Compositions and methods for neurological diseases

Abstract

It provides non-invasive treatment options for various neurological disorders, including pain management. [Solution] Compositions and methods are provided for modulating cell activity using a gene therapy vector comprising an engineered receptor, a polynucleotide encoding the engineered receptor, and a polynucleotide encoding the engineered receptor. These compositions and methods are particularly useful for modulating neuronal activity, for example, in the treatment of disease or in the study of neural circuits.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the interests of U.S. Provisional Application No. 63 / 068,890, filed on 21 August 2020, the entirety of which is incorporated herein by reference.

[0002] Description of text files submitted electronically The sequence listing relating to this application is provided in text format instead of as a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is SWCH_034_01WO_SeqList_ST25.txt. The text file is approximately 297kb in size, was created on August 19, 2021, and submitted electronically via EFS-Web.

[0003] field This disclosure relates to engineered receptors, as well as the use of engineered receptors and small molecule ligands to modulate cell activity and treat diseases. [Background technology]

[0004] background Intractable neurological disorders are often associated with abnormally functioning neurons. Attempts to develop treatments to address these symptoms have been hampered by the lack of readily available target proteins associated with the diseases. For example, unresolved chronic pain is a significant health problem in the United States and worldwide. The U.S. Institute of Medicine reports that 116 million Americans suffer from pain lasting weeks to years, resulting in an estimated annual cost exceeding $560 billion. There is no adequate long-term treatment for chronic pain sufferers, which is costly for both society and individuals. Pain often leads to physical disability, and even without disability, it significantly impacts quality of life. Pain management often fails, even when the healthcare supply environment is optimal, including attentive and well-trained physicians, easy access to opioids, the use of adjuvant analgesics, the availability of patient-managed analgesia, and evidence-based use of nerve blocks and IT pumps.

[0005] The most commonly used treatments for chronic pain are the application of opioid analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs), but these drugs can be addictive and can cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive impairment, hallucinations, and other systemic side effects. Despite the widespread use of these drugs, the success rate for their effectiveness in pain relief is remarkably low. Large randomized trials using various drugs have found that only one in two or three patients achieves at least 50% pain relief (Finnerup et al., 2005). Follow-up studies using the most developed pharmacological treatments have found the same results, showing no improvement in the effectiveness of pharmacotherapy for pain (Finnerup et al., Pain, 150(3):573-81, 2010).

[0006] More invasive options for pain management include nerve blocks and electrical stimulation. Nerve blocks are typically local anesthetic injections into the spinal cord to block pain signals to the brain, and their effects last only a few weeks to months. Nerve blocks are not the recommended treatment option in most cases (Mailis and Taenzer, Pain Res Manag. 17(3):150-158, 2012). Electrical stimulation involves supplying an electric current to block pain signals. The effects may last longer than nerve blocks, but complications can occur in the conductor itself, namely dislocation, infection, breakage, or battery failure. One review found that 40% of patients treated with electrical stimulation for neuropathy experienced one or more of these problems with the device (Wolter, 2014).

[0007] The most invasive and least desirable method of managing pain is the complete surgical removal of the nerve or portion of the nerve causing the pain. This option is only recommended when the patient has exhausted the former and other less invasive procedures and they have proven 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 lesions of the DRG for chronic lumbosacral radiculopathy (Geurts et al., 2003). Other surgical methods for surgically removing painful nerves have similar drawbacks and have serious long-term side effects, including sensory or motor loss, or cause pain elsewhere.

[0008] Methods for treating neurological disorders should be safe, efficient, and cost-effective. Gene therapy can offer non-invasive treatment options for various neurological diseases, including pain management. However, to date, gene therapy has not been widely used for treating neurological diseases. This disclosure addresses these needs. This disclosure will be best understood from the following detailed description in conjunction with the attached drawings. The patent or application file includes at least one drawing drawn in color. It should be emphasized that, according to common practice, various features of the drawing are not to scale. Conversely, the dimensions of various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures: [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Finnerup et al., Pain, 150(3):573-81, 2010. [Non-Patent Document 2] Mailis and Taenzer, Pain Res Manag. 17(3):150-158, 2012. [Brief explanation of the drawing]

[0010] [Figure 1A-1]Figures 1A–1J show heatmaps of YFP fluorescence quenching percentages in variants of the engineered chimeric receptor of Sequence ID No. 33, including the indicated amino acid substitutions, after stimulation with either acetylcholine or the indicated non-native ligands at various doses. Ligand doses are indicated at the top of each chart. The numbers in the boxes indicate the relative amount of quenching observed. Higher levels of quenching indicate higher levels of receptor activation by the ligand at the indicated dose. Quenching levels are also indicated by a color gradient. Dark = more than 70% of the maximum quenching of the YFP reporter (i.e., the majority of the engineered receptor is activated). White = less than 10% quenching (i.e., most of the engineered receptor is not activated). Negative values ​​represent unresponsive individuals with negative quenching due to stimulation artifacts. Sequence ID No. 29 is an unresponsive chimeric cell used as a negative control. CODA283 contains an extra N379K mutation introduced during cloning, located outside the ligand-binding domain and therefore not affecting 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 / bladanicline, 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 fascinicline, Figure 1I shows YFP fluorescence quenching using TC-6987, and Figure 1J shows YFP fluorescence quenching using varenicline. Abbreviations for non-natural ligand names: abt: ABT-126; ach: acetylcholine; azd: AZD-0328; brd: TC-5619 (bladanicline); fac: RG3487 (fascinicline); tc6: TC-6987; var: varenicline. [Figure 1A-2] Same as above. [Figure 1B-1] Same as above. [Figure 1B-2] Same as above. [Figure 1C] Same as above. [Figure 1D-1] Same as above. [Figure 1D-2] Same as above. [Figure 1E-1] Same as above. [Figure 1E-2] Same as above. [Figure 1F-1] Same as above. [Figure 1F-2] Same as above. [Figure 1G-1] Same as above. [Figure 1G-2] Same as above. [Figure 1H-1] Same as above. [Figure 1H-2] Same as above. [Figure 1I-1] Same as above. [Figure 1I-2] Same as above. [Figure 1J-1] Same as above. [Figure 1J-2] Same as above.

[0011] [Figure 2] Figures 2A and 2B show the concentration-response curves of CR-11 (chemically induced receptor-11, an engineered receptor containing an amino acid sequence with amino acid substitutions at Y115D and L131Q in SEQ ID NO: 33) expressed in HEK293 cells to acetylcholine and the non-natural ligand RG-3487 (SA-2, synthetic agonist-2). Responses were evaluated using manual patch-clamp electrophysiology. Current was normalized to 1 for the maximum response. The continuous line through the data points is the best fit obtained by Hill's equation, and the EC50 of each ligand is estimated from the concentration-response curves. Figure 2A shows the concentration-response curves of the wild-type and CR-11 receptors to acetylcholine. Figure 2B shows the concentration-response curves of the wild-type and CR-11 receptors to RG-3487 (SA-2).

[0012] [Figure 3]Figure 3 shows exemplary chloride currents induced by RG-3487(SA-2) in adult rat DRG neurons transduced with a lentivirus expressing CR-11 (a modified receptor containing an amino acid sequence with amino acid substitutions at Y115D and L131Q in SEQ ID NO: 33).

[0013] [Figure 4A] Figure 4A shows evoked action potentials of transduced DRG neurons expressing CR-11 (engineered receptor containing an amino acid sequence with amino acid substitutions at Y115D and L131Q in SEQ ID NO: 33) or control DRG neurons (without CR-11 expression) at different current injections (50 pA to 700 pA). The top panel shows evoked action potentials of control DGF neurons. The bottom left panel shows evoked action potentials of transduced DRG neurons expressing CR-11 in the presence of 3 μM RG-3487(SA-2). The bottom right panel shows evoked action potentials of transduced DRG neurons expressing CR-11 after rinsing off RG-3487(SA-2). [Figure 4B] Figure 4B shows the baseline current values ​​(the current required to induce an action potential) for control DRG neurons and transduced DRG neurons expressing CR-11, in the absence or presence of the indicated ligand.

[0014] [Figure 5-1]Figure 5 shows the percentage of HA-tagged cells expressing the engineered receptor, normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33 ("Normalized HA%"), and the percentage of α-bungarotoxin-positive cells expressing the engineered receptor, normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33 ("Normalized AB%"). Figure 5 also shows the median fluorescence intensity (MFI) of cells expressing the engineered receptor, normalized to control cells expressing the amino acid sequence of SEQ ID NO: 33, as assessed using anti-HA antibody ("Normalized HA MFI") or fluorescently labeled α-bungarotoxin conjugated with Alexa Fluor 647 ("Normalized AB MFI"). [Figure 5-2] Same as above.

[0015] [Figure 6A] Figure 6A shows the relative surface and total expression of the manipulated receptors in cultured DRG neurons or HEK cells. [Figure 6B] Figure 6B shows the relative surface and total expression of the manipulated receptors in cultured hippocampal neurons. [Overview of the project] [Means for solving the problem]

[0016] Abstract This disclosure provides an engineered receptor comprising a ligand-binding domain derived from the human α7-nicotinic acetylcholine receptor (α7-nAChR), wherein the ligand-binding domain contains amino acid mutations in amino acid residues corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172, or Y210 of SEQ ID NO: 4. In some embodiments, the ligand-binding domain comprises an amino acid sequence having at least 85% identity to amino acid residues 23-220 of SEQ ID NO: 4. In some embodiments, the ligand-binding domain contains amino acid mutations in two or more amino acid residues selected from the amino acid residues corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172, and Y210 of SEQ ID NO: 4. In some embodiments, the ligand-binding domain comprises amino acid mutations in the indicated positions of SEQ ID NO: 4: a) Y140, b) R101 and L131, c) Y115 and Y210, d) R101 and Y210, e) R101, Y115 and Y210, f) W77, R101 and L131, g) R101, L131 and S172, h) Q139 and S172D, i) S172 and Y210, j) L131 and S172, k) Y115 and S170, or l) Y115 and L131 The mutation is present in the corresponding amino acid residue.

[0017] In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand-binding domain has an amino acid substitution corresponding to the position shown in SEQ ID NO: 4 a) Y140I, b) R101F and L131G, c) R101F and L131D, d) Y115E and Y210W, e) R101W and Y210V, f) R101F and Y210V, g) R101F and Y210F, h) R101M and L131A, i) R101M and L131F, j) R101W, Y115E and Y210W, k) R101F, Y115E and Y210W, l) W77F, R101F and L131D, m) R101F, L131N, and S172D, n) Q139E and S172D, o) S172D and Y210W, p) L131S and S172D, q) L131T and S172D, r) L131D and S172D, s)Y115D and S170T, t) Y115D and L131Q, u) Y115D and L131E, v)L131E, w)Y140C, x)R101W, y)Y210V, or z)Q139E Includes.

[0018] In some embodiments, the engineered receptor is a chimeric ligand-opening ion channel (LGIC) receptor containing an ion pore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3. In some embodiments, the ion pore domain contains an amino acid sequence having at least 85% identity to amino acids 255-457 of SEQ ID NO: 2, 260-452 of SEQ ID NO: 83, 259-464 of SEQ ID NO: 85, or 259-449 of SEQ ID NO: 87. In some embodiments, the ligand-binding domain of the engineered receptor contains a Cys-loop domain derived from a human glycine receptor. In some embodiments, the Cys-loop domain contains amino acids 166-172 of SEQ ID NO: 2. In some embodiments, the Cys-loop domain contains amino acids 166-180 of SEQ ID NO: 2. In some embodiments, the ligand-binding domain of the engineered receptor contains a β1-2 loop domain derived from the human glycine receptor α1 subunit. In some embodiments, the β1-2 loop domain includes amino acids 81-84 of SEQ ID NO: 2. In some embodiments, the engineered receptor includes the amino acid sequence of any one of SEQ ID NOs. 58-78 and 88.

[0019] This disclosure provides an engineered chimeric LGIC comprising a ligand-gated ion channel (LGIC) derived from a first ligand-gated ion channel (LGIC) and an ion pore domain derived from a second LGIC, the first LGIC being a human α7-nicotinic acetylcholine receptor (α7-nAChR) and containing amino acid mutations in amino acid residues corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172 or Y210 of SEQ ID NO: 4.

[0020] In some embodiments, the ligand-binding domain is located at the position indicated in SEQ ID NO: 4: a.Y140, b.R101 and L131, c.Y115 and Y210, d.R101 and Y210, e.R101, Y115 and Y210, f.W77, R101 and L131, g.R101, L131 and S172, h.Q139 and S172D, i.S172 and Y210, j.L131 and S172, k.Y115 and S170, or l.Y115 and L131 The mutation is present in the corresponding amino acid residue.

[0021] In some embodiments, the ligand-binding domain has an amino acid substitution corresponding to the position shown in SEQ ID NO: 4: a.Y140I, b. R101F and L131G, c.R101F and L131D, d.Y115E and Y210W, e.R101W and Y210V, f.R101F and Y210V, g.R101F and Y210F, h.R101M and L131A, i.R101M and L131F, j.R101W, Y115E and Y210W, k.R101F, Y115E and Y210W, l.W77F, R101F and L131D, m.R101F, L131N and S172D, n.Q139E and S172D, o.S172D and Y210W, p.L131S and S172D, q.L131T and S172D, r.L131D and S172D, s.Y115D and S170T, t.Y115D and L131Q, u.Y115D and L131E, v.L131E, w.Y140C, x.R101W, y.Y210V, or z.Q139E Includes.

[0022] In some embodiments, the second LGIC is a human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1. In some embodiments, the engineered chimeric LGIC contains a polypeptide sequence having at least 85% sequence identity with SEQ ID NO: 33.

[0023] In some embodiments, the potency of the engineered receptor against acetylcholine is lower than that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor against acetylcholine. In some embodiments, the potency of the engineered receptor against acetylcholine is at least twice as low as that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor against acetylcholine. In some embodiments, the potency of the engineered receptor against non-natural ligands is approximately the same as that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor against non-natural ligands. In some embodiments, the potency of the engineered receptor against non-natural ligands is higher than that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor against non-natural ligands. In some embodiments, the potency of the engineered receptor against non-natural ligands is at least twice as high as that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor against non-natural ligands. In some embodiments, the potency of the engineered receptor against the ligand is determined by the EC50 of the receptor against the ligand according to a YFP fluorescence quenching assay using Lenti-X 293T cells.

[0024] In some embodiments, the efficacy of the engineered receptor in the presence of a non-natural ligand is higher than that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or 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 twice as high as that of the human α7-nicotinic acetylcholine receptor (α7-nAChR) or control receptor in the presence of a non-natural ligand. In some embodiments, determining efficacy involves determining the amount of current that passed through the engineered receptor in vitro in the presence of a non-natural ligand.

[0025] In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, CNL002, TC-5619, CNL001, TC-6683, varenicline, and fascinicline / RG3487. In some embodiments, the non-natural ligand is selected from the group consisting of ABT-126, RG3487, and CNL002. In some embodiments, the non-natural ligand is TC-5619.

[0026] This disclosure provides polynucleotides encoding engineered receptors. In some embodiments, the polynucleotide encodes an engineered receptor comprising one of the amino acid sequences of SEQ ID NOs. 58-78 and 88. In some embodiments, the polynucleotide includes a promoter operably ligated to the nucleic acid encoding the engineered receptor. In some embodiments, the promoter is a regulatory promoter. In some embodiments, the regulatory promoter is active in an excitable cell. In some embodiments, the excitable cell is a neuron or a muscle cell. In some embodiments, the excitable cell is a neuron.

[0027] This disclosure provides vectors 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 adenovirus vectors, retrovirus vectors, adeno-associated virus (AAV) vectors, and herpes simplex virus-1 (HSV-1) vectors. In some embodiments, the viral vector is an AVV vector, and the AAV vector is AAV5 or a variant thereof, AAV6 or a variant thereof, or AAV9 or a variant thereof.

[0028] This disclosure provides compositions comprising any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, or any one of the vectors disclosed herein. This disclosure further provides pharmaceutical compositions 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.

[0029] This disclosure provides a method for expressing an engineered receptor in a neuron, comprising contacting the neuron with any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the neuron is a neuron of the peripheral nervous system. In some embodiments, the neuron is a neuron of the central nervous system. In some embodiments, the neuron is a nociceptive neuron. In some embodiments, the neuron is a non-nociceptive neuron. In some embodiments, the neuron is a dorsal root ganglion (DRG) neuron, a trigeminal ganglion (TG) neuron, a motor neuron, an excitatory neuron, an inhibitory neuron, or a sensory neuron. In some embodiments, the neuron is an Aδ afferent fiber, a C fiber, or an Aβ afferent fiber. In some embodiments, the neuron is an Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is a damaged Aβ afferent fiber. In some embodiments, the Aβ afferent fiber is an undamaged Aβ afferent fiber. In some embodiments, the neurons express neurofilament 200 (NF200), piezo2, and TLR-5. In some embodiments, the neurons do not express TrpV1, prostatic acid phosphatase, or NaV1.1.

[0030] In some embodiments, contact is performed in vitro, ex vivo, or in vivo. In some embodiments, contact is performed in vivo in a subject. In some embodiments, contact involves administering a polynucleotide, vector, composition, or pharmaceutical composition to a subject. In some embodiments, contact is performed in vitro or ex vivo. In some embodiments, contact includes lipofection, nanoparticle delivery, microparticle gun, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor can be localized to the cell surface of a neuron.

[0031] This disclosure provides a method for inhibiting neuronal activity, comprising (a) contacting a neuron with any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein, and (b) contacting a neuron with a non-native 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 fibers are undamaged Aβ afferent fibers. In some embodiments, the neurons express neurofilament 200 (NF200), piezo2, and TLR-5. In some embodiments, the neurons do not express TrpV1, prostatic acid phosphatase, or NaV1.1.

[0032] In some embodiments, contact (a) is performed in vitro, ex vivo, or in vivo. In some embodiments, contact (b) is performed in vitro, ex vivo, or in vivo. In some embodiments, contact (a) and / or (b) are performed in vivo in the subject. In some embodiments, contact (a) includes administering an engineered receptor, polynucleotide, vector, or pharmaceutical composition to a subject, and / or contact (b) includes administering a non-natural ligand to a subject. In some embodiments, contact (a) and / or (b) include lipofection, nanoparticle delivery, microparticle gun, electroporation, sonication, or microinjection. In some embodiments, the engineered receptor can be localized to the cell surface of a neuron.

[0033] This disclosure provides a method for treating and / or delaying the onset of neurological disorders in subjects requiring treatment of neurological disorders, comprising administering a therapeutically effective amount of any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein to a subject, and administering a non-natural ligand of an engineered receptor to a subject. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand concurrently with step (a).

[0034] In some embodiments, the neurological disorder is paroxysmal disorder, motor disorder, feeding disorder, spinal cord injury, neurogenic bladder, allodynia, spastic disorder, pruritus, Alzheimer's disease, Parkinson's disease, post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction, anxiety, depression, amnesia, dementia, sleep apnea, stroke, narcolepsy, urinary incontinence, essential tremor, trigeminal neuralgia, burning mouth syndrome, or atrial fibrillation. In some embodiments, the neurological disorder is allodynia. In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, varenicline, and fascinicline / RG3487.

[0035] In some embodiments, a non-natural ligand is administered orally, subcutaneously, topically, or intravenously. In some embodiments, a non-natural ligand is administered orally. In some embodiments, an engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglion, intraneuronally, intracranially, intraspinally, or into the cisterna magna. In some embodiments, an engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered transforaminal injection or intrathecally. In some embodiments, the subject suffers from trigeminal neuralgia, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the subject's trigeminal ganglion (TG). In some embodiments, the subject suffers from neuropathic pain, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the subject's dorsal root ganglion (DRG). In some embodiments, the subject is human.

[0036] In some embodiments, the therapeutically effective dose reduces the severity of signs and / or symptoms of neuropathy. In some embodiments, the therapeutically effective dose delays the onset of signs and / or symptoms of neuropathy. In some embodiments, the therapeutically effective dose eliminates signs and / or symptoms of neuropathy. In some embodiments, signs of neuropathy are nerve injury, nerve atrophy, and / or seizures. In some embodiments, nerve injury is peripheral nerve injury. In some embodiments, the symptom of neuropathy is pain.

[0037] This disclosure provides a method for treating and / or delaying the onset of pain in subjects requiring treatment of pain and / or delaying the onset of pain, comprising administering a therapeutically effective amount of any one of the engineered receptors disclosed herein, any one of the polynucleotides disclosed herein, any one of the vectors disclosed herein, any one of the compositions disclosed herein, or any one of the pharmaceutical compositions disclosed herein to a subject, and administering a non-natural ligand of an engineered receptor to a subject. In some embodiments, the subject is administered the non-natural ligand after step (a). In some embodiments, the subject is administered the non-natural ligand concurrently with step (a). In some embodiments, the non-natural ligand is selected from the group consisting of AZD-0328, ABT-126, TC6987, CNL002, TC-5619, CNL001, TC-6683, varenicline, and fascinicline / RG3487.

[0038] In some embodiments, non-natural ligands are administered orally, subcutaneously, topically, or intravenously. In some embodiments, non-natural ligands are administered orally. In some embodiments, engineered receptors, polynucleotides, vectors, compositions, or pharmaceutical compositions are administered subcutaneously, orally, intrathecally, topically, intravenously, intraganglionically, intraneuronally, intracranially, intraspinally, or into the cisterna magna. In some embodiments, engineered receptors, polynucleotides, vectors, compositions, or pharmaceutical compositions are administered transforaminally or intrathecally.

[0039] In some embodiments, the subject suffers from trigeminal neuralgia, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the subject's trigeminal ganglion (TG). In some embodiments, the subject suffers from neuropathic pain, and the engineered receptor, polynucleotide, vector, composition, or pharmaceutical composition is administered to the subject's dorsal root ganglion (DRG).

[0040] In some embodiments, the subject is human. In some embodiments, the pain is neuropathic pain. In some embodiments, the pain is related to, caused by, or results from chemotherapy. In some embodiments, the pain is related to, caused by, or results from trauma. In some embodiments, the subject suffers from allodynia. In some embodiments, the pain appears after a medical procedure. In some embodiments, the pain is related to, caused by, or results from childbirth or cesarean section. In some embodiments, the pain is related to, caused by, or results from migraine. In some embodiments, the therapeutically effective dose temporarily relieves the subject's pain, permanently relieves the subject's pain, prevents the onset of the subject's pain, and / or eliminates the subject's pain. In some embodiments, steps (a) and (b) are performed before the onset of pain in the subject.

[0041] This disclosure provides a kit comprising (a) the vector of this disclosure and (b) a non-native ligand for the engineered receptor encoded by the vector. This disclosure provides a kit comprising (a) the engineered receptor of this disclosure and (b) a non-native ligand for the engineered receptor. In some embodiments, the combination of engineered receptor and non-native ligand follows one of the combinations provided in Table 29. In some embodiments, the kit includes a device adapted for administering the vector. [Modes for carrying out the invention]

[0042] Detailed explanation A. Overview Compositions and methods are provided for modulating cellular activity using gene therapy vectors comprising engineered ligand-gated ion channel (LGIC) receptors, polynucleotides encoding engineered LGIC receptors, and polynucleotides encoding engineered LGIC receptors. These compositions and methods are particularly useful for modulating neuronal activity, for example, in the treatment of diseases or in the study of neural circuits. Furthermore, reagents, apparatus and kits used to carry out the methods in question are also provided.

[0043] In particular, this disclosure provides engineered receptors that bind to ligands and signal in response to ligands. 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 exhibit increased affinity for known agonist ligands. In some embodiments, the engineered receptors described herein exhibit affinity for antagonist or modulator ligands and respond to antagonist and / or modulator ligands as if they were agonist ligands. This disclosure further provides methods for treating neurological disorders in subjects requiring treatment of neurological disorders. This 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 wild-type endogenous receptors.

[0044] Before describing the methods and compositions of the present invention, it should be understood that this disclosure is not limited to the specific methods or compositions described and is therefore naturally subject to change. Since the scope of this disclosure is limited only by the appended claims, it should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0045] Where a range of values ​​is provided, unless explicitly indicated in the context, each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit, is also specifically disclosed. Each smaller range between any stated value or intervening value within the stated range and any other stated value or intervening value within that stated range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which any, neither, or both limits are included in the smaller range, subject to any specifically excluded limits within the stated range, is also included in this disclosure. If a stated range includes one or both limits, the range excluding one or both of the limits that they include is also included in this disclosure.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but several possible preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe the relevant methods and / or materials from which the publications are cited. This disclosure is understood to supersede any disclosure in the incorporated publications to the extent that a conflict exists.

[0047] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any of the listed methods may be performed in the order of the listed events, or in any other logically possible order.

[0048] The publications discussed herein are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that this disclosure does not have prior rights to such publications by prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified. B. Definition

[0049] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly indicates otherwise. Thus, for example, a reference to “cell” includes multiple such cells, and a reference to “peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0050] Where used herein, the term "and / or" is used in this disclosure as either "and" or "or" unless otherwise specified.

[0051] Throughout this specification, unless otherwise specified by context, the word “comprise,” or variations such as “comprises” or “comprising,” means to include the element or integer, or group of elements or integers, that is described, but not to exclude any other element or integer, or group of elements or integers. Furthermore, descriptions of numerical ranges throughout this specification specifically include all integers and the decimal points between them.

[0052] Throughout this specification, unless otherwise required by context, the phrase “essentially derived from” means to limit the scope of a composition, method or kit described in a particular material or process that does not substantially affect the basic and novel features of the disclosure. For example, a ligand-binding domain “essentially derived from” the disclosed sequence has approximately 5 amino acid residues plus or minus 5 amino acid residues of the disclosed sequence at its sequence boundary, for example, approximately 5, 4, 3, 2, or 1 residue fewer than the enumerated boundary amino acid residues, or approximately 1, 2, 3, 4, or 5 residues more than the enumerated boundary amino acid residues.

[0053] Throughout this specification, unless otherwise specified in the context, the phrase “consisting of” means the exclusion of any element, process, or component not expressed in the claims from a composition, method, or kit. For example, a ligand-binding domain “consisting of” a disclosed sequence consists only of the disclosed amino acid sequence.

[0054] Where used in this application, the terms “about” and “approximately” are used interchangeably. Any numbers used in this application are meant to cover any normal variation as understood by those skilled in the art, with or without “about” or “approximately.” In certain embodiments, the terms “approximately” or “about” mean a range of values ​​that, unless otherwise specified or evident from the context (except where such numbers exceed 100% of the possible values), fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value.

[0055] As used herein, the term “isolated” means a material that is substantially or essentially free from the components that would normally accompany it, as found in its natural state. In some embodiments, the terms “obtained” or “derived” are used synonymously with “isolated.”

[0056] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates such as mammals. Mammals may be, for example, mice, rats, rabbits, cats, dogs, pigs, sheep, horses, non-human primates (e.g., cynomolgus macaques, chimpanzees), or humans. Tissues, cells, or derivatives thereof of subjects obtained in vivo or cultured in vitro are also included. Human subjects may be adults, teenagers, children (2 to 14 years), infants (1 to 24 months), or neonates (up to 1 month). In some embodiments, adults are older than approximately 65 years or older, or older than approximately 60 years. In some embodiments, subjects are pregnant women or women intending to become pregnant.

[0057] The term “sample” refers to the volume and / or mass of biological material subjected to analysis. In some embodiments, a sample includes tissue samples, cell samples, body fluid samples, etc. In some embodiments, a sample is taken from or provided by a subject (e.g., a human subject). In some embodiments, a sample includes a portion of tissue taken from any internal organ, cancerous, precancerous or noncancerous tumors, the brain, skin, hair (including hair follicles), eyes, muscles, bone marrow, cartilage, white adipose tissue and / or brown adipose tissue. In some embodiments, fluid samples include buccal swabs, blood, umbilical cord blood, saliva, semen, urine, ascites, pleural fluid, cerebrospinal fluid, lung lavage fluid, tears, sweat, etc. In some embodiments, those skilled in the art will understand that a “sample” is a “primary sample” in that it is obtained directly from a source (e.g., a subject). In some embodiments, a “sample” is the result of processing a primary sample, for example, to remove certain potentially contaminating components, to isolate specific components, and / or to purify a specific component of interest. In some embodiments, the sample is a cell or a population of cells (e.g., nerve cells). The cell sample may be directly derived from the subject (e.g., a primary sample) or it may be a cell line. The cell line may include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0058] As used herein, “to treat” or “treatment” means to deliver a composition (e.g., an engineered receptor and / or ligand) to a subject and / or cell population in order to affect a physiological outcome. In certain embodiments, treatment results in one or more improvements (e.g., reduction, recovery, or correction) of disease symptoms. The improvement may be an observable or measurable improvement, or it may be an improvement in the subject’s overall well-being. Treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to a reduction in the severity of disease symptoms to a level comparable to the level before the onset of the disease. In some embodiments, treatment may refer to a short-term (e.g., temporary or acute) and / or long-term (e.g., persistent or chronic) reduction of disease symptoms. In some embodiments, treatment may refer to remission of disease symptoms. In some embodiments, treatment may refer to a prophylactic treatment of a subject at risk of developing a particular disease in order to prevent the onset of the disease. Prevention of disease onset may refer to complete prevention of disease symptoms, delay of disease onset, reduction of the severity of symptoms in a later-developing disease, or reduction of the likelihood of developing the disease.

[0059] As used herein, “manage” or “control” refers to the use of a composition or method 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.

[0060] The "therapeutic dose" is the amount of composition necessary to achieve the desired therapeutic outcome. The therapeutic dose is not limited but can vary depending on the patient's disease state and factors such as age, sex, and weight. Generally, the therapeutic dose is also the amount in which the therapeutically beneficial effects of the composition outweigh any toxic or adverse effects. The "therapeutic dose" includes the amount of composition effective in treating the patient.

[0061] An "increase" refers to an increase of at least 5% in value compared to a reference or control level (e.g., an increase in binding affinity, an increase in physiological response, an increase in therapeutic effect, etc.). For example, an increase may include increases 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 times, 1000 times) compared to a reference or control level.

[0062] "Decrease," "reduce," "diminish," or their synonyms refer to a decrease of at least 5% in value compared to a reference or control level (e.g., a decrease in binding affinity, a decrease in physiological response, a decrease in therapeutic effect, a decrease in pain in a subject, etc.). For example, a decrease may include decreases 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 a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (e.g., 500 times, 1000 times), compared to a reference or control level.

[0063] "Maintain," "preserve," "maintain," "no change," "substantial change," or "substantial decrease" generally refer to a physiological and / or therapeutic effect comparable to the effect caused by either the vehicle or the control molecule / composition. A comparable response is one that is not significantly different from or measurably different from the reference response.

[0064] The terms “reference” or “control” level are used interchangeably herein and refer to a specific physiological and / or therapeutic effect value in a subject or sample not treated with the composition described herein, or in a subject or sample treated with a vehicle control. In some embodiments, the reference level refers to a specific physiological and / or therapeutic effect value (e.g., baseline level) measured in the subject or sample before administration of the composition described herein.

[0065] As used herein, “ligand” refers to a molecule that binds to another larger molecule. In some embodiments, ligands bind to receptors. In some embodiments, ligand binding to a receptor alters the function of the receptor, activating or inhibiting its function. In some embodiments, ligand binding to a receptor, such as a ligand-gated ion channel (LGIC), results in the opening or closing of the ion channel.

[0066] The terms “receptor-ligand binding” and “ligand binding” are used interchangeably herein and refer to the physical interaction between a receptor (e.g., LGIC) and a ligand. The term “ligand” as used herein may refer to an endogenous or naturally occurring ligand. For example, in some embodiments, ligands may refer to neurotransmitters (e.g., λ-aminobutyric acid (GABA), acetylcholine, serotonin, etc.) and signaling intermediates (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), amino acids (e.g., glycine), or nucleotides (e.g., ATP). In some embodiments, ligands may refer to non-natural ligands, i.e., synthetic or non-natural ligands. For example, in some embodiments, ligands may refer to small molecules. Ligand binding can be measured by various methods known in the art (e.g., detection of association with radiolabeled ligands).

[0067] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a receptor and a ligand. Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d Affinity can be expressed by ). Affinity can be measured by common methods known in the art, including those described herein.

[0068] The terms “specific binding affinity” or “specific binding” are used interchangeably throughout this specification and the claims and refer to the binding that occurs between a pair of species of molecules, for example, a receptor and a ligand. When the interaction of two species produces a non-covalent complex, the resulting binding is typically the result of electrostatic, hydrogen, or lipophilic interaction. In various embodiments, specific binding between one or more species is direct. In one embodiment, the affinity of a specific binding is about twice that of background binding (non-specific binding), about five times that of background binding, about ten times that of background binding, about twenty times that of background binding, about fifty times that of background binding, about one hundred times that of background binding, or about one thousand times or more that of background binding.

[0069] "Signal transduction" refers to the generation of a biochemical or physiological response as a result of ligand binding to a receptor.

[0070] The terms “wild-type” or “natural” are terms of the art as understood by those skilled in the art, and refer to a naturally occurring characteristic of an organism, strain, gene, or protein that distinguishes it from the typical form or variant form. For example, a wild-type protein is the typical form of that protein as it exists in nature.

[0071] The terms “unnatural,” “mutant,” and “variant” are used interchangeably throughout this specification and the claims and refer to variants of a natural or wild-type composition, such as variant polypeptides having less than 100% sequence identity with the natural or wild-type sequence.

[0072] "Amino acid modification" or "amino acid mutation" may be an amino acid substitution, amino acid deletion, and / or amino acid insertion. Amino acid substitutions may be conservative or non-conservative. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is an amino acid substitution in a protein that changes a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). As used herein, "conservative variation" refers to the substitution of an amino acid residue with another biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue with another, such as isoleucine, valine, leucine, or methionine; the substitution of one polar residue with another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Other examples of conservative substitutions include the change from 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 (praline), histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, glutamine or glutamic acid, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine.

[0073] The terms “parent” or “starter” are used interchangeably throughout this specification and the claims and refer to an initial composition for creating an engineered composition having novel properties, or a mutated, modified, or derivatized protein. In some embodiments, the parent protein is a chimeric protein.

[0074] The term "manipulated" is used throughout this specification and the claims to refer to a composition that does not exist in nature, or a protein having properties different from those of the parent composition, or a protein that has been derivatized from such a composition.

[0075] Generally, “sequence identity” or “sequence homology” refers to the nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their “percent identity.” Whether nucleic acid sequences or amino acid sequences, the percentage identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Percent identity can also be determined 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 methods described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as well as those discussed by Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). In short, the BLAST program defines identity as the number of identically aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percentage identity of proteins being compared across their entire length. Default parameters are provided, for example, to optimize searches using short query sequences in the blastp program.This program also allows for the use of a SEG filter to mask off segments of the query sequence, as determined by the SEG program in Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired range of sequence identity is approximately 80% to 100% and an intervening integer value. Typically, the percentage of 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%.

[0076] As used herein, “substantially identical” means having sequence identity of 85% or more, for example, 90% or more, for example, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, such that the activity of the composition is not altered by the sequence modifications resulting in the difference in sequence identity.

[0077] Where used herein with respect to the position of an amino acid or nucleotide, the terms "corresponding to" or "correspond to" refer to an amino acid in the first polypeptide sequence that aligns with a given amino acid in the reference polypeptide sequence when the first polypeptide sequence and the reference polypeptide sequence are aligned, or a nucleotide in the first polynucleotide sequence that aligns with a given nucleotide in the reference polynucleotide sequence when the first polynucleotide sequence and the reference polynucleotide sequence are aligned. Alignment is performed by those skilled in the art using software designed for this purpose, for example, BLAST program version 2.2.9 with its default parameters.

[0078] As used herein, the term “promoter” refers to one or more nucleic acid regulatory sequences that direct the transcription of a functionally ligated nucleic acid. A promoter may include a nucleic acid sequence near the transcription start site, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A “constituent” 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 “functionally ligated” refers to a functional ligation between a nucleic acid expression regulatory sequence (e.g., a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0079] As used herein, the terms “virus vector,” “viral vector,” or “gene delivery vector” refer to a viral particle that functions as a nucleic acid delivery vehicle and contains nucleic acids (e.g., an AAV expression cassette) packaged within a virion. Illustrative viral vectors of this disclosure include adenovirus vectors, adeno-associated virus vectors (AAVs), lentiviral vectors, and retroviral vectors.

[0080] As used herein, “neuronal activity,” “neuronal activation,” “neuronal firing,” and their variations and synonyms refer to the electrical activity resulting from the stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch-clamp techniques. In some embodiments, determining neuronal activity involves determining the neuron’s excitatory postsynaptic potential (EPSP), inhibitory postsynaptic potential (IPSP), and / or action potential. In some embodiments, the neuron’s activity level depends on or is influenced by the excitatory postsynaptic potential (EPSP), inhibitory postsynaptic potential (IPSP), and / or action potential.

[0081] As used herein, “neurological disorder” or “neurological impairment” refers to a disorder or impairment of the nervous system. In some embodiments, a neurological disorder is related to, caused by, or resulting from a structural, biochemical, and / or electrical abnormality of the brain, spinal cord, nerves, or any component of the nervous system.

[0082] As used herein, “signs” of a disease refer to physical or mental characteristics that are considered to indicate symptoms of the disease. In some embodiments, signs are objective signs of the disease. In some embodiments, signs are objectively evaluated, examined, observed or measured by a person other than the patient, such as a physician.

[0083] As used herein, “symptoms” of a disease refer to the symptoms of a disease, in particular physical or mental features that are considered to exhibit such characteristics as evident to the patient. In some embodiments, symptoms are subjectively assessed by the patient. For example, in some embodiments, the symptom is pain.

[0084] As used herein, “potency” refers to the ability of a receptor described herein to respond to a particular ligand. Therefore, increased potency refers to an increase in the receptor’s responsiveness to a particular ligand. Conversely, decreased potency refers to a decrease in the receptor’s responsiveness to a particular ligand. Receptor potency is generally determined herein by the median effect concentration (EC50) of a particular receptor for a particular ligand. EC50 refers to the concentration of the ligand that induces an intermediate response between baseline and maximum after a certain exposure time. In some embodiments, the response is the opening and closing of ion channels in the receptor.

[0085] As used herein, “substantially retaining potency” for a ligand means an engineered receptor having an EC50 for a particular ligand that remains unchanged or changes and increases / decreases by less than twofold compared to the parent or control receptor.

[0086] As used herein, the “efficacy” of a ligand-related receptor refers to a measure of the receptor’s activity in the presence of the ligand. In some embodiments, efficacy refers to the amount of current that passes through the receptor under specific conditions, for example, in the presence of a specific concentration of the ligand. In some embodiments, determining efficacy includes determining the amount of current that has passed through the receptor and / or the receptor’s base current. C. Manipulated receptors

[0087] This disclosure relates to engineered receptors, engineered receptor variants, and methods of use thereof. As used herein, the term “receptor” refers to any protein located on the surface of a cell that can mediate signaling to and from the cell. The term “engineered receptor” is used herein to refer to a receptor that has been experimentally modified to be physically and / or functionally different from the corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. The term “wild-type receptor” is used herein to refer to a receptor having a polypeptide sequence identical to the polypeptide sequence of a protein found in nature. Wild-type receptors include receptors naturally occurring in humans, as well as orthologues naturally occurring in other eukaryotes, e.g., protists, fungi, plants, or animals, e.g., yeast, insects, nematodes, sponges, mammals, and non-mammalian vertebrates. In some embodiments, the parent receptor is a non-native receptor, i.e., a receptor that does not exist in nature, e.g., an engineered receptor derived from a wild-type receptor. For example, the parent receptor may be an engineered receptor comprising one or more subunits derived from one wild-type receptor and one or more subunits derived from a second wild-type receptor. Therefore, the resulting protein consists of two or more subunits derived from the wild-type receptor. Thus, in some embodiments, the parent receptor is a chimeric receptor. Manipulated receptors in this disclosure include, for example, parent receptor mutants and switch receptors.

[0088] In some embodiments, the engineered receptor of the Disclosure comprises at least one amino acid mutation with respect to the corresponding parental receptor, e.g., one or more mutations in one or more domains of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptors share about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity with the corresponding parental receptor, including all values ​​and subranges between them. In some embodiments, the parental receptor variants have 85% or more sequence identity with the corresponding parental receptor, e.g., 90% or more, or 95% or more sequence identity, e.g., about 96%, about 97%, about 98%, or about 99% sequence identity with the corresponding parental receptor, including all values ​​and subranges between them. In some embodiments, the engineered receptor (e.g., parental receptor variant) is generated by error-prone PCR.

[0089] In some embodiments, the ligand-binding domain (LBD) of the engineered receptor in this disclosure contains at least one amino acid mutation with respect to the corresponding ligand-binding domain of the parent receptor, for example, one or more mutations in the ligand-binding domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand-binding domain of the engineered receptor has sequence identity of 85% or more, 90% or more, or 95% or more, including all values ​​and subranges present between them, with respect to the corresponding ligand-binding domain of the parent receptor, for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with respect to the corresponding ligand-binding domain of the parent receptor. In some embodiments, the ligand-binding domain of the engineered receptor shares 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%, including all values ​​and subranges present between them, with respect to the corresponding ligand-binding domain of the parent receptor.

[0090] In some embodiments, the ionpore domain (IPD) of the engineered receptor of the Disclosure includes at least one amino acid mutation with respect to the corresponding ionpore domain of the parent receptor, for example, one or more mutations in the ionpore domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ionpore domain of the engineered receptor has 85% or greater sequence identity with respect to the corresponding ionpore domain of the parent receptor, 90% or greater, or 95% or greater sequence identity, for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with respect to the corresponding ionpore domain of the parent receptor, including all values ​​and subranges present between them. In some embodiments, the ionpore domains of the manipulated receptor share sequence identity with the corresponding ionpore domain of the parent receptor of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, including all values ​​and subranges present between them.

[0091] 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 disable the function of the engineered receptor compared to the parent receptor, for example, by reducing the binding of endogenous ligands to the engineered receptor compared to the binding of endogenous ligands to the parent receptor, or by reducing the activity of downstream signaling pathways of the engineered receptor that are typically activated in response to ligand binding to the corresponding parent receptor. In some embodiments, the mutation is an amino acid substitution.

[0092] In some embodiments, the amino acid mutation is a gain-of-function amino acid mutation relative to the corresponding parent receptor. A “gain-of-function” amino acid mutation refers to one or more mutations that modify the function of the engineered receptor compared to the parent receptor, for example, by altering or enhancing the affinity of the engineered receptor to a ligand compared to the binding of an endogenous ligand to the parent receptor, or by altering or enhancing the activity of a signaling pathway that is activated in response to the binding of a ligand to the engineered receptor compared to the binding of an endogenous ligand to the corresponding parent receptor. In some embodiments, the gain-of-function mutation results in an increased affinity of the engineered receptor to a ligand. In certain embodiments, the gain-of-function mutation results in an increased affinity of the engineered receptor to an agonist ligand. In some embodiments, the gain-of-function mutation results in an antagonist ligand that acts as an agonist ligand when bound to the engineered receptor (e.g., resulting in activation of the agonist signaling pathway instead of the antagonist signaling pathway). In some embodiments, the gain-of-function mutation results in a modulator ligand that acts as an agonist ligand when bound to the engineered receptor. In some embodiments, the mutation is an amino acid substitution.

[0093] In some embodiments, the engineered receptor subject to this disclosure, or its ligand-binding domain and / or ionpore domain, contains one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations compared to the corresponding parent receptor. In some embodiments, the mutations are amino acid substitutions.

[0094] In some embodiments, the loss-of-function mutation and the gain-of-function mutation are located at the same residue, i.e., they are the same mutation. In other embodiments, the loss-of-function mutation and the gain-of-function mutation are mutations at different amino acid residues. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptor of a subject (or its ligand-binding domain and / or ion-pore domain) containing the loss-of-function mutation and / or gain-of-function mutation shares approximately 99%, approximately 98%, approximately 95%, approximately 90%, approximately 85%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, or less sequence identity with the corresponding parent receptor, e.g., the wild-type receptor or a non-native receptor (or its ligand-binding domain and / or ion-pore domain), including all and partial ranges between them. In some embodiments, the manipulated receptor of interest (or its ligand-binding domain and / or ionpore domain) shares 85% or more sequence identity with the corresponding parent receptor (or its ligand-binding domain and / or ionpore domain), including all values ​​and subranges between them, e.g., 85%, 90%, or 95% or more sequence identity, and in some examples, 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity.

[0095] In some embodiments, the engineered receptors of the Disclosure include receptors produced by a combination of one or more amino acid sequences, e.g., subunits, derived from one wild-type receptor and one or more amino acid sequences, e.g., subunits, derived from a second wild-type receptor. In other words, the engineered receptors include amino acid sequences that are heterogeneous, meaning that they do not exist together in nature. Such receptors are referred to herein as “chimeric receptors.” In some embodiments, the chimeric receptors act as the parent receptors from which the engineered receptors of the Disclosure are produced.

[0096] In some embodiments, the parent receptor mutant exhibits increased affinity for the agonist ligand. In some embodiments, a ligand that functions as an antagonist or modulator when bound to the wild-type receptor functions as an agonist when bound to the parent receptor mutant.

[0097] In some embodiments, the manipulated receptor is a "ligand-dependent ion channel," or LGIC. LGICs refer to a large group of transmembrane proteins that allow ions to pass through when activated by a specific ligand. An LGIC consists of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Ligand binding to an LGIC results in its activation and the opening of the ion pore. Ligand binding causes a dramatic change in the channel's permeability to one or more specific ions; effectively, when the channel is inactive or closed, ions cannot pass through, but when the ligand is bound, up to 10 7 Ions / second can pass through. In some embodiments, LGIC responds to extracellular ligands (e.g., neurotransmitters) and facilitates the influx of ions into the cytosol. In some embodiments, LGIC responds to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and facilitates the efflux of ions from the cytosol to the extracellular environment. Importantly, activation of LGIC facilitates the passage of ions across the cell membrane (e.g., Ca 2+ na + , K + Cl - It leads to the transport of (etc.) but not the transport of the ligand itself.

[0098] LGIC receptors are composed of multiple subunits and may be either homomeric or heteromeric receptors. Homomeric receptors are composed of all subunits of the same type. Heteromeric receptors are composed of at least one subunit that is different from at least one other subunit that is present within the receptor. For example, the glycine receptor is composed of five subunits of two types: an α subunit of which four isoforms (α1-α4) exist, and a β subunit of which one known isoform exists. An exemplary homomeric GlyR is GlyR, which is composed of five α1-GlyR subunits. Similarly, homomeric GABA A The receptor is β3-GABA A The receptor can be composed of subunits, and the nAchR receptor can be composed of the α7-nAchR subunit. An exemplary heteromer GlyR can be composed of one or more α subunits and one or more β subunits (e.g., α1β-GlyR). Examples of LGIC receptor subunits are shown in Table 1. [Table 1-1] [Table 1-2]

[0099] Examples of the LGIC family suitable for use in specific embodiments include, but are not limited to, Cys-loop receptors such as glycine receptors (GlyR), serotonin receptors (e.g., 5-HT3 receptor), λ-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchR), as well as acid-sensing (proton-dependent) ion channels (ASIC), epithelial sodium channels (ENaC), ion channel-type glutamate receptors, IP3 receptors, P2X receptors, ryanodine receptors, and zinc-activated channels (ZAC).

[0100] 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, GR Examples include IA4, GRID1, GRID2, GRIK1, GRIK2, GRIK3, GRIK4, GRIK5, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, ITPR1, ITPR2, ITPR3, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG, CHRND, CHRNE, P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, RYR1, RYR2, RYR3, and ZACN.

[0101] TRPV1, TRPM8, and P2X2 are members of a larger LGIC family that share structural characteristics and gating principles. For example, like TRPV1, TRPV4 is heat-induced but not by capsaicin, and P2X3 is ATP-induced but desensitizes more rapidly than P2X2. Therefore, TRPV1, TRPM8, and P2X2 are non-limiting examples of LGICs suitable for use in specific embodiments.

[0102] In one embodiment, the manipulated receptor is the TRPV1 or TRPM8 receptor or its mutaine. TRPV1 and TRPM8 are vanilloid and menthol receptors expressed by nociceptive neurons in the peripheral nervous system. Both channels are thought to function as non-selective sodium-permeable and calcium-permeable homotetramers. Furthermore, the channels and their major agonists (capsaicin and cooling compounds, e.g., menthol, respectively) are substantially absent in the central nervous system. Capsaicin, as well as several cooling compounds including menthol and icillin, contain potential acceptor sites for photosensitive blocking groups. The association of a photosensitive blocking group with such an acceptor results in a ligand-gated ion channel in which light acts as an indirect trigger by releasing an active ligand.

[0103] In one embodiment, the engineered receptor is the P2X2 receptor or its mutaine. P2X2 is an ATP-dependent non-selective cation channel distinguished by its slow desensitization rate. P2X2 can be used as a selectively addressable source of depolarization current and can provide a platform for generating engineered channel-ligand combinations that completely lack native agonists.

[0104] Non-limiting examples of wild-type LGIC receptor sequences found to be used in the generation of the engineered receptors of this disclosure include the following: In the sequence, the signal peptide is italicized, the ligand-binding domain is bold, and the ionpore domain is underlined.

[0105] In some embodiments, the wild-type LGIC receptor is the human α1-glycine receptor (GlyRα1) (GenBank accession number NP_001139512.1, SEQ ID NO: 2) encoded by the GLRA1 gene (GenBank accession number NM_001146040.1 (SEQ ID NO: 1)). [ka] [ka]

[0106] In some embodiments, the wild-type LGIC receptor is the human α2-glycine receptor (GlyRα2) (GenBank accession number NP_001112357.1, SEQ ID NO. 83) encoded by the GLRA2 gene (GenBank accession number NM_001118885.1, SEQ ID NO. 82). [ka]

[0107] In some embodiments, the wild-type LGIC receptor is the human α3 glycine receptor (GlyRα3) isoform L (GenBank accession number NP_006520.2, SEQ ID NO: 85) encoded by the GLRA3 gene (GenBank accession number NM_006529.3, SEQ ID NO: 84). [ka] [ka]

[0108] In some embodiments, the wild-type LGIC receptor is the human α3 glycine receptor (GlyRα3) isoform K (GenBank accession number NP_001036008.1, SEQ ID NO: 87) encoded by the GLRA3 gene (GenBank accession number NM_001042543.3, SEQ ID NO: 86). [ka]

[0109] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor α7 subunit (α7-nAchR) (GenBank accession number NP_000737.1, SEQ ID NO: 4) encoded by the CHRNA7 gene (GenBank accession number NM_000746.5 (SEQ ID NO: 3)). [ka] [ka]

[0110] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3A (5HT3A, GenBank accession number NP_998786.2, SEQ ID NO: 6) encoded by the HTR3A gene (GenBank accession number NM_213621.3, SEQ ID NO: 5). (a) [ka]

[0111] In some embodiments, the wild-type LGIC receptor is the human 5-hydroxytryptamine receptor 3B (5HT3B, GenBank accession number NP_006019.1, SEQ ID NO: 57) encoded by the HTR3B gene (GenBank accession number NM_006028.4, SEQ ID NO: 56). (a) [ka]

[0112] In some embodiments, the wild-type LGIC receptor is the human γ-aminobutyric acid receptor A (GABA-A), subunit β-3 (GABA-Aβ3) (GenBank accession number NP_000805.1, SEQ ID NO: 8), encoded by the GABRB3 gene (GenBank accession number NM_000814.5, SEQ ID NO: 7). (a) [ka]

[0113] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit ρ1(ρ1)(GABA-Aρ1)(GenBank accession number NP_002033.2, SEQ ID NO: 10), encoded by the GABRR1 gene (GenBank accession number NM_002042.4, SEQ ID NO: 9). (a) [ka]

[0114] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit ρ2(ρ2)(GABA-Aρ2)(GenBank accession number NP_002034.3, SEQ ID NO: 12), encoded by the GABRR2 gene (GenBank accession number NM_002043.4, SEQ ID NO: 11). (a) [ka] [ka]

[0115] In some embodiments, the wild-type LGIC receptor is human GABA-A, subunit ρ3(ρ3)(GABA-Aρ3)(GenBank accession number NP_001099050.1, SEQ ID NO: 14), encoded by the GABRR3 gene (GenBank accession number NM_001105580.2, SEQ ID NO: 13). (a) [ka]

[0116] In some embodiments, the manipulated receptor is a chimeric LGIC receptor. In some embodiments, the chimeric receptor includes a ligand-binding domain sequence derived from at least a first LGIC and an ion-pore conduction domain sequence derived from at least a second LGIC, or more simply, an "ion-pore domain sequence". In some embodiments, the derived amino acid sequence is identical to the corresponding region of the LGIC from which it originates. In some embodiments, the derived amino acid sequence may include a change at least one amino acid position compared to the corresponding region of the LGIC from which it originates. In some embodiments, the amino acid sequence derived from the LGIC sequence differs from the corresponding region of the original amino acid sequence by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the derived amino acid sequence has sequence identity of 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 and partial ranges in between) with the corresponding region of the LGIC amino acid sequence.

[0117] In some embodiments, the first and second LGICs are Cys-loop receptors. The ligand-binding domain sequences and ionpore domain sequences of Cys-loop receptors are publicly known in the art and can be readily identified from the literature 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 ionpore 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 ionpore domain of the second LGIC. In the sequence shown above, the ligand-binding domain is in bold, and the ionpore domain is underlined.

[0118] In some embodiments, the ligand-binding domain of the chimeric receptor is derived from the ligand-binding domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO: 2). In some embodiments, the ligand-binding domain includes 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 approximately 29-235 amino acids of GlyRα1, for example, amino acids 29-235, 29-240, 29-246, 29-248, 29-250, or 29-252 of SEQ ID NO: 2. In certain such embodiments, the ligand-binding domain essentially consists of amino acids 29-235 of SEQ ID NO: 2, amino acids 29-240 of SEQ ID NO: 2, amino acids 29-246 of SEQ ID NO: 2, amino acids 29-248 of SEQ ID NO: 2, amino acids 29-250 of SEQ ID NO: 2, and amino acids 29-252 of SEQ ID NO: 2. In some embodiments, the ionpore domain sequence is derived from a Cys-loop receptor other than human GlyRα1.

[0119] In some embodiments, the ligand-binding domain of the chimeric receptor includes the ligand-binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is human α7-nAChR. In some embodiments, the ligand-binding domain includes approximately amino acids 23-220 of human α7-nAChR (SEQ ID NO: 4), for example, amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of SEQ ID NO: 4. In some embodiments, the ligand-binding domain essentially consists of amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of SEQ ID NO: 4. In some embodiments, the ionpore domain sequence is derived from a Cys-loop receptor other than human α7-nAChR.

[0120] 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 human α7-nAChR. In some embodiments, the ligand-binding domain includes 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 amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of human α7-nAChR (SEQ ID NO: 4). In some embodiments, the ionpore domain sequence is derived from a Cys-loop receptor other than human α7-nAChR.

[0121] In some embodiments, the ligand-binding domain of the chimeric receptor is derived from the ligand-binding domain sequence of the human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ligand-binding domain includes approximately amino acids 23-247 of 5HT3A (SEQ ID NO: 6), e.g., amino acids 23-240, 30-245, 23-247, 23-250, and in some examples, amino acids 30-255 of SEQ ID NO: 6. In certain embodiments, the ligand-binding domain is essentially composed of amino acids 23-240 of SEQ ID NO: 6, essentially composed of amino acids 23-245 of SEQ ID NO: 6, essentially composed of amino acids 30-247 of SEQ ID NO: 6, essentially composed of amino acids 23-250 of SEQ ID NO: 6, and essentially composed of amino acids 23-255 of SEQ ID NO: 6. In some such embodiments, the ligand-binding domain comprises approximately 21-239 amino acids of 5HT3B (SEQ ID NO: 57), for example, amino acids 21-232, 21-235, 21-240, 21-245, and in some examples, amino acids 21-247 of SEQ ID NO: 57. In certain embodiments, the ligand-binding domain is essentially composed of amino acids 21-239, 21-232, 21-235, 21-240, and 21-245 of SEQ ID NO: 57. In some embodiments, the ionpore domain sequence is derived from a Cys-loop receptor other than human 5-hydroxytryptamine receptor 3.

[0122] In some embodiments, the ligand-binding domain of the chimeric receptor is derived from the ligand-binding domain sequence of the human GABA receptor. In some embodiments, the human GABA receptor is human GABA-Aβ3. In some such embodiments, the ligand-binding domain includes approximately amino acids 26-245 of GABA-Aβ3 (SEQ ID NO: 8), for example, amino acids 26-240, 26-245, 26-248, 26-250, and in some examples, amino acids 26-255 of SEQ ID NO: 8. In certain such embodiments, the ligand-binding domain is essentially derived from amino acids 26-240, 26-245, 26-248, 26-250, or 26-255 of SEQ ID NO: 8. In some embodiments, the ionpore domain sequence is derived from a Cys-loop receptor other than the human GABA-A receptor.

[0123] In some embodiments, the ion pore domain to which the ligand-binding domain is fused conducts anions and includes, for example, an ion pore domain sequence of a human glycine receptor or a human serotonin receptor. In other embodiments, the ion-conducting pore domain to which the ligand-binding domain is fused conducts cations and includes, for example, an ion pore domain sequence of a human acetylcholine receptor or a human γ-aminobutyric acid receptor A.

[0124] In some embodiments, the ionpore domain of the manipulated receptor is derived from the ionpore domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ionpore domain includes 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 amino acids approximately 245–457 of GlyRα1 (SEQ ID NO: 2), for example, amino acids 240–457, 245–457, 248–457, 249–457, 250–457, 255–457, or 260–457 of SEQ ID NO: 2. In some embodiments, the ionpore domain is essentially composed of amino acids 245-457 of SEQ ID NO: 2, or essentially composed of amino acids 248-457 of SEQ ID NO: 2, or essentially composed of amino acids 249-457 of SEQ ID NO: 2, or essentially composed of amino acids 250-457 of SEQ ID NO: 2.

[0125] In some embodiments, the ionpore domain of the chimeric receptor includes the ionpore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ionpore domain of the chimeric receptor includes, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ionpore domain of the chimeric receptor includes, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ionpore domain of the chimeric receptor includes, essentially consists of, or comprises an amino acid sequence identical to the ionpore domain sequence of human GlyRα2 (SEQ ID NO: 83). In some embodiments, the ion pore domain sequence of human GlyRα2 contains, essentially consists of, or comprises amino acids 254-452 of SEQ ID NO: 83. In some embodiments, the ion pore domain sequence of human GlyRα2 contains, essentially consists of, or comprises amino acids 254-452 of SEQ ID NO: 83. In some embodiments, the ion pore domain sequence of human GlyRα2 contains, essentially consists of, or comprises amino acids 258-452 of SEQ ID NO: 83. In some embodiments, the ion pore domain sequence of human GlyRα2 contains, essentially consists of, or comprises amino acids 260-452 of SEQ ID NO: 83.

[0126] In some embodiments, the ionpore domain of the chimeric receptor contains the ionpore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence identical to the ionpore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 85). In some embodiments, the ionpore domain sequence of human GlyRα3 isoform L contains, essentially consists of, or comprises amino acids 253-464 of SEQ ID NO: 85. In some embodiments, the ionpore domain sequence of human GlyRα3 isoform L contains, essentially consists of, or comprises amino acids 257-464 of SEQ ID NO: 85. In some embodiments, the ionpore domain sequence of human GlyRα3 isoform L contains, essentially consists of, or comprises amino acids 259-464 of SEQ ID NO: 85.

[0127] In some embodiments, the ionpore domain of the chimeric receptor contains the ionpore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises the same amino acid sequence as the ionpore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 87). In some embodiments, the ionpore domain sequence of human GlyRα3 isoform K contains, essentially consists of, or comprises amino acids 253-449 of SEQ ID NO: 87. In some embodiments, the ionpore domain sequence of human GlyRα3 isoform K contains, essentially consists of, or comprises amino acids 257-449 of SEQ ID NO: 87. In some embodiments, the ionpore domain sequence of human GlyRα3 isoform K contains, essentially consists of, or comprises amino acids 259-449 of SEQ ID NO: 87.

[0128] In some embodiments, the ionpore domain is derived from the ionpore 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 ionpore domain includes 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 approximately 230-502 amino acids of the α7-nAChR (SEQ ID NO: 4), for example, amino acids 227-502, 230-502, 231-502, 232-502, or 235-502. In certain such embodiments, the ionpore domain is essentially composed of amino acids 227-502 of SEQ ID NO: 4, or essentially composed of amino acids 230-502 of SEQ ID NO: 4, or essentially composed of amino acids 231-502 of SEQ ID NO: 4, or essentially composed of amino acids 232-502 of SEQ ID NO: 4, or essentially composed of amino acids 235-502 of SEQ ID NO: 4.

[0129] In some embodiments, the ionpore domain is derived from the ionpore domain sequence of a human serotonin receptor. In some embodiments, the human serotonin receptor is human 5HT3A or 5HT3B. In some such embodiments, the ionpore domain includes 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 amino acids approximately 248–516 of 5HT3A (SEQ ID NO: 6), for example, amino acids 240–516, 245–516, 248–516, 250–516, or 255–516 of SEQ ID NO: 6. In certain such embodiments, the ionpore domain essentially consists of amino acids 240-516 of SEQ ID NO: 6, or essentially consists of amino acids 245-516 of SEQ ID NO: 6, or essentially consists of amino acids 248-516 of SEQ ID NO: 6, or essentially consists of amino acids 250-516 of SEQ ID NO: 6, or essentially consists of amino acids 253-516. In some embodiments, the ionpore domain includes 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 approximately 240-441 amino acids of 5HT3B (SEQ ID NO: 57), for example, amino acids 230-441, 235-441, 240-441, 245-441, or 250-441 of SEQ ID NO: 57. In certain such embodiments, the ionpore domain essentially consists of amino acids 230-441 of SEQ ID NO: 57, or essentially consists of amino acids 235-441 of SEQ ID NO: 57, or essentially consists of amino acids 240-441 of SEQ ID NO: 57, or essentially consists of amino acids 245-441 of SEQ ID NO: 57, or essentially consists of amino acids 250-441 of SEQ ID NO: 57.

[0130] In some embodiments, the ionpore domain is derived from the ionpore domain sequence of a human GABA receptor. In some embodiments, the human GABA receptor is human GABA-Aβ3. In some embodiments, the ionpore domain includes 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 approximately 246-473 amino acids of GABA-Aβ3 (SEQ ID NO: 8), for example, amino acids 240-473, 245-473, 247-473, 250-473, or 253-473 of SEQ ID NO: 8. In certain such embodiments, the ionpore domain essentially consists of amino acids 240-473, 245-473, 247-473, 250-473, or 253-473 of SEQ ID NO: 8.

[0131] In some embodiments, the ionpore domain of the chimeric receptor contains the ionpore domain sequence of human GABA-Aρ1 (GABRR1, SEQ ID NO: 10). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises the same amino acid sequence as the ionpore domain sequence of human GABA-Aρ1 (SEQ ID NO: 10). In some embodiments, the ion pore domain sequence of human GABA-Aρ1 contains, essentially consists of, or comprises amino acids 284-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-Aρ1 contains, essentially consists of, or comprises amino acids 288-479 of SEQ ID NO: 10. In some embodiments, the ion pore domain sequence of human GABA-Aρ1 contains, essentially consists of, or comprises amino acids 290-479 of SEQ ID NO: 10.

[0132] In some embodiments, the ionpore domain of the chimeric receptor contains the ionpore domain sequence of human GABA-Aρ2 (GABRR2, SEQ ID NO: 12). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises the same amino acid sequence as the ionpore domain sequence of human GABA-Aρ2 (SEQ ID NO: 12). In some embodiments, the ionpore domain sequence of human GABA-Aρ2 contains, essentially consists of, or comprises amino acids 265-466 of SEQ ID NO: 12. In some embodiments, the ionpore domain sequence of human GABA-Aρ2 contains, essentially consists of, or comprises amino acids 269-466 of SEQ ID NO: 12. In some embodiments, the ionpore domain sequence of human GABA-Aρ2 contains, essentially consists of, or comprises amino acids 271-466 of SEQ ID NO: 12.

[0133] In some embodiments, the ionpore domain of the chimeric receptor contains the ionpore domain sequence of human GABA-Aρ3 (GABRR3, SEQ ID NO: 14). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence derived from the ionpore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or 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%, or at least 99.5% sequence identity with the ionpore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ionpore domain of the chimeric receptor contains, essentially consists of, or comprises an amino acid sequence identical to the ionpore domain sequence of human GABA-Aρ3 (SEQ ID NO: 14). In some embodiments, the ion pore domain sequence of human GABA-Aρ3 contains, essentially consists of, or comprises amino acids 271-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-Aρ3 contains, essentially consists of, or comprises amino acids 275-468 of SEQ ID NO: 14. In some embodiments, the ion pore domain sequence of human GABA-Aρ3 contains, essentially consists of, or comprises amino acids 277-467 of SEQ ID NO: 14.

[0134] In some embodiments, the ionpore domain of the chimeric ligand-opening ion channel in question contains an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ionpore domain. “M2-M3 linker domain” or “M2-M3 linker” refers to the sequence within the ionpore domain of the LGIC where the C-terminus of the receptor transmembrane domain 2 (M2) is adjacent to its amino (N) terminus, and the N-terminus of the receptor transmembrane domain 3 (M3) is adjacent to its carboxyl (C) terminus. 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, if the ionpore domain of the chimeric receptor contains a heterologous M2-M3 linker, the M2-M3 linker originates from the same receptor as the ligand-binding domain of the chimeric receptor. For example, if the ligand-gated ion channel in question includes a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the ion pore domain sequence may include an M2-M3 linker sequence derived from AChR. In some embodiments, the ion pore domain is derived from GlyRα1, and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the native M2-M3 linker sequence extracted from the ion pore domain corresponds to approximately amino acids 293-313 of GlyRα1 (SEQ ID NO: 2), for example, 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 281-295 amino acids of α7-nAChR (SEQ ID NO: 4), for example amino acids 290-295, 281-290, 281-295, 283-295, 287-292, etc., or 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.

[0135] In some embodiments, the ligand-binding domain of the target chimeric ligand-opening ion channel includes a Cys-loop domain sequence heterogeneous to the Cys-loop sequence of the ligand-binding domain. “Cys-loop domain sequence” or “Cys-loop sequence” refers to a domain within the ligand-binding domain of a Cys-loop LGIC that forms a loop structure adjacent to cysteine ​​at the N-terminus and C-terminus. While we do not wish to be constrained by theory, it is thought that upon ligand binding to the ligand-binding domain, the Cys-loop structurally moves closer to the M2-M3 loop, and this movement mediates signaling in the ionpore domain via biophysical translation of ligand binding in the extracellular domain (as outlined in Miller and Smart, Trends in Pharmacological Sci 2009:31(4)). By substituting an endogenous Cys-loop sequence with a heterologous Cys-loop sequence, the conductivity of LGIC can be increased by 1.5 times or more, for example, at least 2, 3, or 4 times, at least 5 or 6 times in some cases, and at least 7, 8, 9, or 10 times at certain doses. The Cys-loop domain of a Cys-loop receptor can be readily determined from the art and / or by using any publicly available protein analysis tool, e.g., Expasy, uniProt, etc. Typically, if the ligand-binding domain of a chimeric receptor contains 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, if the chimeric ligand-opening ion channel of interest contains a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the ligand-gated ion channel of interest may instead contain the ligand-binding domain sequence derived from AChR, except for the Cys-loop domain sequence derived from 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 extracted from the ligand-binding domain corresponds to approximately amino acids 150–164 of α7-nAChR (SEQ ID NO: 4), for example, amino acids 150–157 of α7-nAChR. In some embodiments, the inserted Cys-loop sequence is derived from approximately amino acids 166–180 of GlyRα1 (SEQ ID NO: 2), for example, amino acids 166–172 of GlyRα1, or from 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.

[0136] In some embodiments, the inserted Cys loop sequence is derived from approximately amino acids 172–186 of GlyRα2 (sequence number 83), for example amino acids 172–178 of GlyRα2, or from 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 approximately amino acids 171–185 of GlyRα3 (sequence number 85 or 87), for example amino acids 171–177 of GlyRα3, or from 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 approximately amino acids 198-212 of GABA-Aρ1 (SEQ ID NO: 10), for example amino acids 198-204 of GABA-Aρ1, or from 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-Aρ1. In some embodiments, the inserted Cys loop sequence is derived from approximately amino acids 178-192 of GABA-Aρ2 (SEQ ID NO: 12), for example amino acids 178-184 of GABA-Aρ2, or from 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-Aρ2. In some embodiments, the inserted Cys loop sequence is derived from amino acids 184-198 of GABA-Aρ3 (SEQ ID NO: 14), for example, amino acids 184-190 of GABA-Aρ3, or from 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.

[0137] In some embodiments, the ligand-binding domain of the target chimeric ligand-dependent ion channel includes a β1-2 loop domain sequence that is heterogeneous to the β1-2 loop domain sequence of the ligand-binding domain. “β1-2 loop domain sequence” or “β1-2 loop or β1-β2 loop” refers to the domain within the ligand-binding domain of the Cys-loop LGIC where the C-terminus of the β1 sheet is adjacent to its N-terminus and the N-terminus of the β2 sheet is adjacent to its C-terminus. While we do not wish to be constrained by theory, the β1-2 loop is thought to play a role in mediating the biophysical translation of ligand binding in the extracellular domain to the ionpore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). Upon ligand binding, the β1-2 loop, along with the Cys-loop, is thought to migrate to the M2-M3 loop, mediating the biophysical translation of ligand binding in the extracellular domain and signal transduction in the ionpore domain where the M2-M3 loop resides (as outlined by Miller and Smart above). By substituting the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence, the conductivity of LGIC can be increased by 1.5 times or more, for example, at least 2, 3, or 4 times, at least 5 or 6 times in some cases, and at least 7, 8, 9, or 10 times at certain doses. The β1-2 loop of the Cys-loop receptor can be readily determined from the art and / or by using any publicly available protein analysis tool, e.g., Expasy, uniProt, etc. Typically, if the ligand-binding domain of the chimeric receptor contains 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, if the chimeric ligand-gated ion channel of interest contains a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the sequence of the β1-2 loop domain of the ligand-binding domain may be derived from GlyR. In some embodiments, the ligand-binding domain is derived from α7-nAChR.In some embodiments, the β1-2 loop sequence extracted from the ligand-binding domain corresponds to approximately amino acids 64-72 or 67-70 of α7-nAChR (SEQ ID NO: 4), for example, amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the inserted β1-2 loop sequence corresponds to approximately amino acids 79-85 of GlyRα1 (SEQ ID NO: 2), for example, amino acids 80-85, 81-84, 79-85, or 81-84 of GlyRα1, having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the ionpore domain is derived from GlyRα2, and the inserted β1-2 loop corresponds to approximately amino acids 86-91 of GlyRα2 (SEQ ID NO: 83), having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the ionpore domain is derived from GlyRα3, and the inserted β1-2 loop corresponds to approximately amino acids 85-90 of GlyRα3 (SEQ ID NO: 85 or 87) having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the ionpore domain is derived from GABA-Aρ1, and the inserted β1-2 loop corresponds to approximately amino acids 112-117 of GABA-Aρ1 (SEQ ID NO: 10) having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the ionpore domain is derived from GABA-Aρ2, and the inserted β1-2 loop corresponds to approximately amino acids 92-97 of GABA-Aρ2 (SEQ ID NO: 12) having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the ionpore 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), having up to 3, 2, or 1 amino acid mutations, or having no amino acid mutations. In some embodiments, the mutations are amino acid substitutions.

[0138] In some embodiments, the Disclosure provides a chimeric LGIC receptor comprising a ligand-binding domain derived from human α7-nAChR, comprising a ligand-binding domain comprising one or more amino acid substitutions of the Disclosure, and an ionpore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or 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.

[0139] In some embodiments, the Disclosure provides a chimeric LGIC receptor comprising a ligand-binding domain derived from human α7-nAChR, comprising a ligand-binding domain comprising one or more amino acid substitutions of the Disclosure, and an ionpore domain derived from a human GABA receptor. 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-loop domain derived from a human GABA receptor. In some embodiments, the ligand-binding domain comprises a β1-2 loop domain derived from a human GABA receptor.

[0140] Non-limiting examples of the sequences of the chimeric LGIC receptors of this disclosure include the sequences disclosed herein as SEQ ID NOs. 15 to 52. In some embodiments, the chimeric LGIC receptor, or the polynucleotide encoding it, has 85% or more sequence identity with the sequences provided in SEQ ID NOs. 15 to 52 herein, for example, 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 with the sequences provided in SEQ ID NOs. 15 to 52. In the sequences, the signal peptide is italicized, the ligand-binding domain is bold, and the ionpore domain is underlined.

[0141] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 junction) that includes a human α7-nAChR signal peptide (italicized) and ligand-binding domain (boldface) fused to a human GlyRα1 ion pore domain (underlined).

Chemical formula

[0142] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (R228 junction) chimera that includes a human α7-nAChR signal peptide (italicized) and ligand-binding domain (boldface) fused to a human GlyRα1 ion pore domain (underlined).

Chemical formula

[0143] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (V224 junction) chimera that includes a human α7-nAChR signal peptide (italicized) and ligand-binding domain (boldface) fused to a human GlyRα1 ion pore domain (underlined).

Chemical formula

[0144] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 (Y233 junction) chimera that includes a human α7-nAChR signal peptide (italicized) and ligand-binding domain (boldface) fused to a human GlyRα1 ion pore domain (underlined).

Chemical formula

Chemical formula

[0145] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 junction) containing a human α7-nAChR signal peptide (italicized) and a ligand-binding domain (bold), fused to a human GlyRα1 ionpore domain (underlined) containing α7-nAChR M2-M3 (lowercase). (a) [ka] (b) [ka] (c) [ka] [ka] (d) [ka] (e) [ka] (f) [ka]

[0146] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a ligand-binding domain (bold) containing a human α7-nAChR signal peptide (italicized) fused to a human GlyRα1 ionpore domain (underlined), and a GlyRα1 Cys-loop sequence (lowercase). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with SEQ ID NO: 33. [ka] (a)

Chem.

[0147] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7 - nAChR signal peptide (italicized), a ligand - binding domain (bold) comprising a GlyRα1 β1 - 2 loop sequence (lower case) and fused to a human GlyRα1 ion - pore domain (underlined). (a)

Chem.

Chem.

[0148] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7 - nAChR signal peptide (italicized), a ligand - binding domain (bold) comprising a GlyRα1 β1 - 2 loop sequence (lower case) and a Cys - loop sequence (lower case) and fused to a human GlyRα1 ion - pore domain (underlined). (a)

Chem.

Chem.

Chem.

Chem.

Chem.

[0149] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italicized) fused to a human GlyRα1 ionpore domain (underlined) containing a human α7-nAChR M2-M3 linker (lowercase), and a ligand-binding domain (bold) containing a GlyRα1 β1-2 loop sequence (lowercase). (a) [ka]

[0150] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera comprising a human α7-nAChR signal peptide (italicized) fused to a human GlyRα1 ionpore domain (underlined) containing a human α7-nAChR M2-M3 linker (lowercase), and a ligand-binding domain (bold) containing a GlyRα1 Cys-loop sequence (lowercase). (a) [ka] [ka]

[0151] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (R241 junction) containing a human 5HT3A serotonin receptor signal peptide (italicized) and a ligand-binding domain (bold) fused to a human GlyRα1 ion pore domain (underlined). (a) [ka]

[0152] In some embodiments, the chimeric LGIC receptor is an HTR3A / GLRA1 chimera (V236 junction) containing a human 5HT3A serotonin receptor signal peptide (italicized) and a ligand-binding domain (bold) fused to a human GlyRα1 ion pore domain (underlined). (a) [ka]

[0153] In some embodiments, the chimeric LGIC receptor is a GABRB3 / GLRA1 chimera (Y245 junction) containing a human GABA-A β3 signaling peptide (italicized) and a ligand-binding domain (bold) fused to a human GlyRα1 ion pore domain (underlined). (a) [ka] C1. Amino acid mutation

[0154] As discussed above, in some embodiments, the engineered receptor of interest contains at least one amino acid mutation that alters the ligand's potency to the engineered receptor compared to its potency to the unmutated parent receptor. In other words, one or more amino acid mutations, such as loss-of-function or gain-of-function mutations, shift the potency of the engineered receptor to the ligand compared 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 located in the ligand-binding domain of the engineered receptor. In some embodiments, as is the case when the ligand-binding domain of the manipulated receptor is a Cys-loop receptor protein, one or more amino acid mutations are substitutions of residues corresponding to residues of α7-nAChR (SEQ ID NO: 4) selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213, and Y217. In some embodiments, one residue is substituted. In some embodiments, two, three, four, or five or more residues are substituted, for example, six, seven, eight, nine, or ten residues are substituted. In certain embodiments, the residues correspond to residues 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 present within the α7-nAChR sequence.

[0155] In some embodiments, one or more substitutions reduce the potency of the manipulated receptor against acetylcholine and non-native ligands by, for example, two times or more, three times or more, four times or more, five times or more, ten times or more, twenty times or more, thirty times or more, fifty times or more, or 100 times. 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 the α7-nAChR. In other examples, one or more substitutions selectively reduce the potency of acetylcholine on the manipulated receptor. In other words, one or more substitutions reduce the efficacy of the engineered receptor against acetylcholine while essentially maintaining efficacy against non-native ligands, or otherwise reducing the efficacy of the engineered receptor against acetylcholine by two or more times than reducing the efficacy of the engineered receptor against non-native ligands, for example, three, four, five or more times, and in some cases, ten, twenty, fifty or 100 or more times. In some embodiments, the substitutions correspond to L131E, L131S, L131T, L131D, or S172D of α7-nAChR. In yet other embodiments, one or more substitutions selectively reduce the efficacy of non-native ligands against the engineered receptor.In other words, one or more substitutions reduce the efficacy of the engineered receptor against non-native ligands while essentially maintaining efficacy against acetylcholine, or otherwise reducing the efficacy of the engineered receptor against non-native ligands by a factor of two or more, e.g., three, five or more, and in some cases, ten, twenty or fifty or more, than reducing the efficacy of the engineered receptor against acetylcholine. In some embodiments, the substitutions correspond to W77M, Y115W, S172T, or S172C of the α7-nAChR. In certain embodiments, one or more substitutions are located within the α7-nAChR sequence. In certain embodiments, the non-native ligands are selected from AZD-0328, TC6987, ABT-126, and fasciniclin / RG3487.

[0156] In other embodiments, one or more substitutions increase the potency of the manipulated receptor against acetylcholine and / or non-native ligands by, for example, 2x or more, 3x or more, 4x or more, 5x or more, 10x or more, 20x or more, 30x or more, 50x or more, or 100x. 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 potency of both acetylcholine and non-native ligands. In some embodiments, the substitutions correspond to L131N, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q, or S188T of α7-nAChR. In other examples, one or more substitutions selectively increase the potency of acetylcholine to the engineered receptor. In other words, one or more substitutions increase the potency of the engineered receptor to acetylcholine by two or more times, e.g., three, four, or five times, or more, in some examples, ten, twenty, fifty, or 100 times, compared to the increase in the potency of the engineered receptor to the non-native ligand. In some embodiments, the substitutions correspond to L141W, S172T, S172C, S188P, or S188W of the α7-nAChR. In certain embodiments, one or more substitutions are located within the α7-nAChR sequence. In certain embodiments, the non-native ligand is selected from AZD-0328, TC6987, ABT-126, and fasciniclin / RG3487. In yet another example, one or more substitutions selectively increase the potency of the non-native ligand against the manipulated receptor.In other words, one or more substitutions increase the potency of the engineered receptor against its non-native ligand by two or more times, e.g., three times, five times or more, and in some cases, ten times, twenty times, or fifty times or more, compared to increasing the potency of the engineered receptor against acetylcholine.

[0157] In some embodiments, the mutated amino acid residue in the engineered receptor of interest 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 mutated amino acid residue in the engineered receptor of interest 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 substitutions are not those 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 wild-type α7nAChR. In some embodiments, the substitutions are those 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 wild-type α7nAChR. In some embodiments, if such substitutions are present in the manipulated receptor, they are present in combination with one or more amino acid mutations of the amino acid mutations described herein.

[0158] In some embodiments, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 4) mediate the binding of the native ligand acetylcholine. In some embodiments, mutations in these residues may reduce acetylcholine binding and may therefore be 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 may mediate the binding of the non-native ligand AZD0328 to this receptor, and mutations in these residues may increase the affinity of AZD0328 and / or other ligands to this receptor and may therefore be considered gain-of-function mutations. In some embodiments, the manipulated receptor in question comprises a mutation in one or more amino acid residues of the ligand-binding domain region of α7-nAChR (SEQ ID NO: 4), or the ligand-binding domain of a chimeric receptor containing the ligand-binding domain region of α7-nAChR, where 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, a mutation in one or more amino acid residues of the ligand-binding domain region of α7-nAChR (SEQ ID NO: 4), or the ligand-binding domain of a chimeric receptor including the ligand-binding domain region of α7-nAChR, is a substitution of 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.

[0159] In some embodiments, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 4) may mediate the binding of acetylcholine and / or nicotine, and one or more mutations in these residues may reduce the binding of acetylcholine and / or nicotine. In some embodiments, R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR may mediate the binding of the non-natural ligand ABT126, and one or more mutations in these residues may increase the affinity of ABT126 and / or other ligands to α7-nAChR. In some embodiments, the mutations are amino acid substitutions. In some embodiments, R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of α7-nAChR may mediate the binding of the non-natural ligand TC6987, and one or more mutations in these residues may increase the affinity of TC6987 and / or other ligands to α7-nAChR. In some embodiments, R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of α7-nAChR may mediate the binding of the non-natural ligand fascinicrin / RG3487, and one or more mutations in these residues may increase the affinity of fascinicrin / RG3487 and / or other ligands to α7-nAChR. In some embodiments, the manipulated receptor in question comprises a mutation in one or more amino acid residues of the ligand-binding domain region of α7-nAChR, or the ligand-binding domain of a chimeric receptor including the ligand-binding domain region of α7-nAChR, where 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, one or more amino acid residues alter the binding of acetylcholine and / or nicotine to α7-nAChR, and the amino acids are selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR. In certain such embodiments, the amino acids are selected from C212 and S170. In some embodiments, mutations of one or more amino acid residues alter the binding of ABT126 to α7-nAChR, and 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 acids are 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, and one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of α7-nAChR. In certain such embodiments, the amino acids are selected from R101, T128, N129, Y210, and C213. In some embodiments, mutations in one or more amino acid residues alter the binding of fascinicrin / RG3487 to α7-nAChR, and 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 acids are selected from Y210, R101, and N129.

[0160] In some embodiments, the residues W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3 (SEQ ID NO: 6) may mediate serotonin binding, and one or more mutations in these residues may reduce serotonin binding to 5HT3. The residues D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3 may mediate the binding of the non-natural ligand silancetron, and one or more mutations in these residues may increase the affinity of silancetron and / or other ligands to 5HT3. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the manipulated receptor in question has a mutation in one or more amino acid residues of the ligand-binding domain of a chimeric receptor including the ligand-binding domain region 5HT3A, or the ligand-binding domain region of 5HT3, the one or more amino acid residues being selected from the group consisting of D64, I66, W85, R87, Y89, N123, Y136, Y138, G146, N147, Y148, K149, T176, S177, S178, L179, W190, R191, F221, E224, Y228, Y229, and E231. In some embodiments, mutations in one or more amino acid residues alter the binding of serotonin to 5HT3, and the amino acids are selected from the group consisting of W85, R87, Y136, Y138, G146, N147, Y148, K149, S177, S178, L179, Y228, and Y229 of 5HT3A. In certain such embodiments, the amino acids are selected from Y136, Y138, N147, K149, and L179. In some embodiments, mutations in one or more amino acid residues alter the binding of silancetron to 5HT3, and the one or more amino acid residues are selected from the group consisting of D64, I66, W85, R87, Y89, N123, G146, Y148, T176, S177, S178, W190, R191, F221, E224, Y228, Y229, and E231 of 5HT3A.In certain such embodiments, the amino acids are selected from D64, I66, Y89, N123, T176, W190, R191, F221, E224, and E231.

[0161] In some embodiments, one or more mutations affecting the ability of a ligand to modulate LGIC activity are located in the ionpore domain of 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 a ligand modulates the channel's activity, and consequently, a mutation of this residue to, for example, serine (T279S) converts its effect from antagonistic (i.e., reducing LGIC activity) to agonistic (i.e., promoting channel activity). In some embodiments, the ligand-dependent ion channel in question involves a mutation in one or more amino acid residues in the ionpore domain of human 5HT3A (SEQ ID NO: 6), or the ionpore domain of a chimeric LGIC receptor containing the ionpore domain of 5HT3A, where the substitution is at the amino acid corresponding to 279 in SEQ ID NO: 6. In certain embodiments, the substitution is a T279S substitution for SEQ ID NO: 6.

[0162] This 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 includes a ligand-binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR). In some embodiments, the parent receptor is a chimeric receptor. In some embodiments, the parent receptor includes an ionpore domain derived from the human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3. In some embodiments, the ligand-binding domain of the engineered receptor includes a Cys-loop domain derived from the human glycine receptor. In some embodiments, the parent receptor includes the amino acid sequence of SEQ ID NO: 33. In some embodiments, the engineered receptor includes two amino acid substitutions compared to the parent receptor containing the amino acid sequence of SEQ ID NO: 33. In some embodiments, the ligand-binding domain of the engineered receptor includes a β1-2 loop domain derived from the human glycine receptor α1 subunit.

[0163] In some embodiments, the ligand-binding domain of the manipulated receptor includes amino acid substitutions in two or more amino acid residues selected from those corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172, and Y210 of human α7-nAChR (SEQ ID NO: 4).

[0164] In some embodiments, the two amino acid substitutions are in pairs of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand-binding domain includes two amino acid substitutions in pairs of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand-binding domain includes an amino acid substitution at residue L131 and an amino acid substitution at S172D. In some embodiments, the ligand-binding domain includes an amino acid substitution at residue L131 and an amino acid substitution at Y115D. In some embodiments, the ligand-binding domain includes pairs of amino acid substitutions selected from the group consisting of L131S and S172D, L131T and S172D, L131D and S172D, Y115D and S170T, Y115D and L131Q, and Y115D and L131E. In some embodiments, the ligand-binding domain includes the L131E amino acid substitution.

[0165] In some embodiments, the ligand-binding domain includes one or more amino acid substitutions in an amino acid residue selected from the group consisting of Y140, R101, L131, Y115, and Y210, where the amino acid residue corresponds to an α7-nAChR amino acid residue. In some embodiments, the ligand-binding domain includes the amino acid substitution of R101W and / or Y210V. In some embodiments, the ligand-binding domain includes two or more amino acid substitutions in an amino acid residue selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand-binding domain includes two amino acid substitutions in an amino acid residue selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand-binding domain includes two amino acid substitutions in a pair of amino acid residues selected from the group consisting of R101 and L131, Y115 and Y210, and R101 and Y210. In some embodiments, the ligand-binding domain includes pairs 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 includes three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand-binding domain includes amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0166] In some embodiments, the ligand-binding domain includes an amino acid substitution at residue L131 and an amino acid substitution at R101F or R101M. In some embodiments, the amino acid substitution at residue L131 is L131G, L131D, L131A, L131F, or L131N.

[0167] In some embodiments, the ligand-binding domain includes a hydrophobic amino acid substitution at residue Y210 and an amino acid substitution at R101W or R101F. In some embodiments, the amino acid substitution at residue Y210 is Y210V, Y210F, or Y210W.

[0168] Those skilled in the art will readily recognize suitable control receptors for comparison with the engineered receptors of the present disclosure. In some embodiments, the control receptor is sequence-identical to the engineered receptor, except for one or more prominent amino acid mutations (e.g., substitutions). In all cases, reference to a control receptor means that the enumerated property changes (e.g., ligand potency) are the result of amino acid mutations in the engineered receptor of the present disclosure.

[0169] This disclosure provides an engineered receptor, which is a chimeric ligand-gated ion channel (LGIC) receptor comprising (a) a ligand-binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR) and including a Cys-loop domain derived from the human glycine receptor α1 subunit, and (b) an ionpore domain derived from the human glycine receptor α1 subunit. In some embodiments, the engineered receptor is derived from a parent engineered receptor comprising or derived from the amino acid sequence of SEQ ID NO: 33, and further comprises one or more amino acid substitutions based on the parent engineered receptor.

[0170] In some embodiments, the potency of the engineered receptor to acetylcholine is lower than that of the human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine. In some embodiments, the potency of the engineered receptor to acetylcholine is at least about 1.5 times lower than that of the human α7 nicotinic acetylcholine receptor (α7-nAChR) to acetylcholine (e.g., about 2 times lower, 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 values ​​and partial ranges that exist between them). In some embodiments, the potency of the engineered receptor to acetylcholine is evaluated by its EC50 to acetylcholine based on a cell reporter assay using YFP fluorescence quenching as described in Example 2 of this disclosure. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 100 μM, at least 200 μM, at least 300 μM, at least 500 μM, at least 700 μM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 1 mM. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 3 mM. In some embodiments, having a higher EC50 for acetylcholine allows for high levels of expression of the engineered receptor intracellularly or on the cell surface without passing a substantial amount of current through the cell in the presence of physiologically occurring concentrations of acetylcholine.

[0171] In some embodiments, the potency of the engineered receptor against non-natural ligands is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) against non-natural ligands. In some embodiments, the potency of the engineered receptor against non-natural ligands is higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) against non-natural ligands. In some embodiments, the potency of the engineered receptor against non-natural ligands is at least about 1.5 times higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) against non-natural ligands (e.g., about 2 times lower, 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). In some embodiments, determining efficacy involves determining EC50 based on a cell reporter assay using YFP fluorescence quenching as described in Example 2 of this disclosure. In some embodiments, the EC50 of the engineered receptor against a 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 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1 μM, less than 2 μM, less than 3 μM, less than 4 μM, less than 5 μM, less than 6 μM, less than 7 μM, less than 8 μM, less than 9 μM, or less than 10 μM. In some embodiments, the EC50 of the engineered receptor for a non-natural ligand is less than 10 nM. In some embodiments, the EC50 of the engineered receptor for a non-natural ligand is less than 100 nM. In some embodiments, the EC50 of the engineered receptor for a non-natural ligand is less than 1 uM.

[0172] In some embodiments, the efficacy of the engineered receptor in the presence of a non-natural ligand is higher than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) 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 about 1.5 times (e.g., about 2 times lower, 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 values ​​and partial ranges that exist between them) higher than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of a non-natural ligand. In some embodiments, determining efficacy involves determining the amount of current that passed through the engineered receptor in vitro in the presence of a non-natural ligand.

[0173] In some embodiments, the ligand-gated ion channel in question contains one or more non-desensitizing mutations. In some embodiments, the mutations are amino acid substitutions. When used in the context of ligand-gated ion channels, "desensitization" refers to a gradual decrease in ion flux in the prolonged presence of an agonist. This results in a gradual loss of the neuron's potency towards the ligand. Non-desensitizing mutations are amino acid mutations that prevent the LGIC from being desensitized to the ligand, thereby preventing the neuron from becoming underresponsive or unresponsive to the ligand. Non-desensitizing mutations can be readily identified by introducing an LGIC containing the mutation into a neuron and analyzing the current flux over time during prolonged exposure to the ligand. If the LGIC does not contain non-desensitizing mutations, the current returns from a peak to a steady state during prolonged exposure; however, if the LGIC contains non-desensitizing mutations, the current remains at a peak flux throughout the period of exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (V294L after proprotein post-treatment to remove the signal peptide) and the L321V mutation in human GABA-A receptor GABRB3 (L296V after proprotein post-treatment to remove the signal peptide). In some embodiments, the desensitization mutation is a substitution of amino acid residues in or near the C-terminus of LGIC with a sequence having 90% or more identity to a desensitization sequence, for example, IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53) derived from the C-terminus of the protein encoded by GABAR1, for example, a substitution of residues 455-479 in GABRR1 with IDRLSRIAFPLLFGIFNLVYWATYLNREPQL (SEQ ID NO: 53). LGIC desensitization, methods for measuring LGIC desensitization, and non-desensitizing mutations are publicly known in the art, 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 full disclosure of which is incorporated herein by reference.

[0174] In some embodiments, the ligand-gated ion channel in question contains one or more conversion mutations. In some embodiments, the mutations are amino acid substitutions. Conversion mutations mean mutations that alter the permeability of the ion pore domain of the LGIC to allow conductance of non-native ions, i.e., ions that cannot pass through naturally. In some cases, the mutation converts permeability from cation to anion, for example, by substituting amino acid residues 260-281 of human α7-nAChR(CHRNA7)(EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 54) or the corresponding amino acids of another cation-permeable LGIC with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 55). In some cases, mutations convert permeability from cationic to anionic, for example, by substituting amino acid residue 279 of GLRA1 or the corresponding amino acid of another anion-permeable LGIC with glutamic acid (E) (this converts the LGIC from anion-tolerant to calcium-tolerant as the A293E substitution in GLRA1), or by deleting amino acid residue 278 of GLRA1 or the corresponding amino acid of another anion-permeable LGIC and substituting amino acid 279 of GLRA1 or the corresponding amino acid of another anion-permeable LGIC with glutamic acid, or by substituting amino acid residue 293 of GLRA1 or the corresponding amino acid of another anion-permeable LGIC with valine (V) (this converts the LGIC from anion-tolerant to cation-tolerant as the P278Δ, A279E, T293V in GLRA1).

[0175] Further engineered receptors beyond those described herein can be readily identified by in vitro screening and validation methods. In some embodiments, a library of parental receptor variants is prepared from a limited number of parental receptors. The parental receptors can be mutated using methods known in the art, including error-prone PCR. In some embodiments, the library of parental receptor variants is then transfected into yeast or mammalian cells and screened in high throughput (e.g., to identify parental receptor variants that can signal in response to a ligand) to identify functional receptors. In some embodiments, the functional parental receptor variants identified in this primary screening are then expressed in mammalian cells and screened for ligand efficacy by, for example, a plate reader and / or electrophysiological assays described herein. Parental receptor variants that show increased binding affinity to agonist ligands or that show in secondary screening that an antagonist ligand or modulator ligand can be used as an agonist can then be selected and subjected to further in vitro and / or in vivo validation and characterization assays.Such screening assays are publicly known in the art, for example, 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; Elfson 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. C2. Exemplary Chimera LGIC

[0176] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to L131S and S172D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor that has no substitutions or only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-native ligand compared to a control receptor that has no such substitutions or only one of such substitutions. In some embodiments, the non-native ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC including a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0177] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to L131T and S172D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor that has no substitutions or only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 3 mM against acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-native ligand compared to a control receptor that has no such substitutions or only one of such substitutions. In some embodiments, the non-native ligand is CNL001. In some embodiments, the engineered receptor is a chimeric LGIC including a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1. In some embodiments, the manipulated receptor is CODA535 (SEQ ID NO: 60), and the control receptor is CODA71, CODA335, or CODA377.

[0178] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to L131D and S172D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency to acetylcholine (as determined, e.g., by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency) to a non-native ligand compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has higher potency (lower EC50) to CNL002 compared to a control receptor that does not have such substitutions and / or has only one of such substitutions. In some embodiments, the non-native ligand for such an engineered receptor is CNL002, AZD-0328, or fascinicrin. In some embodiments, the non-native ligand is fascinicrin. 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 containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0179] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to Y115D and S170T in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions and / or has only one of such substitutions (e.g., S170T only). In some embodiments, such an engineered receptor has an EC50 greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions (e.g., S170T only). In some embodiments, the non-native ligand of such an engineered receptor is fasciniclin or TC-6987. In some embodiments, the non-native ligand is fasciniclin. In some embodiments, the non-native ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0180] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to Y115D and L131Q in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions (e.g., only L131Q). In some embodiments, such an engineered receptor has an EC50 greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-native ligand for such an engineered receptor is AZD-0328, fasciniclin, or TC-6987. In some embodiments, the non-native ligand is fasciniclin. In some embodiments, the non-native ligand is AZD-0328. In some embodiments, the non-natural ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0181] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to Y115D and L131E in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-natural ligand compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-natural ligand for such an engineered receptor is TC-5619, AZD-0328, fasciniclin, or TC-6987. In some embodiments, the non-natural ligand is TC-5619. In some embodiments, the non-natural ligand is fasciniclin. 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 containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0182] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F and L131G in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-native ligand for such an engineered receptor is CNL001, TC-5619, CNL002, AZD-0328, TC-6987, or varenicline. In some embodiments, the non-native ligand is CNL001. In some embodiments, the non-native ligand is TC-5619. In some embodiments, the non-native 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 containing 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.

[0183] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F and L131D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-native ligand for such an engineered receptor is CNL001, TC-5619, CNL002, or TC-6987. In some embodiments, the non-native ligand is CNL001. In some embodiments, the non-native ligand is TC-5619. In some embodiments, the non-native ligand is CNL002. In some embodiments, the non-native ligand is TC-6987. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0184] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to Y115E and Y210W in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-natural ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-natural ligand for such an engineered receptor is TC-5619, ABT-0126, or CNL002. In some embodiments, the non-natural ligand is TC5619 / bladaniclinicrin. In some embodiments, the non-natural ligand is ABT-0126. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0185] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101W and Y210V in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (as determined, e.g., by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, the non-native ligand is TC-5619 / bladanicline. In some embodiments, the engineered receptor is a chimeric LGIC including a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1. In some embodiments, the manipulated receptor is CODA1045 (SEQ ID NO: 68), and the control receptor is CODA71, CODA238, or CODA405.

[0186] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F and Y210V in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions (e.g., only R101F). In some embodiments, such an engineered receptor has an EC50 greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions (e.g., only Y210V). In some embodiments, the non-native ligand is TC-5619 / bladanicline. In some embodiments, the engineered receptor is a chimeric LGIC including a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1. In some embodiments, the manipulated receptor is CODA1047 (SEQ ID NO: 69), and the control receptor is CODA71, CODA236, or CODA405.

[0187] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F and Y210F in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 1 mM against acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 10 nM against CNL001 and / or less than or equal to about 30 nM against TC5619 / bladaniclinn. In some embodiments, the non-native ligand for such an engineered receptor is CNL001 or TC5619 / bladaniclinn. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / bladaniclinicrin. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0188] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101M and L131A in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 1 mM against acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 of about 10 nM or less against CNL001, an EC50 of about 3 nM or less against TC5619 / bladanicline, and / or an EC50 of about 3 nM or less against varenicline. In some embodiments, the non-natural ligand for such an engineered receptor is CNL001, TC5619 / bladaniclinicline, or varenicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / bladaniclinicline. 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 ionpore 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.

[0189] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101M and L131F in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 1 mM or greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 1 nM for CNL001 and / or less than or equal to about 3 nM for varenicline. In some embodiments, the non-native ligand is CNL001, TC5619 / bladanicline, or varenicline. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC5619 / bladaniculin. In some embodiments, the non-natural ligand is varenicline. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0190] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101W, Y115E, and Y210W in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against 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 such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 3 mM or greater than 10 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-native ligand compared to a control receptor that does not have such substitutions or has only one or two such substitutions. In some embodiments, the non-native ligand is TC-5619 / bladaniclinn. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 1 nM for TC-5619 / bladaniclinn. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0191] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F, Y115E, and Y210W in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against 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 such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 3 mM or greater than 10 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-native ligand compared to a control receptor that does not have such substitutions or has only one or two such substitutions. In some embodiments, the non-native ligand is TC-5619 / bladaniclinn. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 10 nM for TC-5619 / bladaniclinn. In some embodiments, the engineered receptor is a chimeric LGIC containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0192] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to W77F, R101F, and L131D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against 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 such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 1 mM or greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one or two such substitutions. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 10 nM for CNL002. In some embodiments, the non-native 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 containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0193] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to R101F, L131N, and S172D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against 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 such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 1 mM or greater than 3 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one or two such substitutions. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 1 nM for CNL001, or less than or equal to about 10 nM for CNL002. In some embodiments, the non-native 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 containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0194] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to Q139E and S172D in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions (e.g., only S172D). In some embodiments, such an engineered receptor substantially retains (or has higher potency against) a non-natural ligand compared to a control receptor that does not have such substitutions 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 including a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1. In some embodiments, the manipulated receptor is CODA1157 (SEQ ID NO: 77), and the control receptor is CODA71, CODA945, or CODA377.

[0195] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR, as well as amino acid substitutions corresponding to S172D and Y210W in the LBD of human α7-nAChR. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., as determined by a higher EC50) compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 greater than 3 mM or greater than 10 mM for acetylcholine. In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor that does not have such substitutions or has only one of such substitutions. In some embodiments, such an engineered receptor has an EC50 less than or equal to about 10 nM for CNL001. In some embodiments, the non-native ligand for such an engineered receptor is CNL001, CNL002, or ABT-126. In some embodiments, the non-native 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 containing a ligand-binding domain derived from human α7-nAChR and an ionpore 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.

[0196] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR and an amino acid substitution at the amino acid residue corresponding to Y140 of human α7-nAChR. In some embodiments, the amino acid substitution is Y140I. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor without such substitution. In some embodiments, the engineered receptor is a chimeric LGIC (e.g., CODA952, SEQ ID NO: 64) containing a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1, with the control receptor being CODA71 (SEQ ID NO: 33). In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor without such substitution. In some embodiments, non-native ligands against such an engineered receptor are CNL001, TC-5619 / bladaniclinn, CNL002, or fascinicrin. In some embodiments, the non-natural ligand is CNL001. In some embodiments, the non-natural ligand is TC-5619 / bladaniclinn. In some embodiments, the non-natural ligand is CNL002. In some embodiments, the non-natural ligand is fascinicrin.

[0197] In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR and an amino acid substitution at the amino acid residue corresponding to Y140 of human α7-nAChR. In some embodiments, the amino acid substitution is Y140C. In some embodiments, such an engineered receptor has lower potency against acetylcholine (e.g., determined by a higher EC50) compared to a control receptor without such substitution. In some embodiments, the engineered receptor is a chimeric LGIC (e.g., CODA965, SEQ ID NO: 88) containing a ligand-binding domain derived from human α7-nAChR and an ionpore domain derived from human GlyRα1, with the control receptor being CODA71 (SEQ ID NO: 33). In some embodiments, such an engineered receptor substantially retains (or has higher potency against) non-native ligands compared to a control receptor without such substitution. In some embodiments, the non-natural ligand for such an engineered receptor is CNL001, TC-5619 / bladaniclinn, 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 / bladaniclinn. 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.

[0198] Table 10 below outlines these exemplary manipulated receptors. [Table 10] D. Ligand

[0199] In some embodiments, the ligands of this disclosure refer to exogenous drugs or compounds having a known mechanism of action on mammalian cells (e.g., known to act as receptor agonists, antagonists, or modulators). Such ligands are sometimes referred to as “binding agents.” The ligands of this disclosure may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the ligands include drugs or compounds approved by the U.S. Food and Drug Administration (FDA) for clinical use in the treatment of certain diseases (e.g., neurological disorders). 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 have not been approved by the FDA for clinical use but are routinely used in laboratory studies. In some embodiments, the ligand is an analogue of one of the aforementioned ligands. In certain embodiments, the ligand is selected from 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, silancetron, PH-399733, fascinicline E / 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, CNL002, RG3487, TC-6987, CNL001, TC-6683, varenicline, and TC-5619.

[0200] In certain embodiments, the ligand is an analog of silancetron, such as one described by formula 2 to 7, which is a compound in either its R or S enantiomer. [ka]

[0201] 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.

[0202] In some embodiments, the ligand is AZD-0328, which has the following formula. [ka]

[0203] In some embodiments, the ligand is TC-6987, defined by the following formula. [ka]

[0204] In some embodiments, the ligand is ABT-126, which is given by the following formula. [ka]

[0205] In some embodiments, the ligand is TC-5619 / bladanicline, which has the following formula. [ka]

[0206] In some embodiments, the ligand is TC-6683, defined by the following formula. [ka]

[0207] In some embodiments, the ligand is varenicline, which has the following formula: [ka]

[0208] In some embodiments, the ligand is fascinicrin / RG3487, which is defined by the following formula. [ka]

[0209] In some embodiments, the ligand is CNL001.

[0210] In some embodiments, the ligand is CNL002.

[0211] In some embodiments, the ligand is an anxiolytic, anticonvulsant, antidepressant, antipsychotic, antiemetic, nootropic, antibiotic, antifungal, antiviral, or antiparasitic drug. [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 5-3] [Table 6] [Table 7] [Table 8] [Table 9] E. Polynucleotides

[0212] In various exemplary embodiments, the disclosure partially intends to include polynucleotides, manipulated receptor polypeptides containing LGIC and their subunits and mutaines, as well as fusion polypeptides, viral vector polynucleotides, and compositions comprising them.

[0213] As used herein, the terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” or “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) defined from ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be conferred before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components. Polynucleotides may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), synthetic RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.Polynucleotides refer to nucleotide lengths 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 (including all ranges and subranges in between) or greater, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, as well as polymeric forms of all intermediate-length nucleotides. In this context, “intermediate length” will be readily understood to mean any length between the cited values ​​such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has sequence identity of 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 and partial ranges in between) with a reference sequence described herein or known in the art, and typically, unless otherwise specified, the variant retains at least one biological activity of the reference sequence.

[0214] As used herein, the term “gene” may refer to a polynucleotide sequence including enhancers, promoters, introns, exons, etc. In certain embodiments, the term “gene” refers to a polynucleotide sequence encoding a polypeptide, whether or not the polynucleotide sequence is identical to a genome sequence encoding a polypeptide.

[0215] As used herein, “cis-acting sequence,” “cis-acting regulatory sequence,” or “cis-acting nucleotide sequence” or equivalent refers to a polynucleotide sequence associated with gene expression, e.g., transcription and / or translation. In one embodiment, a cis-acting sequence is a polynucleotide sequence associated with a polypeptide binding site that represses or reduces transcription, or a transcription factor binding site that contributes to transcriptional repression, thus regulating transcription. Examples of cis-acting sequences that regulate the expression of polynucleotide sequences and can be operably ligated to the polynucleotides of this disclosure to regulate the expression of a manipulated receptor of interest are 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 polyadenylated sequences.

[0216] As a non-limiting example, a promoter sequence is a DNA regulatory region that can bind to RNA polymerase within a cell and initiate transcription of a downstream (3' direction) coding sequence. For the purpose of defining the present invention, a promoter sequence is bound to a transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Within the promoter sequence, a transcription start site and protein binding domains involved in binding of RNA polymerase are found. Eukaryotic promoters often, but not always, contain a "TATA" box and a "CAT" box. A variety of promoters can be used to drive the various vectors of the present invention. For example, the promoter may be a constitutively active promoter, i.e., a promoter that is active in the absence of an externally applied ligand, such as the CMV IE1 promoter, the SV40 promoter, the GAPDH promoter, the Actin promoter. The promoter may be an inducible promoter, i.e., a promoter whose activity is regulated upon application of a ligand to the cell, such as doxycycline, the tet-on or tet-off promoter, the estrogen receptor promoter, etc. The promoter may be a tissue-specific promoter, i.e., a promoter that is active in a particular type of cell.

[0217] In some embodiments, the promoter is active in excitatory cells. "Excitatory cells" means cells that are activated by a change in membrane potential, such as neurons or muscle cells, such as dorsal root ganglion neurons, motor neurons, excitatory neurons, inhibitory neurons, or sensory neurons. Promoters that are active in excitatory cells, which find use in the polynucleotide compositions of the present invention, include neuron promoters such as synapsin (SYN), TRPV1, Na v 1.7, Na v 1.8, Na v1.9 These include 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.

[0218] As used herein, “inducible expression regulatory element” refers to a polynucleotide sequence that is a promoter, enhancer, or functional fragment that is operably ligated to an expressed polynucleotide and increases (turns on) or decreases (turns off) the expression of the polynucleotide operably ligated to that element in response to the presence or absence of a molecule bound to the element. Exemplary regulatory elements for inducible expression include, but are not limited to, tetracycline-responsive promoters, ecdysone-responsive promoters, kmet-responsive promoters, glucocorticoid-responsive promoters, estrogen-responsive promoters, RU-486-responsive promoters, PPAR-γ promoters, and peroxide-inducible promoters.

[0219] "Modifying elements for transient expression" refer to polynucleotide sequences that can be used to express a polynucleotide sequence for a short period of time or transiently. In certain embodiments, one or more regulatory elements for transient expression can be used to limit the duration of the polynucleotide. In certain embodiments, the preferred duration of polynucleotide expression is several minutes, several hours, or several days. Exemplary regulatory elements for transient expression include, but are not limited to, nuclease target sites, recombinase recognition sites, and inhibitory RNA target sites. Furthermore, to some extent, in certain embodiments, regulatory elements for inducible expression may also contribute to controlling the duration of polynucleotide expression.

[0220] As used herein, terms such as “polynucleotide variant” and “variant” refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, modified, or substituted with different nucleotides. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, thereby allowing the modified polynucleotide to retain the biological function or activity of the reference polynucleotide. In certain embodiments, a polynucleotide or variant has sequence identity of 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 and partial ranges in between) with a reference sequence described herein or known in the art, and typically, unless otherwise specified, the variant retains at least one biological activity of the reference sequence.

[0221] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. Hybridizing under “stringent conditions” describes a hybridization protocol in which at least 60% identical nucleotide sequences remain hybridized to one another. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at specified ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probe is occupied at equilibrium.

[0222] As used herein, “sequence identity” or enumerations including, for example, “50% identical sequences” refer to the degree to which sequences are identical nucleotide-wise or amino acid-wise across a comparison window. Thus, “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in 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) occur in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is at least 12 monomer units long, including nucleotides and amino acid residues, but often 15 to 18, and frequently at least 25. Since each of two polynucleotides may contain (1) sequences similar to the other polynucleotide (i.e., only a portion of the complete polynucleotide sequence) and (2) sequences different from the other polynucleotide, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides across a “comparison window” to identify and compare local regions of sequence similarity. The “comparison window” refers to a conceptual segment of at least six consecutive positions, usually about 50 to about 100, more typically about 100 to about 150, where the sequences are compared to the reference sequence at the same number of consecutive positions after the two sequences have been optimally aligned. The comparison window may contain about 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences.The optimal alignment of sequences for aligning the comparison window can be achieved by a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0 from Genetics Computer Group, 575 Science Drive Madison, Wisconsin, USA) or by the best alignment (i.e., the one that yields the highest percentage of homology across the comparison window) generated by any of the various methods of inspection and selection. See also the BLAST family of programs, for example, disclosed in Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994–1998, Chapter 15, Unit 19.3.

[0223] As used herein, “isolated polynucleotide” refers to a polynucleotide purified from an adjacent sequence in its naturally occurring state, for example, a DNA fragment isolated from a sequence normally adjacent to the fragment. In certain embodiments, “isolated polynucleotide” refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and are artificially produced.

[0224] Terms describing the orientation of polynucleotides 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 (OH) group). Polynucleotide sequences can be annotated in either the 5'-to-3' direction or the 3'-to-5' direction. For DNA and mRNA, the 5'-to-3' strand is called the “sense,” “plus,” or “coding” strand because its sequence is identical to the pre-messenger (pre-mRNA) sequence [without thymine (T) in DNA, and uracil (U) in RNA]. For DNA and mRNA, the complementary 3'-to-5' strand, which is the strand transcribed by RNA polymerase, is called the “template,” “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse” refers to the 5'-to-3' sequence when written in the 3'-to-5' direction, or to the 3'-to-5' sequence when written in the 5'-to-3' direction.

[0225] The term “adjacent” refers to polynucleotide sequences located between an upstream and / or downstream polynucleotide sequence, i.e., at the 5' and / or 3' positions relative to the sequence. For example, a sequence “adjacent” by two other elements (e.g., ITRs) indicates that one element is located at the 5' position of the sequence and the other at the 3' position, however, intervening sequences may exist between them.

[0226] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG3' is 3'TCAGTAC5'. The latter sequence is often written as its reverse complement, 5'CATGACT3', with the 5' end on the left and the 3' end on the right. A sequence equal to its reverse complement is said to be a palindrome. Complementarity may be "partial," where only some of the bases of nucleic acids match according to base pairing rules. Alternatively, "complete" or "whole" complementarity may exist between nucleic acids.

[0227] As used herein, the terms “nucleic acid cassette” or “expression cassette” refer to a polynucleotide sequence within a larger polynucleotide, such as a vector, sufficient to express one or more RNAs from the polynucleotide. The expressed RNA may be translated into a protein and may function as a guide RNA or inhibitory RNA targeting other polynucleotide sequences for cleavage and / or degradation. In one embodiment, the nucleic acid cassette comprises one or more polynucleotides of interest. In another embodiment, the nucleic acid cassette comprises one or more expression regulatory sequences operably ligated to one or more polynucleotides of interest. The polynucleotides include the polynucleotides of interest. As used herein, the term “polynucleotides of interest” refers to a polynucleotide encoding a polypeptide or fusion polypeptide, or an inhibitory polynucleotide as intended herein, e.g., LGIC, and a polynucleotide that acts as a template for transcription of its subunits and muteins. In certain embodiments, the polynucleotides of interest encode a polypeptide or fusion polypeptide having one or more enzymatic activities, such as nuclease activity and / or chromatin remodeling or epigenetic modification activity.

[0228] The vector may comprise one, two, three, four, five, six, seven, eight, nine, or ten or more nucleic acid cassettes. In preferred embodiments of the present disclosure, the nucleic acid cassette comprises one or more expression regulatory sequences (e.g., promoters or enhancers operable in neuronal cells) operably ligated to a manipulated receptor, e.g., LGIC, or a polynucleotide encoding its subunit or mutaine. The cassette may be isolated as a single unit from or inserted into other polynucleotide sequences, e.g., plasmids or viral vectors.

[0229] In one embodiment, the polynucleotides contemplated herein comprise one, two, three, four, five, six, seven, eight, nine, or more nucleic acid cassettes, any number or combination thereof, which may be in the same or opposite orientation.

[0230] Furthermore, as a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that, as intended herein, there are many nucleotide sequences that can encode fragments of polypeptides or their variants. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon use, e.g., polynucleotides optimized for human and / or primate codon selection, are specifically envisioned in this disclosure. In one embodiment, a polynucleotide comprising a particular allele sequence is provided. The allele is an endogenous polynucleotide sequence that varies as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0231] In some embodiments, this disclosure provides polynucleotides encoding the engineered receptors described herein. In some embodiments, this disclosure provides polynucleotides encoding the chimeric engineered LGIC receptors described herein.

[0232] In some embodiments, the Disclosure provides polynucleotides encoding an engineered receptor comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences of SEQ ID NOs. 58-78 and 88. In some embodiments, the Disclosure provides polynucleotides encoding an engineered receptor comprising an amino acid sequence of any of the amino acid sequences of SEQ ID NOs. 58-78 and 88. In some embodiments, the Disclosure provides polynucleotides encoding an engineered receptor comprising an amino acid sequence of any of the amino acid sequences of SEQ ID NOs. 58-78 and 88. F. Vector

[0233] In some aspects of this disclosure, a nucleic acid molecule, i.e., a polynucleotide encoding an engineered receptor, is delivered to a target. In some cases, the nucleic acid molecule encoding the engineered receptor is delivered to the target by a vector. In various embodiments, the vector comprises one or more polynucleotide sequences as intended herein. The term “vector” is used herein to mean a nucleic acid molecule capable of importing or transporting another nucleic acid molecule. The imported polynucleotide is generally ligated into the vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within a cell, or may contain sequences sufficient to enable integration into host cell DNA. The vector can deliver the target polynucleotide to an organism, cell, or cellular component. In some cases, the vector is an expression vector. As used herein, “expression vector” refers to a vector, e.g., a plasmid, capable of promoting the expression and replication of the polynucleotide incorporated therein. Typically, the nucleic acid sequence to be expressed is operably ligated to cis-acting regulatory sequences, e.g., a promoter and / or enhancer sequence, and is subject to transcriptional regulatory control by the promoter and / or enhancer. In certain cases, a vector is used to deliver a nucleic acid molecule encoding the manipulated receptor described herein to a target.

[0234] In certain embodiments, any vector suitable for introducing an expression cassette or polynucleotide encoding the engineered receptor into nerve cells can be used. Examples of suitable vectors include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some cases, the vector is a circular nucleic acid, such as plasmids, BACs, PACs, YACs, cosmids, or fosmids. In some cases, a circular nucleic acid molecule can be used to deliver a nucleic acid molecule encoding the engineered receptor to a target. For example, a plasmid DNA molecule encoding the engineered receptor can be introduced into a target cell, thereby transcribing the DNA sequence encoding the engineered receptor into mRNA, and the mRNA "message" is translated into a protein product. Circular nucleic acid vectors generally contain regulatory elements that modulate the expression of the target protein. For example, a circular nucleic acid vector may contain any number of promoters, enhancers, terminators, splice signals, origins of replication, initiation signals, etc.

[0235] In some cases, the vector may contain a replicon. The replicon may be any nucleic acid molecule capable of self-replication. In some cases, the replicon may be an RNA replicon derived from a virus. A variety of suitable viruses (e.g., RNA viruses) are available, including but not limited to alphaviruses, picornaviruses, flaviviruses, coronaviruses, pestiviruses, rubiviruses, caliciviruses, and hepaciviruses.

[0236] In some embodiments, the vector is a non-viral vector. “Non-viral vector” means any delivery vehicle that does not contain a viral capsid or envelope, such as lipid nanoparticles (anionic (negatively charged), neutral or cationic (positively charged)), heavy metal nanoparticles, polymer-based particles, plasmid DNA, minicircle DNA, minivector DNA, ccDNA, synthetic RNA, exosomes, etc. Non-viral vectors may be delivered by any suitable method well understood in the art, including, for example, nanoparticle delivery, particle guns, electroporation, sonication, or microinjection. See, for example, Chen et al., Mol. Therapy, Methods and Clinical Development. 2016 Jan; Vol 3, issue 1, and Hardy, CE et al., Genes (Basel). 2017 Feb; 8(2):65.

[0237] In other embodiments, the vector is a viral vector. “Viral vector” means a delivery vehicle comprising a viral capsid or envelope surrounding a polynucleotide encoding the RNA or polypeptide of interest. In some cases, the viral vector is derived from a replication-deficient virus. Non-limiting examples of viral vectors suitable for delivery to the nucleic acid molecules of this disclosure include those derived from adenoviruses, retroviruses (e.g., lentiviruses), adeno-associated viruses (AAVs), and herpes simplex virus-1 (HSV-1). Examples of suitable viral vectors include, but are not limited to, retroviral vectors (e.g., lentiviral vectors), herpesvirus-based vectors, and parvovirus-based vectors (e.g., adeno-associated virus (AAV)-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors).

[0238] As used herein, the term “parvovirus” encompasses all parvoviruses, including autonomously replicating parvoviruses and dependviruses. Autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, mouse microvirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline pancytopenia 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, Vol. 2, Chapter 69 (3rd edition, Lippincott-Raven Publishers).

[0239] The Dependovirus genus includes adeno-associated viruses (AAVs), including, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV.

[0240] In preferred embodiments, the vector is an AAV vector. In specific cases, the viral vector is an AAV5, AAV-6, or AAV-9 vector.

[0241] The genomic composition of all known AAV serotypes is similar. The AAV genome is a linear single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Terminal inversion sequences (ITRs) are adjacent to the intrinsic coding nucleotide sequences of the non-structural replication (Rep) protein and the structural (VP) protein. The VP proteins (VP1, -2, and -3) form a capsid and contribute to viral affinity. The terminal 145nt ITRs are self-complementary and are organized to form energetically stable intramolecular double helices that form a T-shaped hairpin. These hairpin structures function as origins for viral DNA replication and act as primers for cellular DNA polymerase complexes. Following wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and functions in viral genome replication.

[0242] In some cases, the outer protein of a viral vector, the "capsid," is naturally occurring, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In specific cases, the capsid is synthetically engineered to have specific intrinsic features not found in nature, such as altered affinity, increased transduction efficiency, or immune evasion (e.g., through directed evolution or rational design). An example of a rationally designed capsid is a mutation in one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid protein. Non-limiting examples of viral vectors whose VP3 capsid protein is synthetically manipulated and suitable for use with the compositions and methods provided herein include AAV1(Y705+731F+T492V), AAV2(Y444+500+730F+T491V), AAV3(Y705+731F), AAV5(Y436+693+719F), AAV6(Y705+731F+T492V), AAV8(Y733F), AAV9(Y731F), and AAV10(Y733F). Non-limiting examples of viral vectors manipulated 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 containing amino acid mutations at one or more positions corresponding to T492, Y705, Y731, or any combination thereof of the AAV6 capsid protein, wherein the AAV capsid protein is serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or another AAV serotype. In some embodiments, 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 containing one or more amino acid substitutions corresponding to T492V, Y705F, or Y731F of the 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, one or more substitutions are two or more substitutions, two substitutions, or three substitutions.

[0243] In some embodiments, the viral vector comprises an AAV5 capsid protein containing 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 containing one or more mutations selected from Y693F and Y719F. In some embodiments, the viral vector comprises an AAV5 capsid protein containing the amino acid mutation Y693F+Y719F. In some embodiments, the AAV5 capsid protein comprises or 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%, or 100% sequence identity with SEQ ID NO: 79.

[0244] In some embodiments, the viral vector comprises an AAV6 capsid protein containing mutations at one or more amino acid positions selected from T492, Y705, and Y731. In some embodiments, the viral vector comprises an AAV6 capsid protein containing one or more mutations selected from T492V, Y705F, and Y731F. In some embodiments, the viral vector comprises an AAV6 capsid protein containing the amino acid mutation T492V+Y705F+Y731F. In some embodiments, the AAV6 capsid protein comprises or 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%, or 100% sequence identity with SEQ ID NO: 80.

[0245] In some embodiments, the viral vector comprises an AAV9 capsid protein containing mutations at one or more amino acid positions selected from T492, Y705, and Y731. In some embodiments, the viral vector comprises an AAV9 capsid protein containing one or more mutations selected from T492V, Y705F, and Y731F. In some embodiments, the viral vector comprises an AAV9 capsid protein containing the amino acid mutation T492V+Y705F+Y731F. In some embodiments, the AAV9 capsid protein comprises or 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%, or 100% sequence identity with SEQ ID NO: 81.

[0246] In some embodiments, a viral vector containing the AAV capsid protein (or a variant of the AAV capsid protein) contributes to the targeted expression of the engineered receptor in a subpopulation of cells or neurons of interest. In some embodiments, the neurons are nociceptors.

[0247] In this specification, “recombinant parvovirus or AAV vector” (or “rAAV vector”) refers to a vector containing one or more polynucleotides contended herein, adjacent to one or more AAV ITRs. Such polynucleotides are said to be “heterogeneous” with respect to the ITR because such combinations do not normally occur in nature. Such rAAV vectors can be replicated and packaged into infectious viral particles if present in insect host cells expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g., in a chromosome, or in another vector such as a plasmid or baculovirus used for cloning or transfection), the rAAV vector is typically called a “provector” that can be “rescued” by replication and capsid formation in the presence of AAV packaging function and necessary helper functions.

[0248] In certain embodiments, any AAV ITR, including ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, can be used as an AAV vector. In one preferred embodiment, the AAV vector contemplated herein includes one or more AAV2 ITRs.

[0249] An rAAV vector containing two ITRs has a payload capacity of approximately 4.4 kB. A self-complementary rAAV vector contains a third ITR and packages the double-stranded recombinant portion of the vector, leaving only approximately 2.1 kB for the polynucleotide intended herein. In one embodiment, the AAV vector is an scAAV vector.

[0250] Using a dual rAAV vector strategy, an expanded packaging capacity of nearly double (approximately 9 kB) of the rAAV packaging capacity was achieved. Dual vector strategies useful for rAAV production as intended herein include, but are not limited to, splicing (trans-splicing), homologous recombination (overlap), or a combination of the two (hybrid). In a dual AAV trans-splicing strategy, the splice donor (SD) signal is located at the 3' end of the 5'-half vector, and the splice acceptor (SA) signal is located at the 5' end of the 3'-half vector. Trans-splicing results in the production of mature mRNA and full-size protein during co-infection of the same cells with dual AAV vectors and during head-to-tail chain formation mediated by two half-terminal inversion sequences (ITRs) (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the two halves of the large transgene expression cassette contained in a dual AAV vector may contain homologous overlap sequences (a dual AAV overlap at the 3' end of the 5'-half vector and the 5' end of the 3'-half vector) that mediate the rearrangement of a single large genome by homologous recombination (Duan et al., 2001). This strategy relies on the recombination properties of the transgene overlap sequence (Ghosh et al., 2006). A third dual AAV strategy (hybrid) is based on adding a highly recombinable region derived from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al., 2008, Ghosh et al., 2011) to the transsplicing vector. The added region is positioned downstream of the SD signal in the 5'-half vector and upstream of the SA signal in the 3'-half vector to increase recombination between the dual AAVs.

[0251] A "hybrid AAV" or "hybrid rAAV" refers to an rAAV genome packaged with a capsid of a different AAV serotype (preferably a serotype different from one or more AAV ITRs), otherwise it may be called a pseudotype rAAV. For example, rAAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 type genomes can be capsidized within an AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 type capsid or a variant thereof, provided that the AAV capsid and genome (preferably one or more AAV ITRs) are of different serotypes. In certain embodiments, pseudotyped rAAV particles may be referred to as "x / y" type, where "x" indicates the source of the ITR and "y" indicates the serotype of the capsid, for example, a 2 / 5 rAAV particle having an ITR derived from AAV2 and a capsid derived from AAV6.

[0252] "Host cells" include cells transfected, infected, or transduced in vivo, ex vivo, or in vitro using the recombinant vectors or polynucleotides of this disclosure. Host cells may include virus-producing cells and cells infected with viral vectors. In certain embodiments, host cells are infected with the viral vectors intended herein in vivo. In certain embodiments, the term "target cells" is used interchangeably with "host cells" and refers to infected cells of a desired cell type.

[0253] High-potency AAV preparations are developed using techniques known in the art, for example, U.S. Patent Nos. 5,658,776, 6,566,118, 6,989,264, and 6,995,006, U.S. Patent Application Publication 2006 / 0188484, International Publication 98 / 22607, International Publication 2005 / 072364, and International Publication 1999 / 011764, as well as Viral Vectors for Gene Therapy: Methods and Protocols, edited by Machida, Humana. It can be manufactured as described in Press, 2003, Samulski et al. (1989) J. Virology 63, 3822, Xiao et al. (1998) J. Virology 72, 2224, and Inoue et al. (1998) J. Virol. 72, 7024. Methods for producing pseudotype AAV vectors (e.g., International Publication No. 00 / 28004), as well as various modifications or formulations of AAV vectors to reduce their immunogenicity when administered in vivo, have also been reported (see, for example, International Publication Nos. 01 / 23001, 00 / 73316, 04 / 112727, 05 / 005610, and 99 / 06562). G. Pharmaceutical Compositions

[0254] In some embodiments, the Disclosure provides compositions comprising a polynucleotide encoding an engineered receptor as described herein, or a vector comprising a polynucleotide encoding an engineered receptor as described herein. In some embodiments, the composition further comprises a ligand as described herein. The pharmaceutical comprises a target polynucleotide (RNA or DNA) encoding an engineered receptor, a vector carrying the target polynucleotide (RNA or DNA) encoding an engineered receptor, or a ligand present in a pharmaceutically acceptable vehicle. In some embodiments, the Disclosure provides a first composition comprising a polynucleotide encoding an engineered receptor as described herein, or a second composition comprising a vector comprising a polynucleotide encoding an engineered receptor as described herein, and a ligand as described herein.

[0255] A “pharmaceutically acceptable vehicle” may be a vehicle approved by a federal or state regulatory agency, or a vehicle listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier used to formulate the compounds of this disclosure for administration to mammals. Such pharmaceutical vehicles may be liquids, such as water, and oils, such as petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, or sesame oil. Pharmaceutical vehicles may also be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, or urea. In addition, auxiliaries, stabilizers, thickeners, lubricants, and colorants may be used. When administered to mammals, the compounds and compositions of this disclosure, as well as the pharmaceutically acceptable vehicles, excipients, or diluents, may be sterile. In some cases, when the compounds of this disclosure are administered intravenously, aqueous media such as water, saline solution, and aqueous solutions of dextrose and glycerol are used as vehicles.

[0256] Pharmaceutical compositions may take the form of capsules, tablets, pills, pellets, medicinal drops, powders, granules, syrups, elixirs, liquids, suspensions, emulsions, suppositories, or sustained-release formulations thereof, or any other form suitable for administration to mammals. In some examples, pharmaceutical compositions are formulated for administration according to routine procedures as pharmaceutical compositions suitable for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for their formulation are described in Remington: The Science and Practice of Pharmacy, edited by Alfonso R. Gennaro, Mack Publishing Co., Easton, Pennsylvania, 19th edition, Chapters 86, 87, 88, 91 and 92, which are incorporated herein by reference.

[0257] The selection of excipients is determined in part by the specific vector, as well as the specific method used to administer the composition. Therefore, a wide variety of suitable formulations of the pharmaceutical compositions of this disclosure exist.

[0258] 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 ganglion 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 intraneuronal administration. In some embodiments, the pharmaceutical composition is formulated for delivery to nerve cells.

[0259] For example, vectors can be formulated into injectable formulations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher fatty acids, or propylene glycol, and using conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives as needed.

[0260] As another example, the vector can be formulated into preparations suitable for oral administration, comprising (a) a liquid solution such as an effective amount of the compound dissolved in a diluent such as water or saline, (b) capsules, pouches or tablets, each containing a predetermined amount of the active ingredient as a solid or granule, (c) a suspension in a suitable liquid, and (d) a suitable emulsion. The tablet form may contain lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and one or more other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Medicinal lozenge forms may include lozenges containing a flavor, the active ingredient typically in sucrose and acacia or tragacanth, and the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; emulsions and gels containing the active ingredient plus excipients as described herein.

[0261] As another example, the formulations covered by this disclosure may be aerosol formulations administered by inhalation. These aerosol formulations may be encapsulated in pressurized, acceptable propellants such as dichlorodifluoromethane, propane, or nitrogen. They may also be formulated as pharmaceuticals for use in non-pressurized preparations, such as for use in nebulizers or atomizers.

[0262] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be provided in sealed containers of unit or multiple doses, such as ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid excipient for injection, such as water, immediately before use. Immediate injection solutions and suspensions may be prepared from the aforementioned types of sterile powders, granules, and tablets.

[0263] Formulations suitable for topical administration may be provided as creams, gels, pastes, or foams, containing the active ingredient and, optionally, a carrier. In some embodiments, the topical formulation comprises one or more components selected from structuring agents, thickeners or gelling agents, and emollients or lubricants. Frequently used structuring agents include long-chain alcohols such as stearyl alcohol, as well as glyceryl ethers or their esters and oligo(ethylene oxide) ethers or their esters. Thickeners and gelling agents include, for example, polymers of acrylic acid or methacrylic acid and its esters, polyacrylamides, and naturally occurring thickeners such as agar, carrageenan, gelatin, and guar gum. Examples of emollients include triglyceride esters, fatty acid esters and amides, waxes such as beeswax, whale wax, or carnauba wax, phospholipids such as lecithin, and sterols and their fatty acid esters. The topical formulation may further contain other components, such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (i.e., sunscreens), etc.

[0264] The compounds of this disclosure may be formulated for topical administration. Vehicles for topical application may be in various forms, e.g., lotions, creams, gels, ointments, sticks, sprays, or pastes. They may contain various types of carriers, including but not limited to solutions, aerosols, emulsions, gels, and liposomes. The carrier may be formulated, for example, as an emulsion having an oil-in-water or water-in-oil base. Suitable hydrophobic (oily) components to be used in the emulsion include, for example, vegetable oils, animal fats and oils, synthetic hydrocarbons, and their esters and alcohols, including polyesters, as well as organopolysiloxane oils. Such emulsions may also contain emulsifiers and / or surfactants, e.g., nonionic surfactants, for dispersing and suspending the discontinuous phase within the continuous phase.

[0265] Suppository formulations are also provided by mixing with various bases, such as emulsifying or water-soluble bases. Formulations suitable for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, or foams.

[0266] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, and each dosage unit, such as a teaspoon, a tablespoon, a tablet, or a suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor in the composition as a solution in sterile water, saline solution, or another pharmaceutically acceptable carrier.

[0267] As used herein, the term “unit dosage form” refers to a physically distinct unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of the compound of the Disclosure calculated in an amount sufficient to produce the desired effect when combined with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the novel unit dosage forms of the Disclosure depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0268] The dose level may vary depending on the function of the specific compound, the properties of the delivery vehicle, etc. The desired dose of a given compound can be easily determined by various means.

[0269] In the context of this disclosure, doses administered to animals, particularly humans, should be sufficient to influence a prophylactic or therapeutic response in the animal over a reasonable timeframe, as described in more detail below, for example. The dose depends on a variety of factors, including the potency of the particular compound used, the animal's symptoms, the animal's body weight, and the severity and stage of the disease. The size of the dose is also determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the particular compound.

[0270] In pharmaceutical dosage forms, the compounds may be administered in the form of free bases or pharmaceutically acceptable salts thereof, or the compounds may also be used alone or appropriately associated with other pharmaceutically active compounds. H. Clinical Uses and Treatment Methods

[0271] The compositions and methods disclosed herein can be used to treat neurological disorders or conditions. In some aspects of this disclosure, methods are provided to treat a neurological disorder or condition of interest, comprising introducing an engineered receptor into a nerve cell and providing a ligand that activates the engineered receptor in an amount effective in controlling the cell's activity, thereby reducing pain in the subject. In some aspects, the vectors or compositions disclosed herein are used in the manufacture of pharmaceuticals for treating neurological disorders or conditions.

[0272] In some cases, the methods and compositions of this disclosure are used to treat epilepsy. The compositions described herein may be used to prevent or control epileptic seizures. Epileptic seizures may be classified as tonic-clonic, tonic, clonic, myoclonus, absence, or atonic seizures. In some cases, the compositions and methods described herein may prevent or reduce the number of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%, including the whole range and partial range in between.

[0273] In some cases, the methods and compositions of this disclosure are used to treat eating disorders. Eating disorders may be mental disorders defined by abnormal eating behaviors that adversely affect the physical or mental health of the subject. In some cases, the eating disorder is anorexia nervosa. In other cases, the eating disorder is bulimia nervosa. In some cases, the eating disorder is pica, rumination disorders, avoidant / restrictive food intake disorders, bulimia nervosa (BED), other specified eating and eating disorders (OSFED), compulsive eating, diabetic eating, orthorexia nervosa, selective eating disorder, drunken orexia, anorexia during pregnancy, or Gourmand syndrome. In some cases, the compositions include G protein-coupled receptors that increase or decrease the production of one or more molecules associated with eating disorders. In other cases, the compositions include ligand-gated ion channels that alter the production of one or more molecules associated with eating disorders. One or more molecules associated with eating disorders include, but are not limited to, molecules of the hypothalamic-pituitary-adrenal (HPA) system, including vasopressin, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, epinephrine, or norepinephrine, as well as serotonin, dopamine, neuropeptide Y, leptin, or ghrelin.

[0274] In some cases, 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, motor disorders, atrial fibrillation, cancer (e.g., brain tumors), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological disorders or disorders that can be treated by the compositions and methods herein include adyslexia, agraphia, alcoholism, dyslexia, aneurysm, transient monocular blindness, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, telangiectasia ataxia, attention deficit hyperactivity disorder, auditory processing disorder, autism spectrum disorder, bipolar disorder, Bell's palsy, and brachial plexus. Brain injury, brain damage, brain tumor, Canavan disease, Capgras syndrome, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelin breakdown, central nucleus myopathy, head injury, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, autosomal dominant cerebral arteriovenous disease with subcortical infarction and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebrovascular disease, cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) ), chronic pain, Coffin-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital bilateral facial nerve palsy, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative traumatic disorders, Cushing's syndrome, cyclothymic disorders, giant cell inclusion body disorder (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, de Morchet syndrome, Deujourine-Klumpke palsy, Deujourine-Sotta disease, post-sleep phase Dementia, dementia, dermatomyositis, developmental coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, dysarthria, autonomic neuropathy, acalculia, dysgraphia, dyskinesia, dyslexia, dystonia, syringomyelia syndrome, encephalitis, brain herniation, trigeminal nerve hemangioma, encopresis, enuresis, epilepsy, epileptic intellectual disability in women, Elb's palsy, erythromelalgia, explosive head syndrome, Fabry disease, Fahl's disease, dizziness,Familial spastic paraplegia, febrile seizures, Fisher syndrome, Friedreich's ataxia, fibromyalgia, Foville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy-febrile seizures plus, Gerstmann syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukoatrophy, gray matter ectopic, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, Haller's disease Foldenspätz disease, head trauma, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary polyneurotic ataxia, herpes zoster, shingles, Hirayama disease, Hirschsprung's disease, Holmes-Ardie syndrome, holoprosencephaly, Huntington's disease, anencephaly, hydrocephalus, hypercortisolosis, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile Refsum's disease, infantile spasms, inflammatory muscle disease, intracranial cyst, increased intracranial pressure, isodicentric 15. Joubert syndrome, Karak syndrome, Keens-Sayer syndrome, Kinsbrunn syndrome, Klein-Levin syndrome, Klippel-Feyll syndrome, Krabbe disease, Lafora disease, Lambert-Eaton asymptomatic syndrome, Landau-Klefner syndrome, posterior inferior cerebellar artery (Wallenberg) syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, leukoencephalopathy with disappearing white matter, Lewy body dementia, gyral defects, locked-in syndrome, lumbar disc disease, lumbar spinal stenosis, Lyme disease - neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), cerebrum, macropsia, disembarkation, macrocephaly with subcortical cysts, megacephaly, Melkerson-Rose Meniere's syndrome, Meniere's disease, meningitis, Menkes's disease, metachromatic leukoatrophy, microcephaly, micropsia, migraine, Miller-Fischer syndrome, primary school stroke (transient ischemic attack), phonophobia, mitochondrial myopathy, Möbius syndrome, unilateral lower leg flexor atrophy, motor impairment, Moyamoya disease, mucopolysaccharidosis, multiple stroke dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelin-destroying diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neuro-Behçet's disease, neurofibromatosis, neuroleptic malignant syndrome, neurological symptoms of AIDS, neurological sequelae of lupus,Neurogenic myotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neurosis, Niemann-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal insufficiency, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, recurrent vision, paresthesia, Parkinson's disease, congenital paramyotonia, paraneoplastic disease, seizures, Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, periodic Quadriplegia, peripheral neuropathy, pervasive developmental disorder, photosensitive sneeze reflex, phytanic acid storage, Pick's disease, nerve compression, pituitary tumor, PMG, polyneuropathy, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, postherpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy, progressive multifocal white matter brain damage, progressive supranuclear palsy, prosopagnosia, pseudotumor, quadriopora, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome 1, Ramsay Hunt syndrome 2, Ramsay Hunt syndrome 3, Smussen's encephalitis, reflex sympathetic atrophy, Refsum's disease, REM sleep behavior disorder, repetitive hyperactivity injury, restless limb syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg syndrome, Sydenham's chorea, Sandhoff's disease, Schilder's disease, schizencephaly, sensory processing disorder, septal visual dysplasia, shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, bulbar spinal muscular atrophy, spine Myolocerebellar ataxia, split brain, Steele-Richardson-Olszewski syndrome, generalized rigidity syndrome, stroke, Sturge-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic brain injury, hemosiderin deposition disorder, Sydenham's chorea, syncope, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, tardive dysphonia, Taarov's cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered cord syndrome, Thomsen's disease, thoracic outlet syndrome, painful tics, Todd's palsy, Tourette's syndrome, toxic encephalopathy, transient ischemic attack, infectious spongiform encephalopathy,These include transverse myelitis, traumatic brain injury, tremor, trichotillomania, trigeminal neuralgia, tropical spastic paraplegia, trypanosomiasis, tuberous sclerosis, Unverlicht-Lundborg disease, von Hippel-Lindau disease (VHL), Viliuisk encephalomyelitis (VE), Wallenberg syndrome, West syndrome, whiplash, Williams syndrome, Wilson's disease, or Zellweger syndrome.

[0275] In some cases, the compositions and methods disclosed herein can be used to treat brain cancer or brain tumors. Non-limiting examples of brain tumors or tumors that may be suitable for treatment with the vectors and compositions described herein include anaplastic astrocytoma (grade III glioma), astrocytoma (grade II glioma), brainstem glioma, ependymoma, ganglioglioma, ganglioneuroma, glioblastoma (grade IV glioma), glioma, juvenile pilocytic astrocytoma (JPA), low-grade astrocytoma (LGA), medullablastoma, mixed glioma, oligodendroglioma, optic glioma, pilocytic astrocytoma (grade I glioma), and primitive neuroectodermal tumor (PNET). Examples of brain tumors include gliomas; acoustic neuromas, (vestibular schwannomas), acromegaly, adenomas, chondrosarcomas, chordomas, craniopharyngiomas, epidermal tumors, jugular vein corpuscle tumors, infratentorial meningiomas, meningiomas, pituitary adenomas, pituitary tumors, and skull base tumors including Rathke's fissure cysts; metastatic cancers including brain metastases and metastatic brain tumors; other brain tumors including cerebral cysts, choroid plexus papillomas, CNS lymphomas, colloid cysts, cystic tumors, dermoids, germ cell tumors, lymphomas, nasal carcinomas, nasopharyngeal tumors, pineal gland tumors, pineoblastomas, pineal cell tumors, supratentorial meningiomas, and angiomas; and spinal cord tumors including astrocytomas, ependymomas, meningiomas, and Schwann cell tumors.

[0276] This disclosure, in part, envisions compositions and methods for controlling, managing, preventing or treating pain in a subject. "Pain" refers to an unpleasant perception and / or discomfort in the body of the subject. The perception of pain can range from mild and occasional to severe and constant. Pain can be classified as acute pain or chronic pain. Pain may be nociceptive pain (i.e., pain caused by tissue injury), neuropathic pain, or psychogenic pain. In some cases, pain is caused by or related to a disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by an injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment by the compositions and methods of this specification include neuropathic pain including peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, back pain, cancer-related neuropathy, HIV / AIDS-related neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, chronic alcoholism, hypothyroidism, pain associated with 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, as well as nociceptive pain including central nervous system trauma, contusions / sprains, and burns, myocardial infarction, acute pancreatitis, postoperative pain, post-traumatic pain, renal colic, cancer-related pain, fibromyalgia-related pain, carpal tunnel syndrome-related pain, and back pain.

[0277] The compositions and methods herein may be used to improve the pain level of a subject. In some cases, the pain level in the subject is improved by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 99%, or about 100%, including the whole range and partial range in between. The pain level in the subject can be assessed by various methods. In some cases, the pain level is assessed by self-reporting (i.e., the subject expresses the level of pain they are experiencing through an oral report). In some cases, the pain level is assessed by behavioral indicators of pain, such as facial expressions, limb movements, vocalizations, restlessness, and defensiveness. These types of assessments may be useful, for example, when the subject is unable to self-report (e.g., infants, unconscious subjects, non-human subjects). The level of pain may be assessed after treatment with the composition of this disclosure by comparing it to the level of pain the subject experienced before treatment with the composition.

[0278] In various embodiments, methods for controlling, managing, preventing, or treating pain in a subject include administering an effective amount of the manipulated receptors described herein to the subject. While not intending to be bound by any particular theory, this disclosure aims to reduce pain in a subject by modulating neuronal activity using the vectors disclosed herein.

[0279] In various embodiments, a vector encoding an engineered receptor that activates or depolarizes nerve cells is administered (or introduced) to one or more nerve cells, such as inhibitory interneurons, that reduce pain sensation. In the presence of a ligand, nerve cells expressing the engineered receptor are activated, reducing their sensitivity to pain and enhancing the analgesic effect that stimulates these nerve cells.

[0280] In various embodiments, a vector encoding an engineered receptor that inactivates or hyperpolarizes nerve cells is administered (or introduced) to one or more nerve cells, such as nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc., to increase sensitivity to pain or aches. In the presence of a ligand, nerve cells expressing the engineered receptor are inactivated, reducing sensitivity to pain and enhancing analgesic effects.

[0281] Targeting the expression of manipulated receptors to a subpopulation of nociceptors can be achieved by 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 one or more delivery methods.

[0282] In certain embodiments, the compositions and methods described herein are effective in reducing pain. Examples of pain suitable for treatment with the vectors, compositions and methods described 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.

[0283] Pain can be classified as acute or chronic. “Acute pain” refers to pain that begins suddenly and is usually sharp in nature. Acute pain can be mild and last only a moment, or it can be severe and last for weeks or months. In most cases, acute pain does not last longer than three months and disappears when the underlying cause of the pain is treated or healed. However, unrelieved acute pain can lead to chronic pain. “Chronic pain” refers to persistent or recurrent pain that lasts beyond the normal course of an acute illness or injury, or for more than three to six months, and has adverse effects on an individual’s health. In certain embodiments, the term “chronic pain” refers to pain that persists when it should not. Chronic pain may be nociceptive pain or neuropathic pain.

[0284] In some embodiments, pain is expected or anticipated to occur in relation to, or as a result of, injury, infection, or medical intervention. In some embodiments, infection causes nerve damage. In some embodiments, medical intervention is surgery, such as surgery on the central core of the body. In some embodiments, medical intervention is surgery to remove part or all of one or more tissues, tumors, or organs within the body. In some embodiments, medical intervention is amputation. In certain embodiments, the compositions and methods contemplated herein are effective in reducing acute pain. In certain embodiments, the compositions and methods contemplated herein are effective in reducing chronic pain.

[0285] Clinical pain is present when a patient's symptoms include characteristics of discomfort and abnormal sensitivity. Individuals may present with a variety of pain symptoms. Such symptoms include 1) dull, burning, or stabbing spontaneous pain, 2) an exaggerated pain response to noxious stimuli (hyperalgesia), and 3) pain caused by normally harmless stimuli (allodynia - Meyer et al., 1994, Textbook of Pain, 13-44). Patients suffering from various forms of acute and chronic pain may have similar symptoms, but the underlying mechanisms may differ, and therefore may require different treatment strategies. Thus, pain can also be classified into several different subtypes according to their different pathophysiologies, including nociceptive pain, inflammatory pain, and neuropathic pain.

[0286] In certain embodiments, the compositions and methods described herein are effective in reducing nociceptive pain. In certain embodiments, the compositions and methods described herein are effective in reducing inflammatory pain. In certain embodiments, the compositions and methods described herein are effective in reducing neuropathic pain.

[0287] Nociceptive pain is triggered by tissue damage or strong stimuli that could cause damage. Moderate to severe acute nociceptive pain is a prominent feature of central nervous system trauma, bruises / sprains, burns, pain from myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical procedure), post-traumatic pain, renal colic, cancer pain, and back pain. Cancer pain may also be chronic pain such as tumor-related pain (e.g., bone pain, headache, facial pain, or visceral pain) or pain associated with cancer treatment (e.g., post-chemotherapy syndrome, chronic postoperative pain syndrome, or post-radiation syndrome). Cancer pain can also occur in response to chemotherapy, immunotherapy, hormone therapy, or radiation therapy. Back pain may result from a herniated or ruptured disc, or abnormalities of the lumbar facet joints, sacroiliac joints, paraspinal muscles, or posterior longitudinal ligament. Back pain can resolve spontaneously, but in some patients, if it persists for 12 weeks or more, it can become a chronic condition, which can be particularly debilitating.

[0288] Neuropathic pain can be defined as pain initiated or caused by a primary lesion or dysfunction of the nervous system. Examples of neuropathic pain include peripheral neuropathy, diabetic neuropathy, postherpetic 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 deficiencies.

[0289] Neuropathic pain may be related to pain disorders, a term referring to diseases, disorders, or conditions associated with or caused by pain. Exemplary examples of pain disorders include arthritis, allodynia, typical trigeminal neuralgia, somatoform disorders, hypoesthesis, hyperalgesia, neuralgia, neuritis, neuropathic pain, analgesia, painful analgesia, causalgia, sciatica, osteoarthritis, fibromyalgia, visceral disorders, chronic pain disorders, migraines / headaches, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis, or cancer-related pain.

[0290] The inflammatory process is a complex series of biochemical and cellular events activated in response to tissue damage or the presence of foreign bodies, resulting in swelling and pain. Joint pain is a common type of inflammatory pain.

[0291] Other types of pain suitable for treatment by the vectors, compositions and methods intended herein include, but are not limited to, pain resulting from musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-rheumatoid arthritis, dystrophinopathy, glycogenolysis, polymyositis and suppurative myositis; cardiac and vascular pain, including pain caused by anguina, myocardical infarction, mitral stenosis, pericarditis, digital ischemic phenomenon, edematous sclerosis (scleredoma) and skeletal muscle ischemia; headaches, including migraines (including migraines with aura and migraines without aura), cluster headaches, tension headaches, mixed headaches and headaches associated with vascular disorders; and orofacial pain, including toothache, earache, burning mouth syndrome and temporomandibular myofascial pain.

[0292] The effective amounts of compositions and methods intended herein for reducing the amount of pain experienced by human subjects can be determined using various pain scales. Patient self-reports can be used to assess whether pain has been reduced; see, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231. Alternatively, observational pain scales can be used. The LANSS pain scale can be used to assess whether pain has been reduced; see, for example, Bennett (2001) Pain 92:147. Visual analog pain scales can be used; see, for example, Schmader (2002) Clin. J. Pain 18:350. The Likert pain scale can be used, for example, where 0 is no pain, 5 is moderate pain, and 10 is the worst possible pain. Examples of self-reported pain scales for children include the Faces Pain Scale, the Wong-Baker Faces Pain Rating Scale, and the Colored Analog Scale. Examples of self-reported pain scales for adults include the Visual Analog Scale, the Verbal Numerical Rating Scale, the Verbal Descriptor Scale, and the Brief Pain Inventory. Examples of pain measurement scales include the Alder Hey Triage Pain Score (Stewart et al. (2004) Arch. Dis. Child. 89:625); the Behavioral Pain Scale (Payen et al. (2001) Critical Care Medicine 29:2258); and the Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med.Singapore 23:129); Checklist of Nonverbal Pain Indicators (Feldt (2000) Pain Manag.Nurs. 1:13); Critical-Care 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 27:1783); Dolorimeter Pain Index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Revised Faces Pain Scale Scale-Revised) (Hicks et al. (2001) Pain 93:173); Face, Legs Activity, Cry, Consolability Scale; McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptor Differential Scale (Gracely and Kwilosz (1988) Pain 35:279); Numerical 1-Point Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numeric Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310); Wong-Baker FACES Pain Rating Scale; and Visual Analog Scale One example is the scale (Huskisson (1982) J. Rheumatol. 9:768).

[0293] In certain embodiments, a method is provided to alleviate pain in a subject, comprising introducing an engineered receptor into a nerve cell and controlling the activity of the cell by providing an effective amount of ligand to activate the engineered receptor, thereby alleviating pain in the subject. The method provides significant analgesia for pain without off-target effects such as general central nervous system depression. In certain embodiments, the method provides a reduction of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including all and partial ranges in between) of neuropathic pain in a subject compared to an untreated subject. In some embodiments, the method includes the step of measuring the pain of the subject before and after administration of the ligand, wherein the pain of the subject is reduced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, including all and partial ranges in between. In such cases, the measurement may be taken 4 hours or more after the 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 the administration of the ligand.

[0294] In certain embodiments, the vector intended herein is administered or introduced into one or more nerve cells. The nerve cells may be of the same type or a mixed population of different types. In one embodiment, the nerve cells are nociceptors or peripheral sensory neurons. Examples of sensory neurons include, but are not limited to, dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In one embodiment, the nerve cells are inhibitory interneurons involved in neuronal pain circuits.

[0295] In some cases, a vector encoding a manipulated receptor is administered to a target requiring administration. Non-limiting examples of administration methods include subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, infusion, intracranial, intrathecal, intranasal, intraganglionic, intraspinal, cisterna magna, and intraneuronal administration. In some cases, administration may include injection of a liquid formulation of the vector. In other cases, administration may include oral delivery of a solid formulation of the vector. In some cases, the oral formulation may be administered with food. In certain embodiments, the vector is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneuronally, intraganglionically, intraspinally, or intraventricularly to a target in order to introduce the vector into one or more nerve cells. In various embodiments, the vector is rAAV.

[0296] In one embodiment, AAV is administered intrathecally (IT) or intraganglionicly (IG) to sensory neurons or nociceptors, such as DRG neurons, TGG neurons, etc. The IT route delivers AAV into the cerebrospinal fluid (CSF). This route of administration may be suitable, for example, for the treatment of chronic pain or other peripheral nervous system (PNS) or central nervous system (CNS) indications. In animals, IT administration is achieved by inserting an IT catheter through the cisterna magna and advancing it caudally to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a routine bedside procedure with an excellent safety profile.

[0297] In certain cases, the vector may be administered to the subject by intraganglion administration. Intraganglion administration may include direct injection into one or more ganglia. The IG route may deliver AAV directly to the DRG or TGG parenchyma. In animals, IG administration to the DRG is performed by open neurosurgery, which is undesirable in humans due to the complex and invasive procedure required. In humans, minimally invasive CT imaging-guided techniques can be used to safely target the DRG. AAV can be delivered into the DRG parenchyma using a needle assembly customized for convection-enhanced drug delivery (CED). In an unspecified case, the vector of this disclosure may be delivered to one or more dorsal root ganglia and / or trigeminal ganglia for the treatment of chronic pain. In another unspecified case, the vector of this disclosure may be delivered to the inferior ganglia (vagus nerve) for the treatment of epilepsy.

[0298] In yet another specific case, the vector may be administered to the subject by intracranial administration (i.e., directly into the brain). In a non-limiting example of intracranial administration, the vector of this disclosure may be delivered, for example, to the cerebral cortex to treat an epileptic seizure focus, to the paraventricular region of the hypothalamus to treat satiety disorders, or to the central nucleus of the amygdala to treat satiety disorders. In yet another specific case, the vector may be administered to the subject by intraneuronal injection (i.e., directly into a nerve). The nerve may be selected based on the indication being treated, such as an injection into the sciatic nerve to treat chronic pain, or an injection into the vagus nerve to treat epilepsy or satiety disorders. In yet another specific case, the vector may be administered to the subject by subcutaneous injection, for example, into a sensory nerve terminal, to treat chronic pain.

[0299] The vector dose may be expressed as the number of vector genome units delivered to the target. As used herein, “vector genome unit” refers to the number of individual vector genomes administered in a given dose. The size of individual vector genomes generally depends on the type of viral vector used. The vector genomes of this disclosure may range from approximately 1.0 kilobase, 1.5 kilobase, 2.0 kilobase, 2.5 kilobase, 3.0 kilobase, 3.5 kilobase, 4.0 kilobase, 4.5 kilobase, 5.0 kilobase, 5.5 kilobase, 6.0 kilobase, 6.5 kilobase, 7.0 kilobase, 7.5 kilobase, 8.0 kilobase, 8.5 kilobase, 9.0 kilobase, 9.5 kilobase, 10.0 kilobase, and up to over 10.0 kilobase. Thus, a single vector genome may contain up to 10,000 base pairs or more nucleotides. In some cases, the vector dose may be approximately 1 × 10⁶ 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×10 13 、8×10 13 、9×10 13 、1×10 14 、2×10 14 、3×10 14 、4×10 14 、5×10 14 、6×10 14 、7×10 14 、8×10 14 、9×10 14 、1×10 15 、2×10 15 、3×10 15 、4×10 15 、5×10 15 、6×10 15 、7×10 15 、8×1015 , 9×10 15 , 1 x 10 16 , 2×10 16 , 3 x 10 16 , 4×10 16 , 5×10 16 , 6×10 16 , 7×10 16 , 8×10 16 , 9×10 16 , 1 x 10 17 , 2×10 17 , 3 x 10 17 , 4×10 17 , 5×10 17 , 6×10 17 , 7×10 17 , 8×10 17 , 9×10 17 , 1 x 10 18 , 2×10 18 , 3 x 10 18 , 4×10 18 , 5×10 18 , 6×10 18 , 7×10 18 , 8×10 18 , 9×10 18 , 1 x 10 19 , 2×10 19 , 3 x 10 19 , 4×10 19 , 5×10 19 , 6×10 19 , 7×10 19 , 8×10 19 , 9×10 19 , 1 x 10 20 , 2×10 20 , 3 x 10 20 , 4×10 20 , 5×10 20 , 6×10 20 , 7×10 20 , 8×10 20 , 9×10 20 Alternatively, it may be a vector genome unit exceeding that size.

[0300] In certain embodiments, the vector intended herein is at least about 1 × 10 9 Genome particles / mL, at least about 1 × 10⁶ 10Genome particles / mL, at least approximately 5 × 10⁶ 10 Genome particles / mL, at least about 1 × 10⁶ 11 Genome particles / mL, at least approximately 5 × 10⁶ 11 Genome particles / mL, at least about 1 × 10⁶ 12 Genome particles / mL, at least approximately 5 × 10⁶ 12 Genome particles / mL, at least approximately 6 × 10⁶ 12 Genome particles / mL, at least approximately 7 × 10⁶ 12 Genome particles / mL, at least approximately 8 × 10⁶ 12 Genome particles / mL, at least approximately 9 × 10⁶ 12 Genome particles / mL, at least approximately 10 × 10 12 Genome particles / mL, at least approximately 15 × 10⁶ 12 Genome particles / mL, at least approximately 20 × 10⁶ 12 Genome particles / mL, at least approximately 25 × 10⁶ 12 Genome particles / mL, at least approximately 50 × 10 12 Genome particles / mL, or at least approximately 100 × 10⁶ 12 The target is administered a titer of genome particles / mL. The terms “genome particles (gp),” “genome equivalents,” or “genome copies” (gc) used in relation to viral titer refer to the number of virions containing recombinant AAV DNA genome, regardless of whether they are infectious or functional. The number of genome particles in a particular vector preparation can be measured by methods well understood in the art, such as quantitative PCR of genomic DNA, or as described, for example, Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0301] The vectors of this disclosure can be administered in a certain volume of fluid. In some cases, the vectors can be administered in volumes of approximately 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, 10.0 mL, 11.0 mL, 12.0 mL, 13.0 mL, 14.0 mL, 15.0 mL, 16.0 mL, 17.0 mL, 18.0 mL, 19.0 mL, 20.0 mL, or greater than 20.0 mL. In some cases, the vector dose can be expressed as the concentration or titer of the vector administered to the subject. In this case, the vector dose can be expressed as the number of vector genome units per volume (i.e., genome units / volume).

[0302] In certain embodiments, the vector intended herein is at least about 5 × 10 9 Infection units / mL, at least approximately 6 × 10 9 Infection units / mL, at least approximately 7 × 10 9 Infection units / mL, at least approximately 8 × 10 9 Infection units / mL, at least approximately 9 × 10 9 Infection units / mL, at least about 1 × 10⁻⁶ 10 Infection units / mL, at least approximately 1.5 × 10⁻⁶ 10 Infection units / mL, at least approximately 2 × 10⁻⁶ 10 Infection units / mL, at least approximately 2.5 × 10⁻⁶ 10 Infection units / mL, at least approximately 5 × 10 10 Infection units / mL, at least approximately 1 × 10⁻⁶ 11 Infection units / mL, at least approximately 2.5 × 10⁻⁶ 11 Infection units / mL, at least approximately 5 × 10 11 Infection units / mL, at least about 1 × 10⁻⁶ 12 Infection units / mL, at least approximately 2.5 × 10⁻⁶ 12 Infection units / mL, at least approximately 5 × 10 12 Infection units / mL, at least about 1 × 10⁻⁶ 13Infection units / mL, at least approximately 5 × 10 13 Infection units / mL, at least about 1 × 10⁻⁶ 14 The drug is administered to the subject at a titer of infection units / mL. The terms “infection units (iu),” “infectious particles,” or “replication units” used in relation to viral titer refer to the number of infectious and replicable recombinant AAV vector particles measured by an infectious center assay, also known as a replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0303] In certain embodiments, the vector intended herein is at least about 5 × 10 10 Transduction units / mL, at least about 1 × 10⁻⁶ 11 Transduction units / mL, at least approximately 2.5 × 10⁻⁶ 11 Transduction units / mL, at least approximately 5 × 10 11 Transduction units / mL, at least about 1 × 10⁻⁶ 12 Transduction units / mL, at least approximately 2.5 × 10⁻⁶ 12 Transduction units / mL, at least approximately 5 × 10 12 Transduction units / mL, at least about 1 × 10⁻⁶ 13 Transduction units / mL, at least approximately 5 × 10 13 Transduction units / mL, at least about 1 × 10⁻⁶ 14 The drug is administered to the subject at a titer of transduction units / mL. The term "transduction unit" (tu) used in relation to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured by a functional assay such as Xiao et al. (1997) Exp. Neurobiol., 144:113-124 or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0304] The vector dose is generally determined by the route of administration. In certain cases, intraganglionic injection involves approximately 1 × 10¹⁶ doses in a volume of approximately 0.1 mL to approximately 1.0 mL. 9 ~Approx. 1×10 13The vector genome may contain approximately 1 × 10¹¹ units in a volume of approximately 1.0 mL to approximately 12.0 mL. In other specific cases, intrathecal injection may contain approximately 1 × 10¹¹ units in a volume of approximately 1.0 mL to approximately 12.0 mL. 10 ~Approx. 1×10 15 The vector genome may contain approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. In yet another specific case, intracranial injection may contain approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. 9 ~Approx. 1×10 13 The vector genome may contain approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. In other specific cases, intraneuronal injection may contain approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. 9 ~Approx. 1×10 13 It may contain a vector genome. In another specific example, intrathecal injection contains approximately 1 × 10⁶ in a volume of approximately 0.1 mL to approximately 1.0 mL. 9 ~Approx. 1×10 13 It may contain a vector genome. In yet another specific case, the cisterna mass injection contains approximately 5 × 10 in a volume of approximately 0.5 mL to approximately 5.0 mL. 9 ~Approx. 5×10 13 The vector genome may contain approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. In yet another specific case, the subcutaneous injection contains approximately 1 × 10¹¹ units in a volume of approximately 0.1 mL to approximately 1.0 mL. 9 ~Approx. 1×10 13 It may contain a vector genome.

[0305] In some cases, the vector is delivered to the target by injection. The amount of vector delivered to the target by injection can be measured as the vector injection rate. Non-limiting examples of vector injection rates include 1–10 μL / min for intraganglionic, intraspinal, intracranial, or intraneural administration, and 10–1000 μL / min for intrathecal or cisternal ventricle administration. In some cases, the vector is delivered to the target by MRI-guided convection-enhanced drug delivery (CED). This technique allows for increased viral dispersal and transduction, distributing the virus throughout a large portion of the brain, and reduces vector backflow along the needle pathway.

[0306] In various embodiments, methods are provided that include administering a vector encoding an engineered receptor that inactivates or hyperpolarizes nerve cells to one or more nerve cells to increase sensitivity to pain or aches, and targeting a ligand that specifically binds to nerve cells expressing the engineered receptor, thereby inactivating the cells, reducing sensitivity to pain, and enhancing analgesic effects.

[0307] In various embodiments, methods are provided that include administering a vector encoding an engineered receptor that activates or polarizes nerve cells to one or more nerve cells to reduce their sensitivity to pain or aches, and administering a ligand that specifically binds to nerve cells expressing the engineered receptor to target cells, thereby activating the cells, reducing their sensitivity to pain, and enhancing the analgesic effect.

[0308] Ligand formulations can be administered to a subject via various routes. Non-limiting examples of administration methods include subcutaneous, intravenous, intramuscular, transdermal, intradermal, intraperitoneal, oral, infusion, intracranial, intrathecal, intranasal, ganglionary, and intraneural administration. In some cases, administration may involve injection of a liquid formulation of the ligand. In other cases, administration may involve oral delivery of a solid formulation of the ligand. In certain cases, the ligand is administered orally (e.g., in pills, tablets, capsules, etc.). In some cases, the oral composition may be administered with food. In another specific case, the ligand is administered by intrathecal injection (i.e., into the subarachnoid space of the spinal cord) for delivery to the cerebrospinal fluid (CSF) of the subject. In yet another specific case, the ligand is administered topically (e.g., in skin patches, creams, lotions, ointments, etc.).

[0309] The dosage of ligand administered to a subject is not absolutely limited, but depends on the composition, the properties of its active ingredients and their undesirable side effects (e.g., immune response to antibodies), the type of subject and symptom being treated, and the mode of administration. Generally, the dose is a therapeutically effective dose, such as an amount sufficient to achieve the desired biological effect, for example, an amount effective in reducing or attenuating the level of pain experienced by the subject. In certain embodiments, the dose may also be a prophylactic dose or an effective dose. The therapeutically effective dose of ligand may depend on the route of administration, the indication being treated, and / or the ligand selected for use.

[0310] In one embodiment, the ligand is initially administered to the subject before the vector is administered. A therapeutically effective dose of the ligand can be administered to the subject at some point after the vector has been delivered. Generally, after the vector has been delivered, there is a period of time required for one or more cells of the subject to produce the protein encoded by the vector (i.e., the engineered receptor). During this period, administering the ligand to the subject may not be beneficial to the subject. In this situation, it may be appropriate to administer the ligand after a certain amount of the engineered receptor has been produced by one or more cells of the subject.

[0311] In one embodiment, the ligand is initially administered to the target at almost the same time as the vector is administered to the target.

[0312] In one embodiment, the ligand is initially administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 11 or 12 hours, days, weeks, months, or years after the administration of the vector to the subject. In some cases, a therapeutically effective dose of the ligand may be administered to the subject at least 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, or more than 30 days after the delivery of the vector. In certain examples, a therapeutically effective dose of the ligand is administered to the subject at least one week after the delivery of the vector. In further examples, therapeutically effective doses of the ligand are administered daily for at least three consecutive days.

[0313] The therapeutically effective dose or dosage of the ligand in this disclosure may be expressed as mg or μg of ligand per kg of body weight of the subject. In some examples, the therapeutically effective dose of ligand is approximately 0.001 μg / kg, approximately 0.005 μg / kg, approximately 0.01 μg / kg, approximately 0.05 μg / kg, approximately 0.1 μg / kg, approximately 0.5 μg / kg, approximately 1 μg / kg, approximately 2 μg / kg, approximately 3 μg / kg, approximately 4 μg / kg, approximately 5 μg / kg, approximately 6 μg / kg, approximately 7 μg / kg, approximately 8 μg / kg, approximately 9 μg / kg, approximately 10 μg / kg, approximately 20 μg / kg, approximately 30 μg / kg, approximately 40 μg / kg, approximately 5 0μg / kg, approximately 60μg / kg, approximately 70μg / kg, approximately 80μg / kg, approximately 90μg / kg, approximately 100μg / kg, approximately 120μg / kg, approximately 140μg / kg, approximately 160μg / kg, approximately 180μg / kg, approximately 200μg / kg, approximately 220μg / kg, approximately 240μg / kg, approximately 260μg / kg, approximately 280μg / kg, approximately 300μg / kg, approximately 320μg / kg, approximately 340μg / kg, approximately 360μg / kg, approximately 380μg / kg, approximately 400μ g / kg, approximately 420μg / kg, approximately 440μg / kg, approximately 460μg / kg, approximately 480μg / kg, approximately 500μg / kg, approximately 520μg / kg, approximately 540μg / kg, approximately 560μg / kg, approximately 580μg / kg, approximately 60 0μg / kg, approximately 620μg / kg, approximately 640μg / kg, approximately 660μg / kg, approximately 680μg / kg, approximately 700μg / kg, approximately 720μg / kg, approximately 740μg / kg, approximately 760μg / kg, approximately 780μg / kg, approximately 8 It may be 00 μg / kg, approximately 820 μg / kg, approximately 840 μg / kg, approximately 860 μg / kg, approximately 880 μg / kg, approximately 900 μg / kg, approximately 920 μg / kg, approximately 940 μg / kg, approximately 960 μg / kg, approximately 980 μg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, or more than 10 mg / kg.

[0314] In certain embodiments, the dose of ligand administered to the subject is at least about 0.001 micrograms / kilogram (μg / kg), at least about 0.005 μg / kg, at least about 0.01 μg / kg, at least about 0.05 μg / kg, at least about 0.1 μg / kg, at least about 0.5 μg / kg, 0.001 milligrams / kilogram (mg / kg), at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, or at least about 10 mg / kg or more.

[0315] In certain embodiments, the dose of ligand administered to the subject is at least about 0.001 μg / kg to at least about 10 mg / kg, at least about 0.01 μg / kg to at least about 10 mg / kg, at least about 0.1 μg / kg to at least about 10 mg / kg, at least about 1 μg / kg to at least about 10 mg / kg, at least about 0.01 mg / kg to at least about 10 mg / kg, at least about 0.1 mg / kg to at least about 10 mg / kg, or at least about 1 mg / kg to at least about 10 mg / kg, or any intermediate range therebetween.

[0316] In some embodiments, a therapeutically effective amount of ligand can be expressed as a molar concentration (i.e., M or mol / L). In some cases, a therapeutically effective amount of ligand is approximately 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM , 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or more.

[0317] A therapeutically effective dose of ligand may be administered once or more daily. In some cases, a therapeutically effective dose of ligand may be administered as needed (e.g., if pain relief is required). Ligands may be administered continuously (e.g., daily without interruption for the duration of the treatment regimen). In some cases, the treatment regimen may last less than one week, one week, two weeks, three weeks, one month, or more than one month. In some cases, a therapeutically effective dose of ligand may be administered daily, for at least two consecutive days, for at least three consecutive days, for at least four consecutive days, for at least five consecutive days, for at least six consecutive days, for at least seven consecutive days, for at least eight consecutive days, for at least nine consecutive days, for at least ten consecutive days, or for at least ten consecutive days. In certain cases, a therapeutically effective dose of ligand may be administered for three consecutive days. In some cases, a therapeutically effective dose of the ligand may be administered once, twice, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-five, thirty, thirty-five, forty, or more than forty times per week. In some cases, a therapeutically effective dose of the ligand may be administered once, twice, three, four, five, six, seventeen, eighteen, nineteen, or more than ten times per day. In some cases, a therapeutically effective dose of the ligand may be administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine hours, at least every ten hours, at least every eleven hours, at least every twelve hours, at least every thirteen hours, at least every fourteen hours, at least every fifteen hours, at least every sixteen hours, at least every seventeen hours, at least every eighteen hours, at least every nineteen hours, at least every twenty hours, at least every twenty-one hours, at least every twenty-two hours, at least every twenty-three hours, or at least daily.Ligand doses may be administered to the subject continuously, or 1, 2, 3, 4, or 5 times daily, 1, 2, 3, 4, 5, 6, or 7 times per week, 1, 2, 3, or 4 times per month, 1 every 2 months, 1 every 3 months, 1 every 4 months, 1 every 5 months, or 1 every 6 months, or 1 year, or at longer intervals. Treatment duration may be 1 day, 1, 2, or 3 weeks, 1, 2, 3, 4, 5, 7, 8, 9, 10, or 11 months, 1, 2, 3, 4, or 5 years, or longer.

[0318] The subjects treated by the methods and compositions disclosed herein may be human or non-human animals. As used herein, the term “treat” and its grammatical equivalents generally refer to the use of a composition or method to reduce, eliminate or prevent the symptoms of a disease, including achieving therapeutic and / or preventive benefits. Therapeutic benefits mean delaying, halting, reversing, eradicating, or improving the progression of the symptoms of the disorder or symptom being treated. Preventive benefits of treatment include reducing the risk of a symptom, delaying the progression of a symptom, or reducing the likelihood of a symptom developing.

[0319] Non-human animals include, but are not limited to, non-human primates, domestic animals, household pets, and laboratory animals. For example, non-human animals may be great apes (e.g., chimpanzees, baboons, gorillas, or orangutans), Old World monkeys (e.g., rhesus macaques), New World monkeys, dogs, cats, bison, camels, cattle, deer, pigs, donkeys, horses, mules, llamas, sheep, goats, buffalo, reindeer, yaks, mice, rats, rabbits, or any other non-human animals. The compositions and methods described herein are suitable for the treatment of veterinary animals. Veterinary animals include, but are not limited to, dogs, cats, horses, cattle, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some embodiments, contacting a tissue or cell population with the composition includes administering the composition to the cell population or subject. In some embodiments, administration is carried out in vitro, for example, by adding the composition to a cell culture system. In some embodiments, administration is carried out in vivo, for example, by administration via a specific route. When administering two or more compositions, the compositions may be administered simultaneously (e.g., on the same day) via the same route, or at different times via the same route. Alternatively, the compositions may be administered simultaneously (e.g., on the same day) via different routes, or at different times via different routes.

[0320] The number of times the composition is administered to a subject requiring administration depends on the discretion of the medical professional, the disability, the severity of the disability, and the subject's response to the formulation. In some embodiments, the composition is administered at least once. In further embodiments, administration is performed two or more times within a given period, for example, two, three, four, five, six, seven, eight, nine, ten, or more times. The dose and / or frequency of each administration may be adjusted as necessary based on the patient's symptoms and physiological response.

[0321] In some embodiments, the composition may be administered for a duration sufficient to achieve the desired physiological effect or improvement in the symptom of interest. If the symptom of interest does not improve, the composition may, at the discretion of the physician, be administered chronically, i.e., over a long period including the lifespan of the subject, to improve, otherwise control, or limit the symptoms of the disease or symptom of interest. If the condition of the subject improves, the composition may, at the discretion of the physician, be administered continuously, or the dose of the drug to be administered may be temporarily reduced or temporarily interrupted for a certain period (i.e., a “drug-free period”). The length of the drug-free period may vary between 2 days and 1 year, including, by example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. Dose reductions during drug-free periods may range from 10% to 100%, including, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0322] If the composition is administered more than once, each administration may be performed by the same agent and / or at the same geographical location. Alternatively, each administration may be performed by different parties and / or at different geographical locations. With regard to human and veterinary treatments, the amount of a particular ligand administered may depend on a variety of factors, including the disorder being treated and its severity, the activity of the particular ligand used, the patient's age, weight, overall health, sex, and diet, the timing of administration, route of administration, and rate of excretion of the particular ligand used, the duration of treatment, drugs used in combination with or simultaneously with the particular ligand used, the judgment of the prescribing physician or veterinarian, and similar factors known in the fields of medicine and veterinary medicine. Similarly, the effective concentration of a given composition may depend on a variety of factors, including the patient's or subject's age, sex, weight, genetic condition, and overall health. kit

[0323] In one embodiment, the Disclosure provides a kit comprising a vector comprising a polynucleotide encoding the engineered receptor of the Disclosure. In one embodiment, the Disclosure provides a kit comprising the engineered receptor of the Disclosure.

[0324] In some embodiments, the kit includes (a) a vector comprising a polynucleotide encoding an engineered receptor of the Disclosure, and (b) a non-native ligand of the Disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the kit includes instructions for administering the vector. In some embodiments, the kit includes a device adapted for administering the vector.

[0325] In some embodiments, the kit comprises (a) an engineered receptor of the Disclosure and (b) a non-natural ligand of the Disclosure.

[0326] Table 29 below provides exemplary combinations of engineered receptors and non-native ligands described herein, which may include the kits of this disclosure. Each of the engineered receptors in Table 29 may exist as a protein, a protein-coding polynucleotide, or a vector containing a protein-coding polynucleotide. In some embodiments, the engineered receptor includes a ligand-binding domain derived from human α7-nAChR. In some embodiments, the engineered receptor includes an ionpore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1. In some embodiments, the engineered receptor includes a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 33, except for the mutations shown in Table 29. [Table 29-1] [Table 29-2]

[0327] In some embodiments, the kit further includes packaging material and one or more components therein. The kit may include a label or packaging insert containing a description of the components or instructions for the in vitro, in vivo, or ex vivo use of the components therein.

[0328] The label or insert may include identification information for one or more of its components, dosage, mechanism of action, pharmacokinetics, and pharmacodynamics of the active ingredient. The label or insert may include information identifying the manufacturer, lot number, manufacturer's location and date, and expiration date. The label or insert may include manufacturer information, lot number, manufacturer's location and date. The label or insert may include information about diseases for which the components of the kit may be used. The label or insert may include instructions for clinicians or subjects to use one or more components of the kit in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency, or duration for performing any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein, and instructions.

[0329] Labels or inserts may include information about any benefits that the components may offer, such as preventive or therapeutic benefits. Labels or inserts may also include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which the use of a particular composition is inappropriate. Adverse side effects or complications may also occur if the subject is taking, planning to take, or currently taking one or more other drugs that may be incompatible with the composition, or if the subject is receiving, planning to receive, or currently receiving another incompatible treatment protocol or regimen; therefore, instructions may include information about such incompatibility.

[0330] All articles, publications and patents cited herein are incorporated herein by reference as if each individual article, publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. However, no reference to any reference, article, publication, patent, patent publication and patent application cited herein constitutes, nor does it suggest, that they constitute valid prior art or form part of the common general knowledge in any country of the world.

[0331] Unless the context indicates otherwise, it is particularly intended that the various features described herein may be used in any combination.

[0332] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0333] It should be understood that the above description and the following examples are intended to be illustrative and not to limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art. [Examples]

[0334] Example 1. Discovery of an engineered receptor containing a mutation in the ligand-binding domain. To generate LGICs that conduct anionic currents after exposure of human α7-nAChR to non-natural small molecule agonists, a chimeric ligand-gated ion channel (LGIC) receptor containing a ligand-binding domain derived from human α7 nicotinic acetylcholine receptor (α7-nAChR) and a chloride-conducting ion pore domain derived from human GlyR1α was genetically engineered. The engineered receptor (CODA71) with the amino acid sequence of SEQ ID NO: 33 was identified, which was nearly as sensitive to acetylcholine, ABT-126, and TC-6987 as wild-type α7-nAChR, and TC-6987 showed partial agonist activity against SEQ ID NO: 33 similar to that of wild-type. SEQ ID NO: 33 was approximately twice as sensitive to nicotine as wild-type and approximately three-fold and ten-fold more sensitive to AZD-0328 and fascinicrin / RG3487, respectively. CODA71 is described in detail in International Publication Nos. 2019104307 and 2021035179, which are incorporated in their entirety herein.

[0335] Amino acid substitutions were introduced into the ligand-binding domain of an engineered receptor having the amino acid sequence of SEQ ID NO: 33. The binding pockets for each ligand in α7-nAChR were modeled, and the amino acid residues forming the binding pockets were mapped. Subsequently, libraries of single, double, and triple mutant chimeric LGICs were constructed, with each mutant chimeric LGIC containing substitutions at one or more amino acids in the ligand-binding pocket of SEQ ID NO: 33. The parent chimeric receptor (SEQ ID NO: 33) was cloned into pcDNA3.1(+)(Invitrogen) using standard molecular biology techniques with BamHI and EcoRI sites. Amino acid substitutions were introduced by site-directed mutagenesis. A list of the generated mutants is shown in Table 20 above.

[0336] All of the resulting manipulated receptors were identified with native ligands, acetylcholine (Ach), and non-native ligands, such as AZD-0328 (adisinsight.springer.com / drugs / 800018503), TC-6987 (drugbank.ca / drugs / DB14854), ABT-126 (medchemexpress.com / Nelonicline.html), and TC-5619 (en.wikipedia.org / wiki / Bradanicl). The efficacy against ine, TC-6683 (pubchem.ncbi.nlm.nih.gov / compound / TC-6683_-Azd1446), varenicline (en.wikipedia.org / wiki / Varenicline), fascinicline / RG3487 (researchgate.net / figure / Molecular-structure-of-RG3487_fig1_47499934), CNL001, and CNL002 was analyzed. Example 2: Characterization of manipulated receptors using high-throughput fluorescence-based plate screening

[0337] To screen these mutant LGICs for those with novel response profiles to ligands, we developed an anion reporter assay for evaluating LGIC function in a high-throughput format. In this assay, cells expressing a YFP reporter whose fluorescence is quenched in the presence of an anion are transfected with DNA encoding the channel of interest. Upon exposure to the ligand, the activated channel flows the anion, resulting in dose-dependent quenching of YFP, which can be detected by a plate reader. A stronger quenching signal (i.e., a more positive value) indicates higher ligand activity on the receptor.

[0338] Lenti-X 293T cells (LX293T, Clontech) were maintained in DMEM (Invitrogen) containing 10% FBS and 1% penicillin / streptomycin. For plate reader assays, LX293T cells were infected with lentivirus to produce cells that stably express a mutant YFP (H148Q / I152L) reporter exhibiting enhanced sensitivity to the anion. Two days prior to the assay, cells were divided at a density of 20,000 cells / well in 96-well tissue culture plates coated with poly-d-lysine (Thermo Scientific). The following day, cells were transiently transfected with 0.1 μg / well DNA using the standard Fugene protocol (Promega). On the day of the assay, cells were washed twice with 1× extracellular solution (1×ECS: 140mM NaCl, 5mM KCl, 1mM MgCl2, 2mM CaCl2, 10mM HEPES, 10mM glucose, pH 7.2, mOsms 300). After the final wash, 100 μL of 1×ECS was added to each well, and the plate was incubated at 37°C for 30 minutes. While the plate was incubating, the drug was diluted 2-fold with 1×ECS-NaI (same components as 1×ECS except that 140mM NaCl was replaced with 140mM NaI). The plate was then read using Flexstation3 (Molecular Devices). Each well of the plate, 8 wells at a time, was read for 2 minutes using Flexstation3 (Molecular Devices) as follows: 1) Read baseline YFP fluorescence for 17 seconds, 2) add 100 μL of ligand, and 3) then measure the change in YFP fluorescence every 1.3 seconds for 1 minute and 43 seconds. Quenching is calculated by dividing the average fluorescence over the last 10 seconds of reading by the baseline average over the first 15 seconds before ligand addition.

[0339] As shown in the figures, Figures 1A–1J provide heatmaps of the percentage of YFP fluorescence quenching after stimulation with various doses of acetylcholine or non-native ligands. CODA71 (SEQ ID NO: 33) was used as a control, and CODA75 (SEQ ID NO: 29, a non-responsive chimeric manipulated receptor) was used as a negative control. Quenching of the fluorescence signal, indicated by the blue shaded cells, indicates the level of engineered receptor activation by the non-native ligand at that concentration. The results demonstrate that the engineered receptor has varying potencies against the acetylcholine and non-native ligands tested (see also Section C1, “Amino Acid Variations,” of the above disclosure).

[0340] The values ​​for these heatmaps can be found in Tables 11-20 below. [Table 11-1] [Table 11-2] [Table 11-3] [Table 12-1] [Table 12-2] [Table 12-3] [Table 13] [Table 14-1] [Table 14-2] [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2] [Table 16-3] [Table 17] [Table 18-1] [Table 18-2] [Table 19-1] [Table 19-2] [Table 20-1] [Table 20-2]

[0341] Table 21 below lists the EC50 values ​​from a set of experiments for the shown mutants, comparing the EC50 values ​​for acetylcholine and TC-5619 determined from YFP fluorescence plate reader experiments with the EC50 values ​​from the electrophysiological studies described in Example 3 below. The results in Table 21 demonstrate that the EC50 values ​​obtained from YFP fluorescence plate reader experiments show excellent agreement with values ​​obtained from high-throughput electrophysiological (ephys) studies such as those described in Example 3 below. [Table 21] Example 3: Characterization of manipulated receptors using high-throughput electrophysiology

[0342] EC determined by plate reader 50To confirm and better understand the flow of maximum current, the engineered receptors were subjected to a high-throughput electrophysiology system as described below. For the HEK293T study, the cDNA encoding the ion channel was cloned into pcDNA3.1 using standard recombination techniques. HEK293T cells from Clontech (Lenti-X® 293T cell line) were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin to 40–50% confluence using a standard cell culture protocol, transfected with an ion channel plasmid at a concentration of 18 μg per 15 cm dish using Fugene6, and grown for a further 24 hours. Cells were then assayed in an electrophysiology system (IonFluxHT and / or Mercury, Fluxion Biosciences) that allows for the evaluation of dose-response relationships via a microfluidic-based platform to establish the overall cell composition. Ensemble plates were primed with extracellular buffers (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.2 with NaOH, mOsm 310), intracellular buffers (145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM EGTA, pH 7.2 with CsOH, mOsm 305), and test compounds (freshly prepared stocks) diluted with extracellular buffers, as adapted from Lynagh and Lynch. The compounds were then detached from the plates using accutase, centrifuged, resuspended in extracellular buffers, and loaded onto ensemble plates. Cells were then subjected to standard protocols for priming, capture, disruption, and establishment of total cellular composition, and the cells were maintained at -60 mV throughout the recording. After recording baseline levels, dose-response relationships were evaluated by applying escalating doses of the test compound using IonFlux software.Next, a custom Python script was used to analyze the data offline, converting it to CSV format, replotting the traces, and applying QC measurements to eliminate erroneous recordings (i.e., access resistance and / or baseline standard deviation, as well as thresholding based on artifact removal). Then, the peak current was calculated, and the collective data were fitted using a four-parameter logistic equation, as described by Hill's equation. After adding the drug for 1 second, the current was measured using an automated patch-clamp system (Fluxion Biosciences), and the calculated EC50 values ​​are summarized in Table 22 below and Table 21 above (Example 2). [Table 22-1] [Table 22-2]

[0343] These results indicate that all mutant engineered receptors exhibit reduced potency against acetylcholine compared to wild-type nAchRa7 or the control (SEQ ID NO: 33). For example, some engineered receptors have EC50 values ​​several orders of magnitude higher than those of wild-type nAchRa7. Furthermore, the results show that some engineered receptors exhibit increased potency against specific non-natural ligands compared to the wild-type receptor. For example, engineered receptors containing the L131D, S172D amino acid sequences of SEQ ID NO: 33 show at least a 10-fold increase in potency against AZD-0328 and RG-3487 compared to the wild-type control receptor. These results demonstrate that engineered receptors can be used to reduce potency against acetylcholine while simultaneously maintaining or increasing potency against synthetic small molecule nAChα7 receptor agonists that are recognized as safe and well-tolerated in humans.

[0344] These results from electrophysiological methods provide confirmation of EC50 values ​​from a plate reader and further confirm the decoupling of acetylcholine and non-native ligand responses of the engineered receptors disclosed herein. Example 4: Characterization of manipulated receptors using manual patch-clamp electrophysiology

[0345] Using whole-cell manual patch-clamp electrophysiology in HEK293 cells, rat DRG neurons, and rat hippocampal neurons, the EC50 of various engineered receptors disclosed herein to acetylcholine and non-native ligands was measured using Hill's formula, with peak current measurements from incremental ligand doses. Basal current shifts were also measured in rat DRG neurons to reflect receptor efficacy. To calculate the basal current, the amount of current capable of generating an action potential was first determined. Then, after ligand addition, the injected current was gradually increased up to 700 pA.

[0346] For manual patch-clamp experiments, cells plated on coverslips were visualized at 10x and 40x magnification using an inverted fluorescence microscope (Olympus). A 3–6 MOhm glass patch electrode (Sutter, BF150-86-10) was used, along with an Axopatch 200B amplifier and a Digidata 1550B (Molecular Recordings were made at room temperature using Molecular Devices. Unless otherwise specified, recordings were made in extracellular solution (ECS) containing 140 mM sodium chloride, 4 mM potassium chloride, 1 mM magnesium chloride, 2 mM calcium chloride, 10 mM HEPES, and 10 mM D-(+)-glucose (pH=7.3, molar osmotic pressure=305-315 mOsm using sodium hydroxide). An 8-line reservoir (AutoMate Scientific) combined with an 8-channel zero-dead-volume perfusion penc...

Claims

[Claim 1] The invention described herein.

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