neuropathic pain, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to a dorsal root ganglion (DRG) of the subject. E 95. The method of any one of E 83-94, wherein the subject is a human. E 96. The method of any one of E 83-95, wherein the therapeutically effective amount reduces the severity of the signs and / or symptoms of the neurological disorder. E 97. The method of any one of E 83-96, wherein the therapeutically effective amount delays the onset of signs and / or symptoms of the neurological disorder. E 98. The method of any one of E 83-97, wherein the therapeutically effective amount eliminates signs and / or symptoms of the neurological disorder.

99. The method of any one of claims 96-98, wherein the sign of the neurological disorder is nerve damage, nerve atrophy and / or epilepsy.

100. The method of claim 99, wherein the nerve injury is peripheral nerve injury.

101. The method of any one of E 96-100, wherein the symptom of the nervous system disorder is pain.

102. A method for treating pain and / or delaying the onset of pain in a subject in need thereof, comprising: a administering to the subject a therapeutically effective amount of the engineered receptor of any one of claims 1-33, the polynucleotide of any one of claims 34-40, or the vector of any one of claims 41-44, the composition of claim 45, or the pharmaceutical composition of claim 46, and b. Administering a non-natural ligand for the engineered receptor to the subject.

103. The method of claim 102, wherein the non-natural ligand is administered to the subject after step (a).

104. The method of claim 102, wherein the non-natural ligand is administered to the subject concurrently with step (a).

105. The method of any one of claims 102-104, wherein the non-natural ligand is selected from AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, varenicline, and Facinicline / RG3487.

106. The method of any one of claims 102-105, wherein the non-natural ligand is administered orally, subcutaneously, topically, or intravenously.

107. The method of claim 106, wherein the non-natural ligand is administered orally.

108. The method of any one of claims 102-107, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionally, intraneurally, intracranially, intraspinally, or to the cisterna magna.

109. The method of any one of claims 102-108, wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered by transforaminal injection or intrathecally. E 110. The method of any one of E 102-109, wherein the subject suffers from trigeminal neuralgia, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to the trigeminal ganglion (TG) of the subject.

111. The method of any one of claims 102-110, wherein the subject suffers from neuropathic pain, and wherein the engineered receptor, the polynucleotide, the vector, the composition, or the pharmaceutical composition is administered to a dorsal root ganglion (DRG) of the subject.

112. The method of any one of claims 102-111, wherein the subject is a human.

113. The method of any one of claims 101-112, wherein the pain is neuropathic pain. E 114. The method of any one of E 101-113, wherein the pain is associated with, caused by, or resulted from chemotherapy.

115. The method of any one of claims 101-114, wherein the pain is associated with, caused by, or resulted from trauma. E 116. The method of any one of E 101-115, wherein the subject suffers from allodynia.

117. The method of any one of Claims 101-116, wherein the pain manifests following a medical procedure.

118. The method of any one of claims 101-117, wherein the pain is associated with, caused by, or results from labor or a cesarean section.

119. The method of any one of claims 101-118, wherein the pain is associated with, caused by, or resulted from a migraine.

120. The method of any one of claims 101-119, wherein the therapeutically effective amount temporarily reduces pain in the subject, permanently reduces pain in the subject, prevents the onset of pain in the subject, and / or eliminates pain in the subject.

121. The method of any one of claims 101-120, wherein steps (a) and (b) are performed prior to manifestation of pain in the subject.

122. A kit comprising (a) the vector of any one of claims 41-44, and (b) a non-natural ligand for the engineered receptor.

123. A kit comprising (a) the engineered receptor of any one of claims 1-33, and (b) a non-natural ligand for the engineered receptor.

124. The kit of claim 122 or 123, wherein the engineered receptor and the non-natural ligand are any one of the combinations provided in Table 29.

125. The kit of claim 122 or 124, comprising a device suitable for administering the vector.

126. A method of treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of the polynucleotide of any one of claims 34-40 or the vector of any one of claims 41-44.

127. The method of claim 126, wherein the polynucleotide is delivered to hippocampal neurons in the subject, or wherein the vector transduces hippocampal neurons in the subject.

128. The method of claim 126 or 127, wherein the vector is an AAV9 vector comprising a capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

81.

129. The method of any one of claims 126-128, wherein the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

130. The method of any one of claims 126-129, wherein the polynucleotide or the vector is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cistern magna) administration, intracerebral administration, intraventricular administration, or direct injection into an epileptic focus in the hippocampus.

131. A method of treating neuropathic pain in a subject in need thereof, comprising administering an effective amount of the polynucleotide of any one of claims 34-40 or the vector of any one of claims 41-44.

132. The method of claim 131, wherein the neuropathic pain is peripheral neuropathy or trigeminal neuralgia.

133. The method of claim 131 or 132, wherein the polynucleotide is delivered to dorsal root ganglion neurons or trigeminal ganglion neurons in the subject, or wherein the vector transduces the dorsal root ganglion neurons or the trigeminal ganglion neurons in the subject.

134. The method of any one of claims 131 or 133, wherein the vector is an AAV9 vector comprising a capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO:81, and a non-threonine mutation at a position corresponding to T492 of SEQ ID NO:

81.

135. The method of claim 134, wherein the non-threonine mutation is a valine substitution.

136. The method of any one of claims 131-135, wherein said polynucleotide or said vector is administered by intrathecal (IT) or intraganglionic (IG) administration.

137. The method of any one of claims 126-136, further comprising administering a non-natural ligand for the engineered receptor.

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