Expression cassettes for treating epilepsy and neuropathic pain - Patent Application 20070229933

Abstract

The present disclosure provides expression cassettes, vectors and methods for expressing transgenes in cells, for example, neurons.Furthermore, methods are provided for achieving desired expression levels of transgenes in neuronal cells, for example, for use in combination with small molecule ligands to treat focal epilepsy or neuropathic pain.In one aspect, the present disclosure provides a recombinant nucleic acid comprising an expression cassette, comprising, in the order of 5' to 3', one or more of a 5' enhancer, a promoter, a 5' untranslated region (UTR), a transgene, a 3' enhancer, and a polyadenylation sequence (polyA), wherein the transgene is operably linked to the promoter.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 304,960, filed January 31, 2022, and U.S. Provisional Patent Application No. 63 / 312,480, filed February 22, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (SWCH_038_02WO_SeqList_ST26.xml; size: 383,720 bytes; and creation date: January 29, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Intractable neurological diseases are often associated with abnormally active neurons, and attempts to develop therapies to treat these conditions have been hampered by the lack of tractable target proteins associated with the diseases.

[0004] Focal epilepsy is a chronic, debilitating neurological disorder affecting over 2 million people in the United States and is characterized by unpredictable seizures that initiate from specific locations in the brain. Recurrent seizures result in cognitive and emotional deficits, and current interventions have limited efficacy and multiple side effects. Although focal seizures result from abnormal firing in a subset of neurons, many antiepileptic drugs rely on systemic compound administration to alter whole-brain activity. Thus, patients with epilepsy suffer from substantial side effects due to unintended modulation of neurons involved in normal cognition and undesirable off-target changes in other biological systems. Mesial temporal lobe epilepsy (mTLE) is the most common form of focal epilepsy, and it is commonly associated with hippocampal sclerosis as a histopathological abnormality.

[0005] Neuropathic pain includes, among others, peripheral neuropathy and trigeminal neuralgia. The former is a common neurological disorder resulting from damage to, or dysfunction of, the peripheral nervous system. It is characterized by numbness, tingling and pain, and often begins in the hands and feet, but can affect other areas of the body. Trigeminal neuralgia, also known as suicide disease, is caused by damage to the myelin sheath that protects the trigeminal cranial nerve. It results in extreme, diffuse, shock-like facial pain that can last from seconds to minutes. Current pharmacological and surgical approaches provide little relief and are potentially addictive, while having significant side effects.

[0006] The most commonly used treatment for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive impairment, hallucinations, and other systemic side effects.Despite the widespread use of pharmaceutical drugs, their effectiveness in pain relief has a surprisingly low success rate.More invasive options for pain treatment include nerve blocks and electrical stimulation.The most invasive and least preferred method for managing pain is the complete surgical removal of the nerve or part thereof that is causing pain.

[0007] An ideal pain and focal epilepsy treatment would alter only the activity of the specific neurons responsible for the occurrence of pain and / or seizures. Thus, a new strategy for treating pain or focal epilepsy is to deliver heterologous proteins encoded by recombinant nucleic acids to subpopulations of neurons to control their abnormal activity.

[0008] One consideration of this strategy is the selection of a heterologous protein to control neuronal activity and its expression in target cells. Recently, designer receptors activated only by designer drugs to alter seizure activity (DREADDs) have shown promising results in animal epilepsy models. However, inhibitory DREADDs rely on G protein-coupled receptors that indirectly activate ion channels via second messengers, and can inconsistently alter neuronal potentials.

[0009] An alternative treatment approach is to transduce targeted neurons with a transgene encoding an engineered ligand-gated ion channel (LGIC) that can then respond to exogenous ligands. Oral administration of this small molecule ligand, designed to interact only with the engineered LGIC, can be finely tuned to control abnormal neuronal activity and suppress seizures without adverse effects.

[0010] There is a need in the art to design an optimal expression cassette that allows robust expression of transgene in target cells to control neuronal activity.Such expression cassette can be used to treat neuropathic pain such as peripheral neuropathy and trigeminal neuralgia, or focal epilepsy such as medial temporal lobe epilepsy.The present disclosure provides such expression cassette, vector, method, etc. Summary of the Invention [Means for solving the problem]

[0011] overview In one aspect, the disclosure provides a recombinant nucleic acid comprising an expression cassette comprising, in 5' to 3' order, one or more of a 5' enhancer, a promoter, a 5' untranslated region (UTR), a transgene, a 3' enhancer, and a polyadenylation sequence (polyA), wherein the transgene is operably linked to the promoter.

[0012] In some embodiments, the expression cassette comprises a 5' enhancer comprising a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 37-39.

[0013] In some embodiments, the expression cassette does not comprise a 5' enhancer.

[0014] In some embodiments, the promoter comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 41-51. In some embodiments, the promoter is a neuron-specific promoter.

[0015] In some embodiments, the expression cassette comprises an intron between the promoter and the transgene. In some embodiments, the expression cassette comprises an intron between the 5'UTR and the transgene. In some embodiments, the expression cassette comprises an intron between the promoter and the 5'UTR. In some embodiments, the intron comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 57-61.

[0016] In some embodiments, the expression cassette does not contain an intron.

[0017] In some embodiments, the expression cassette comprises a 5'UTR comprising a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs:52-56.

[0018] In some embodiments, the expression cassette comprises a 3' enhancer comprising a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs:62-65.

[0019] In some embodiments, the expression cassette comprises a polyA comprising a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs:67-70.

[0020] In some embodiments, the expression cassette comprises a non-neuronal silencing element embedded in a promoter, wherein the non-neuronal silencing element comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 40. In some embodiments, the expression cassette comprises a non-neuronal silencing element between the 5' enhancer and the promoter, wherein the non-neuronal silencing element comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 40.

[0021] In some embodiments, the expression cassette comprises a 3'UTR between the 3' enhancer and the polyA, wherein the 3'UTR comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:66.

[0022] In some embodiments, the transgene encodes a ligand-gated ion channel (LGIC). In some embodiments, the ligand-gated ion channel comprises a ligand-binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR). 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:25. In some embodiments, the ligand-binding domain comprises one or more amino acid mutations listed in Table 5. In some embodiments, the ligand-gated ion channel comprises an ion pore domain derived from the human glycine receptor. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity to amino acids 255-457 of SEQ ID NO:26, 260-452 of SEQ ID NO:27, amino acids 259-464 of SEQ ID NO:28, or amino acids 259-449 of SEQ ID NO:29. In some embodiments, the ligand-binding domain of the engineered receptor comprises a Cys loop domain derived from the human glycine receptor. In some embodiments, the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 25-31 and 33. In some embodiments, the human glycine receptor is human glycine receptor alpha 1 and the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the transgene comprises or consists of a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 32. In some embodiments, the transgene is codon-optimized for expression in a human cell. In some embodiments, the human cell is a neuron.

[0023] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (iii) an hCaMKII 5' UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:53; (iv) an hCaMKII 5' UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:58; (v) a transgene; (vi) a WPREx3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:64; (vii) a WPREx3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:66; and (viii) an alpha globin 3'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:68; and (viii) an hGH polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:68.

[0024] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 173 to 1877 of SEQ ID NO:108, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 3237 to 4315 of SEQ ID NO:108.

[0025] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:48; (iii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:54. (iv) an hSyn intron comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:59; (v) a transgene; (vi) a WPREx3' end gene comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:64. (vii) an alpha globin 3'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:66; and (viii) an hGH polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:68.

[0026] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 173 to 1159 of SEQ ID NO:106, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 2519 to 3597 of SEQ ID NO:106.

[0027] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:38; (ii) an hSyn-V2 promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:49; (iii) a transgene; and (iv) a WPREx-V2 promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:65. a 3' enhancer; and (v) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69.

[0028] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 149-946 of SEQ ID NO:125, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 2285-3416 of SEQ ID NO:125.

[0029] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:38; (ii) a hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (iii) a transgene; and (iv) a WPREx-V2 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:65. a 3' enhancer; and (v) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69.

[0030] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 149-1527 of SEQ ID NO:126, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 2848-3979 of SEQ ID NO:126.

[0031] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) an hSyn-V2 promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:49; (ii) a transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:65; and (iv) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69.

[0032] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 153 to 661 of SEQ ID NO:127, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 1982 to 3113 of SEQ ID NO:127.

[0033] In some embodiments, the expression cassette comprises, in order from 5' to 3', (i) an hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (ii) a transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:65; and (iv) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69.

[0034] In some embodiments, the expression cassette comprises, in 5' to 3' order, a transgene sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 153 to 1224 of SEQ ID NO:128, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 2545 to 3676 of SEQ ID NO:128.

[0035] In some embodiments, the expression cassette comprises, in 5' to 3' order, (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:48; (iii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:54. (iv) an hTPI intron comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:60; (v) a transgene; (vi) a FullEES 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:63; and (vii) a rβ globin polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:70.

[0036] In some embodiments, the expression cassette comprises, in 5' to 3' order, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 173 to 1188 of SEQ ID NO:98, a transgene, and a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to nucleotides 2548 to 3824 of SEQ ID NO:98.

[0037] In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 71-93, excluding the sequence of the transgene (SEQ ID NO: 36). In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 121-124, excluding the sequence of the transgene (SEQ ID NO: 32). In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 71-93 and 121-124.

[0038] In some embodiments, the recombinant nucleic acid comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR) flanking each end of the expression cassette. In some embodiments, the recombinant nucleic acid comprises a 5' ITR sequence having at least 90% identity to SEQ ID NO: 94 or 119 and a 3' ITR sequence having at least 90% identity to SEQ ID NO: 95 or 120.

[0039] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 96-118 and 125-128. In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 96-118, excluding the sequence of the transgene (SEQ ID NO: 36). In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 125-128, excluding the sequence of the transgene (SEQ ID NO: 32).

[0040] In one aspect, the present disclosure provides a vector comprising the recombinant nucleic acid of the present disclosure.In some embodiments, the vector is a non-viral vector.In some embodiments, the vector is a viral vector.In some embodiments, the vector comprises or consists of AAV vector genome.

[0041] In one aspect, the present disclosure provides an AAV comprising the vector of the present disclosure.In some embodiments, the AAV is an AAV9 serotype.In some embodiments, the AAV is a self-complementary AAV or a single-stranded AAV.In some embodiments, the AAV is a wild-type AAV or a modified AAV.In some embodiments, the AAV comprises a capsid protein that has at least 95% identity with the AAV9 capsid protein (SEQ ID NO: 8) or the AAV9-TV capsid protein (SEQ ID NO: 9).

[0042] In one aspect, the disclosure provides a host cell comprising a nucleic acid of the disclosure or a vector of the disclosure.

[0043] In one aspect, the disclosure provides a method of producing an AAV of the disclosure.

[0044] In one aspect, the present disclosure provides a kit comprising a recombinant nucleic acid of the present disclosure, a vector of the present disclosure, or an AAV of the present disclosure.

[0045] In one aspect, the present disclosure provides a method of expressing a transgene in a cell comprising delivering a recombinant nucleic acid of this disclosure or a vector of this disclosure to a cell.

[0046] In one aspect, the disclosure provides a method of transducing a cell, comprising contacting the cell with an AAV of the disclosure. In some embodiments, the cell is a neuron. In some embodiments, the neuron is a hippocampal neuron. In some embodiments, the neuron is an excitatory neuron. In some embodiments, the neuron is a CAMK2 positive neuron. In some embodiments, the neuron is an inhibitory neuron. In some embodiments, the neuron is a GABAergic neuron. In some embodiments, the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron. In some embodiments, the neuron comprises an isolectin B4 (IB4) positive nerve fiber. In some embodiments, the neuron comprises an NF200 positive nerve fiber. In some embodiments, the neuron comprises a CGRP positive nerve fiber. In some embodiments, the neuron comprises a C fiber. In some embodiments, the neuron comprises an Aδ fiber. In some embodiments, the cell is an ex vivo cell. In some embodiments, the cell is an in vivo cell of a subject, optionally wherein the subject is a human. In some embodiments, cells comprising the expression cassette have a higher expression level of the transgene compared to a corresponding cell comprising a control expression cassette, optionally said higher expression being at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the transgene expression level of a control expression cassette comprising the polynucleotide sequence of SEQ ID NO: 88 excluding the transgene sequence.In some embodiments, cells comprising the expression cassette have comparable or higher transgene expression levels compared to corresponding cells comprising a control expression cassette, optionally wherein the transgene expression level is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold the transgene expression level of a control expression cassette comprising the polynucleotide sequence of SEQ ID NO: 87 excluding the transgene sequence.

[0047] In one aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant nucleic acid of the present disclosure, a vector of the present disclosure, or an AAV of the present disclosure, wherein the disease or disorder is epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome. In some embodiments, the epilepsy is focal epilepsy. In some embodiments, the epilepsy is mesial temporal lobe epilepsy (mTLE). In some embodiments, the recombinant nucleic acid, vector, or AAV is administered intracranially, intrathecally (spinal), intrathecally (cisternal), intracerebrally, intraventricularly, or by direct injection into the epileptic focus of the hippocampus. In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by direct injection into the epileptic focus of the hippocampus. In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by 1×10 9 ~1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject. In some embodiments, the AAV comprises the capsid protein of AAV9 (SEQ ID NO: 8). In some embodiments, the method reduces the duration, intensity, and / or frequency of epilepsy by at least 10%.

[0048] In one aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant nucleic acid of the present disclosure, a vector of the present disclosure, or an AAV of the present disclosure, wherein the disease or disorder is neuropathic pain, spasticity, spinal cord injury, or avulsion injury. In some embodiments, the disease or disorder is neuropathic pain. In some embodiments, the neuropathic pain is peripheral neuropathy. In some embodiments, the neuropathic pain is trigeminal neuralgia. In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by intrathecal (IT) or intraganglionic (IG) administration. In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by intraganglionic (IG) administration directly to the dorsal root ganglion or trigeminal ganglion. In some embodiments, 1×10 9 ~1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject. In some embodiments, the AAV comprises the capsid protein of AAV9-TV (SEQ ID NO: 9). In some embodiments, the method reduces the level of pain by at least 10%.

[0049] In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by systemic, parenteral, intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputamenal, intrahippocampal, intrastriatal, or intracerebroventricular injection. 9 ~1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

[0050] In some embodiments, the method comprises administering a ligand of the ligand-gated ion channel encoded by the transgene. In some embodiments, the ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487. In some embodiments, the ligand is TC-5619.

[0051] The patent or application file contains at least one drawing executed in color. [Brief description of the drawings]

[0052] [Figure 1-1] Figure 1 shows the design of the 27 expression cassettes. For each cassette, the left element is located upstream (5') of the right element. The "*" symbol in cassette number 20 indicates that the NRSE element is embedded within the CMV promoter instead of being placed upstream of the CMV promoter in this cassette. [Figure 1-2] Same as above.

[0053] [Diagram 2] FIG. 2 shows the results of ddPCR of CODA receptor mRNA ratios relative to the β-actin housekeeping gene in SK-N-AS and HeLa cells transfected with the indicated cassettes.

[0054] [Diagram 3] FIG. 3 shows the results of FLAG ELISA of CODA receptor protein in SK-N-AS and HeLa cells transfected with the indicated cassettes.

[0055] [Figure 4] FIG. 4 shows the results of ddPCR for CODA receptor mRNA expression ratios compared to the β-actin housekeeping gene for nine expression cassettes packaged as AAV9 vectors and transduced in neonatal rat hippocampal mixed cultures.

[0056] [Diagram 5] FIG. 5 shows the results of ddPCR of the CODA receptor mRNA ratio compared to the β-actin housekeeping gene in rat hippocampus directly injected in vivo with AAV9 carrying six expression cassettes.

[0057] [Figure 6]FIG. 6 shows the localization of FLAG-tagged CODA receptors by immunofluorescence from six expression cassettes tested in vivo in the rat hippocampus.

[0058] [Figure 7] FIG. 7 shows seizure frequency in a KA focal epilepsy mouse model injected with AAV containing the indicated expression cassettes followed by repeated administration of TC-5619.

[0059] [Figure 8] FIG. 8 shows the design of an efficacy study using the KA focal epilepsy mouse model injected with AAV containing the indicated expression cassettes.

[0060] [Figure 9] FIG. 9 shows the results of ddPCR for CODA receptor mRNA expression ratios compared to the β-actin housekeeping gene of 10 expression cassettes packaged as AAV6 vectors and transduced into human iPSC-derived sensory neuron progenitor cells.

[0061] [Figure 10] FIG. 10 shows the results of ddPCR of the CODA receptor mRNA ratio compared to the β-actin housekeeping gene in rat L3 and L4 DRG directly injected in vivo with AAV6 carrying the four expression cassettes.

[0062] [Figure 11] FIG. 11 shows the localization of FLAG-tagged CODA receptors by immunofluorescence from the four expression cassettes tested in vivo in rat L5 DRG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Detailed Description Details of the present disclosure are described in the attached description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described here. Other features, objects, and advantages of the present disclosure are clear from the description and claims. In this specification and the appended claims, the singular form includes the plural form unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications cited herein are incorporated herein by reference in their entirety.

[0064] The embodiments according to the present disclosure will be described more completely below. However, aspects of the present disclosure may be embodied in different forms, and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Unless otherwise expressly indicated, all specific embodiments, features, and terms are intended to include both the recited embodiments, features, or terms, and their biological equivalents. The terms used in the description herein are intended to describe only specific embodiments, and are not intended to be limiting.

[0065] Before describing the method and composition of the present invention, it should be understood that the present disclosure is not limited to the specific method or composition described, and therefore may of course vary.It should also be understood that the scope of the present disclosure is limited only by the appended claims, and therefore the terms used herein are intended to describe only specific embodiments and are not intended to be limiting.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.

[0066] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. definition

[0067] Throughout this disclosure, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g., polypeptides known to those of skill in the art, and so forth.

[0068] Throughout this specification, the term "and / or" is used in the present disclosure to refer to either "and" or "or," unless specified otherwise.

[0069] Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of a stated element or integer or group of elements or integers but not to the exclusion of any other element or integer or group of elements or integers. Moreover, the recitation of numerical ranges throughout this specification specifically includes all integers and decimal points therebetween.

[0070] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting essentially of" refers to a limitation of the scope of a described composition, method, or kit to specified materials or steps that do not substantially affect the basic and novel feature(s) of the subject disclosure. For example, a polypeptide "consisting essentially of" a disclosed sequence has an amino acid sequence that is the disclosed sequence plus or minus about 5 amino acid residues of the boundary of the sequence, e.g., about 5, 4, 3, 2, or about 1 residue less than the recited boundary amino acid residue, or about 1, 2, 3, 4, or 5 residues more than the recited boundary amino acid residue.

[0071] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a polypeptide "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0072] The terms "about" and "approximately" are used as equivalents. Any numerical values ​​used in this application, including or not including about / approximately, are meant to encompass any normal variation that would be understood by a person skilled in the relevant technical field. In certain embodiments, the term "about" or "about" refers to a range of values ​​that is within 10% (greater or less) in either direction of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of possible values).

[0073] The term "isolated" means material that is substantially or essentially free from components that normally accompany it as found in its natural state. In some embodiments, the term "obtained" is used synonymously with isolated.

[0074] The terms "subject", "individual" and "patient" are used interchangeably to refer to vertebrates, such as mammals. A mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee) or a human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject may be an adult, a teenager, a child (2-14 years), an infant (1-24 months), or a newborn (up to 1 month). In some embodiments, an adult is about 65 years or older, or an elderly person about 60 years or older.

[0075] The term "sample" refers to a volume and / or mass of biological material that is subjected to analysis. In some embodiments, samples include tissue samples, cell samples, fluid samples, etc. In some embodiments, samples are taken from or provided by a subject (e.g., a human subject). In some embodiments, samples include portions of tissue taken from any internal organs, cancerous, precancerous, or noncancerous tumors, brain, skin, hair (including hair roots), eye, muscle, 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, peritoneal fluid, pleural fluid, cerebrospinal fluid, lung lavage, tears, sweat, etc. One of skill in the art will appreciate that in some embodiments, a "sample" is a "primary sample" in that it is obtained directly from a source (e.g., a subject). In some embodiments, a "sample" is the result of processing of a primary sample, for example, to remove certain potentially contaminating components, to isolate certain components, and / or to purify certain components of interest. In some embodiments, the sample is a cell or a population of cells (e.g., neuronal cells). The cell sample may be directly derived from a subject (e.g., a primary sample) or may be a cell line. The cell line may include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0076] The terms "treating", "treatment" and grammatical equivalents as used herein generally refer to the use of a composition or method to reduce, eliminate, or prevent symptoms of a disease, including achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit means slowing down, stopping, reversing, or eradicating or ameliorating the progression of the symptoms of the disorder or condition being treated. A prophylactic benefit of treatment includes reducing the risk of a condition, delaying the progression of a condition, or reducing the likelihood of occurrence of a condition. In some embodiments, treating refers to delivering a composition to a subject and / or a population of cells to affect a physiological outcome. In some embodiments, treatment results in an improvement (e.g., reduction, improvement, or correction) of one or more disease symptoms. The improvement may be an observable or measurable improvement, or an improvement in the subject's general sense of well-being. Treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to a reduction in the severity of disease symptoms to a level equivalent to that before the onset of the disease. In some embodiments, treatment may refer to short-term (e.g., temporary or acute) and / or long-term (e.g., sustained or chronic) reduction of disease symptoms. In some embodiments, treatment may refer to the amelioration of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of a subject at risk of developing a particular disease to prevent the onset of the disease. Prevention of disease onset may refer to preventing disease symptoms altogether, delaying the onset of the disease, reducing the severity of symptoms in a disease that has subsequently developed, or reducing the likelihood of disease onset.

[0077] The term "management" or "controlling" refers to the use of the compositions or methods contemplated in this disclosure to improve the quality of life of an individual suffering from a particular disease.

[0078] The term "effective amount" refers to an amount capable of achieving a stated result (e.g., transducing a target neuron). In some embodiments, an effective amount for administration in vivo refers to a dose capable of achieving a desired result, such as, for example, transducing a sufficient amount of a target neuron for therapeutic purposes.

[0079] A "therapeutically effective amount" is an amount of a composition that can achieve a desired therapeutic result. The therapeutically effective amount may vary depending on factors such as, but not limited to, the disease state and the subject's age, sex, and weight. In general, a therapeutically effective amount is also one in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes an amount of a composition that is effective for treating a subject.

[0080] "Increase" refers to an increase in value (e.g., an increase in transduction efficiency) of at least 5% compared to a reference or control level. For example, an increase may include an increase of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. Increase also refers to an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) over a reference or control level.

[0081] "Decrease," "reduce," "diminish" or synonyms thereof refer to a decrease in value (e.g., a decrease in transduction efficiency) of at least 5% compared to a reference or control level. For example, a decrease may include a decrease of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. A decrease also refers to a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) lower than a reference or control level.

[0082] The term "reference" or "control" level is used interchangeably throughout this disclosure and refers to the value of a particular physiological effect and / or therapeutic effect in a subject or sample that is not treated with the composition of the present disclosure, or in a subject or sample that is treated with a control. In some embodiments, the control is a vehicle control. In some embodiments, the reference level refers to the value of a particular physiological effect and / or therapeutic effect that is a measure in a subject or sample before administration of the composition of the present disclosure (e.g., baseline level).

[0083] The term "ligand" refers to a molecule that binds to another, larger molecule. In some embodiments, a ligand binds to a receptor. In some embodiments, the binding of a ligand to a receptor alters the function of the receptor to activate or inhibit its function. In some embodiments, the binding of a ligand to a receptor, such as a ligand-gated ion channel (LGIC), results in the opening or closing of an ion channel. The term "ligand" may refer to an endogenous or naturally occurring ligand. For example, in some embodiments, a ligand refers to a neurotransmitter (e.g., lambda-aminobutyric acid (GABA), acetylcholine, serotonin, etc.) and a signaling intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., synthetic, or non-naturally occurring ligand (e.g., a binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by various methods known in the art (e.g., detection of association with a radiolabeled ligand). "Receptor-ligand binding" and "ligand binding" are used interchangeably throughout this disclosure and refer to the physical interaction between a receptor (eg, LGIC) and a ligand.

[0084] "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 throughout this disclosure, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described in this disclosure.

[0085] The term "wild-type" or "native" is a term of the art understood by those of skill in the art and refers to the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of that protein as it occurs in nature.

[0086] The terms "non-naturally occurring," "variant," and "mutant" are used interchangeably throughout the specification and claims to refer to variants of a naturally occurring or wild-type composition, e.g., variant polypeptides having less than 100% sequence identity with the naturally occurring or wild-type sequence.

[0087] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids. The terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, methylation, carboxylation, deamidation, acetylation, or conjugation with a labeling moiety.

[0088] The amino acid modification may be an amino acid substitution, an amino acid deletion, and / or an amino acid insertion. The amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, conservative substitution, or conservative variation) is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used throughout this disclosure, a "conservative variation" refers to the replacement of an amino acid residue with another, biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another; or the replacement of one polar residue for another, such as the replacement of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Other examples of conservative substitutions include alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to praline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, and the like.

[0089] The term "engineered" is used throughout this specification and claims to refer to a non-naturally occurring composition or protein that has properties that differ from the parent composition or protein from which it is derived.

[0090] Generally, "sequence identity" or "sequence homology" refers to the correspondence between nucleotides or amino acids of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity". The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. This program can be used to determine the percent identity over the entire length of the proteins being compared. For example, default parameters are provided for optimizing searches with short query sequences using the BLAST program.The program also allows the use of a SEG filter to mask off segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Desirable ranges of sequence identity are approximately 80% to 100% and intervening integer values. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0091] As used throughout this disclosure, "substantially identical" refers to having a sequence identity that is 85% or greater, e.g., 90% or greater, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, where the activity of the composition is not altered by modifications in the sequence that result in differences in sequence identity.

[0092] As used throughout this disclosure, an "amino acid mutation" refers to any difference in an amino acid sequence compared to the corresponding parent sequence, such as an amino acid substitution, deletion, and / or insertion.

[0093] As used throughout this disclosure, the term "viral vector" or "viral vector" refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid (e.g., an expression cassette) packaged within the virion. Exemplary viral vectors of this disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0094] "AAV virion" or "AAV virus" or "AAV virus particle" or "AAV vector particle" or "AAV vector" refers to a viral particle that contains at least one AAV capsid polypeptide and a polynucleotide (e.g., an AAV vector genome containing an expression cassette) enclosed in the capsid. The polynucleotide may contain heterologous nucleic acid (i.e., a polynucleotide other than a wild-type AAV genome, such as an expression cassette delivered to a mammalian cell). AAV vectors are often named based on the name (serotype) of the capsid polypeptide. For example, an AAV5 vector indicates that the AAV vector contains an AAV5 capsid polypeptide.

[0095] When used in reference to viral titer, the term "vector genome unit", "genomic unit", "genomic particle (gp)" or "genomic copy" (gc) refers to the number of vector genomes encapsidated in virions, regardless of infectivity or functionality. 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, for example, Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0096] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0097] An "infectious" virion, virus or virus particle is one that contains a polynucleotide component that can be delivered into a subject cell for a virus species. This term does not necessarily mean the replicative ability of the virus. As used herein, an "infectious" virus or virus particle is one that can infect a target cell and express heterologous nucleic acid in the target cell when it approaches the target cell. Thus, "infectivity" refers to the ability of a virus particle to approach, enter, and express heterologous nucleic acid in the target cell. Infectivity may refer to in vitro infectivity or in vivo infectivity. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles.

[0098] The term "transducing units (tu)" when used with reference 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 in a functional assay such as that described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0099] The ability of viral particles to express heterologous nucleic acid in cells can be referred to as "transduction".The ability of viral particles to express heterologous nucleic acid in cells can be assayed using several techniques, including marker gene evaluation, such as green fluorescent protein (GFP) assay (for example, when the virus contains the nucleotide sequence encoding GFP), in which GFP is produced and detected and / or measured in the cells infected with viral particles; or measurement of produced protein, for example, by enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).

[0100] The term "tropism" refers to the ability of an AAV vector to infect one or more specified cell types, but can also encompass the manner in which the vector functions to transduce cells in one or more specified cell types; that is, tropism refers to preferential entry of the AAV virion into a particular cell type(s) or tissue type(s) and / or entry into a particular cell or tissue type, and optionally, then, preferably, expression (e.g., transcription and optionally translation) of sequences carried by the AAV virion in the cell, e.g., for recombinant viruses, preferential interaction with the cell surface that facilitates expression of the heterologous nucleotide sequence(s).

[0101] The term "tropism profile" refers to the pattern of transduction of one or more target cells, tissues and / or organs by the AAV vectors described herein.

[0102] Unless otherwise specified, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to a suitable control (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more transduction or tropism, respectively, of a control). A suitable control will depend on a variety of factors, including the desired tropism profile. Similarly, whether a capsid and / or virus "does not efficiently transduce" or "does not have efficient tropism" or similar terms for a target tissue can be determined by reference to a suitable control.

[0103] As used throughout this disclosure, "neuronal activity," "neuronal activity," "neuronal firing," and variations and synonyms thereof, refer to electrical activity resulting from stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch clamp techniques. In some embodiments, determining neuronal activity includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the level of neuronal activity depends on or is influenced by the excitatory postsynaptic potential (EPSP), the inhibitory postsynaptic potential (IPSP), and / or the action potential.

[0104] As used throughout this disclosure, "neurological disease" or "neurological disorder" refers to a disease or disorder of the nervous system. In some embodiments, a neurological disease is associated with, caused by, or results from structural, biochemical, and / or electrical abnormalities in the brain, spinal cord, nerves, or any component of the nervous system.

[0105] As used throughout this disclosure, a "symptom" of a disease refers to a physical or mental characteristic that is considered to be indicative of a disease state, particularly one that is evident to the patient. In some embodiments, a symptom is subjectively assessed by the patient. For example, in some embodiments, a symptom is pain; in some embodiments, a symptom is seizures.

[0106] As used throughout this disclosure with respect to amino acid positions, the term "corresponding to" or "corresponding to" refers to an amino acid in a first polypeptide sequence that aligns with a given amino acid in a reference polypeptide sequence when the first and reference polypeptide sequences are aligned. Alignment is performed by one of skill in the art using software designed for this purpose, such as Clustal Omega version 1.2.4, with the default parameters for that version.

[0107] As used throughout this disclosure, the term "expression cassette" refers to a DNA segment that contains one or more transgenes to be transcribed and one or more regulatory elements, including but not limited to, promoters, enhancers, introns, UTRs, and / or polyA. When introduced into a suitable host cell, the expression cassette can, among other things, direct the cellular machinery to transcribe the transgene into RNA.

[0108] As used throughout this disclosure, the term "promoter" refers to one or more nucleic acid control sequences that direct transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the start site of transcription, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. A promoter may be inducible or repressible. An "inducible" promoter initiates increased levels of transcription from an operably linked nucleic acid in response to certain changes in external conditions (e.g., temperature, concentration of a particular molecule, or oxygen or nutrient levels). In some embodiments, a promoter is "tissue-specific" in a particular cell type(s) or tissue(s) compared to a control cell type or tissue, which causes substantially higher levels of expression of the operably linked nucleic acid. In some embodiments, a promoter may be neuron-specific, conferring substantially more (e.g., 2-fold, 5-fold, or 10-fold higher) expression of an operably linked nucleic acid in neurons than in non-neuronal cells.

[0109] As used throughout this disclosure, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or a collection of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs transcription of the second nucleic acid sequence.

[0110] As used throughout this disclosure, the term "enhancer" refers to a polynucleotide sequence that typically acts on the activity of a promoter in cis position, thus stimulating the transcription of a gene operably linked to the promoter. Usually, the effect of an enhancer is completely independent of position and orientation, and therefore it may be located before or after a promoter, in an intron, or even in a gene coding region. An enhancer may be located in the immediate vicinity of a gene coding region or promoter, or quite far from the promoter. It is also possible for the enhancer to have physical and / or functional overlap with the promoter. Overview

[0111] The present disclosure provides an expression cassette for expressing a transgene in a target cell. In some embodiments, the target cell is a hippocampal neuron. In some embodiments, the target cell is a dorsal root ganglion neuron. In some embodiments, the target cell is a trigeminal ganglion neuron.

[0112] In some embodiments, the expression cassettes of the present disclosure are optimized for one or more of the following properties: Preferential expression in neurons; · Promoting sustained gene expression; Expressed at sufficient levels to be effective without causing significant cellular health issues; -Easily packaged into AAV vectors.

[0113] In some embodiments, expression cassette is further optimized to balance expression level, efficacy and toxicity.In some embodiments, expression cassette of the present disclosure allows medium to low level expression of transgene in target cell, but allows wider therapeutic window in vivo compared with control expression cassette that allows higher expression of transgene (e.g., corresponding small molecule ligand of transgene can be administered in a wider range without causing toxicity).

[0114] In some embodiments, the expression cassette of the present disclosure mediates high-level expression of the encoded transgene in dorsal root ganglion (DRG) and / or trigeminal ganglion (TGG) neurons. In some embodiments, the expression cassette of the present disclosure enhances the expression of the encoded transgene compared to a control expression cassette. In some embodiments, the expression cassette of the present disclosure is delivered to DRG and / or TGG neurons to treat neuropathic pain. In some embodiments, the expression cassette comprises a polynucleotide encoding an engineered ligand-gated ion channel (eLGIC) receptor or a chimeric version thereof.

[0115] In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human dorsal root ganglion and / or trigeminal ganglion neurons in vivo compared to a control expression cassette. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human dorsal root ganglion and / or trigeminal ganglion neurons in vitro compared to a control expression cassette. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human dorsal root ganglion and / or trigeminal ganglion neurons ex vivo compared to a control expression cassette.

[0116] In some embodiments, the expression cassette of the present disclosure mediates high-level expression of the encoded transgene in hippocampal neurons. In some embodiments, the expression cassette mediates high-level expression of the encoded transgene preferentially in excitatory neurons of the hippocampus. In some embodiments, the expression cassette of the present disclosure enhances the expression of the encoded transgene compared to a control expression cassette. In some embodiments, the expression cassette of the present disclosure is delivered to hippocampal neurons to treat focal epilepsy. In some embodiments, the expression cassette comprises a polynucleotide encoding an engineered ligand-gated ion channel (eLGIC) receptor or a chimeric version thereof.

[0117] In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in hippocampal neurons compared to a control expression cassette. In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in hippocampal neurons compared to a control expression cassette.

[0118] In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human hippocampal neurons in vivo compared to a control expression cassette. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human hippocampal neurons in vitro compared to a control expression cassette. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in human hippocampal neurons ex vivo compared to a control expression cassette.

[0119] In some embodiments, the expression cassette is comprised in a vector (e.g., a viral vector or a non-viral vector). In some embodiments, the expression cassette is comprised in an AAV vector. In some embodiments, the expression cassette is a CRISPR / CAS expression system. In some embodiments, the expression cassette encodes a therapeutic protein or antibody.

[0120] The present disclosure provides methods of using the expression cassettes or corresponding vectors of the present disclosure in therapeutic treatment regimens, vaccines, or research tool development modalities.

[0121] The present disclosure also provides a method of using an expression cassette of the present disclosure to reduce the amount of total nucleic acid administered to a subject, the method comprising administering to said subject an amount of total nucleic acid that is less than the amount of nucleic acid that would be administered to said subject if said nucleic acid included a control expression cassette.

[0122] The present disclosure also provides a method of treating neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia, in a subject in need thereof comprising administering an effective amount of a recombinant nucleic acid comprising an expression cassette, wherein the expression cassette comprises a transgene encoding a ligand-gated ion channel or a chimeric version thereof.

[0123] The disclosure also provides a method of treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of a recombinant nucleic acid comprising an expression cassette, wherein the expression cassette comprises a transgene encoding a ligand-gated ion channel or a chimeric version thereof. Expression cassette

[0124] In some embodiments, the expression cassette comprises one or more of the regulatory elements provided below in Table 1. An exemplary arrangement of the various elements is provided in FIG. Table 1. Non-limiting examples of regulatory elements [Table 1-1] [Table 1-2] 5' Enhancer

[0125] In some embodiments, the expression cassette of the present disclosure comprises a 5' enhancer.

[0126] In some embodiments, the 5' enhancer comprises or consists of a CMV enhancer. In some embodiments, the CMV enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 37. In some embodiments, the CMV enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 37.

[0127] In some embodiments, the 5' enhancer comprises or consists of a CMV-V2 enhancer. In some embodiments, the CMV-V2 enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 38. In some embodiments, the CMV-V2 enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 38.

[0128] In some embodiments, the 5' enhancer comprises or consists of the CMV-V3 enhancer. In some embodiments, the CMV-V3 enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 39. In some embodiments, the CMV-V3 enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 39.

[0129] In some embodiments, the expression cassette of the present disclosure does not comprise a 5' enhancer. Non-neuronal silencing elements

[0130] In some embodiments, an expression cassette of the present disclosure comprises a non-neuronal silencing element, also known as a neuron-restrictive silencer element (NRSE).

[0131] NRSEs have been identified in some neuronal genes, and confer neuronal specificity by silencing transcription in non-neuronal cells. For example, neuron-restricted silencer elements exist in the promoter of neuronal nicotinic acetylcholine receptor β2 subunit gene, which determines its neuron-specific expression in the nervous system. In some embodiments, such NRSEs can silence or promote transcription depending on the cellular context in the nervous system. Research has shown that certain NRSEs can activate the transcription of synthetic promoters when located downstream in the 5' untranslated region or less than 50 bp upstream from the TATA box, but switch to silencers when located further upstream. Further discussion of NRSEs can be found in Bessis et al., Proc Natl Acad Sci USA. 1997 May 27; 94(11): 5906-5911, the entire contents of which are incorporated by reference for all purposes.

[0132] In some embodiments, a NRSE of the present disclosure comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 40. In some embodiments, a NRSE comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 40.

[0133] In some embodiments, the NRSE is embedded in a promoter of the present disclosure. In some embodiments, the NRSE is located between 30 and 50 base pairs after the TATA box in the expression cassette.

[0134] In some embodiments, the expression cassette of the present disclosure does not comprise a neuron-restrictive silencer element (NRSE). promoter

[0135] In some embodiments, the expression cassette of the present disclosure comprises a promoter. In some embodiments, the promoter is operably linked to the transgene of the expression cassette. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a neuron-specific promoter.

[0136] In some embodiments, the promoter comprises or consists of a cβ actin promoter. In some embodiments, the cβ actin promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 41. In some embodiments, the cβ actin promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 41.

[0137] In some embodiments, the promoter comprises or consists of a CMV promoter. In some embodiments, the CMV promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 42. In some embodiments, the CMV promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 42.

[0138] In some embodiments, the promoter comprises or consists of the hCaMKIIa promoter. In some embodiments, the hCaMKIIa promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 43. In some embodiments, the hCaMKIIa promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 43.

[0139] In some embodiments, the promoter comprises or consists of the hsCaMKIIa promoter. In some embodiments, the hsCaMKIIa promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 44. In some embodiments, the hsCaMKIIa promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 44.

[0140] In some embodiments, the promoter comprises or consists of the h gamma enolase promoter. In some embodiments, the h gamma enolase promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 45. In some embodiments, the h gamma enolase promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 45.

[0141] In some embodiments, the promoter comprises or consists of the hMecp2 promoter. In some embodiments, the hMecp2 promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 46. In some embodiments, the hMecp2 promoter comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 46.

[0142] In some embodiments, the promoter comprises or consists of the hPDGFβ promoter. In some embodiments, the hPDGFβ promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 47. In some embodiments, the hPDGFβ promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 47.

[0143] In some embodiments, the promoter comprises or consists of an hSyn promoter. In some embodiments, the hSyn promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 48. In some embodiments, the hSyn promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 48.

[0144] In some embodiments, the promoter comprises or consists of the hSyn-V2 promoter. In some embodiments, the hSyn-V2 promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 49. In some embodiments, the hSyn-V2 promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 49.

[0145] In some embodiments, the promoter comprises or consists of the mMecp2 promoter. In some embodiments, the mMecp2 promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 50. In some embodiments, the mMecp2 promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 50.

[0146] In some embodiments, the promoter comprises or consists of the msCaMKIIa promoter. In some embodiments, the msCaMKIIa promoter comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 51. In some embodiments, the msCaMKIIa promoter comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 51.

[0147] In some embodiments, the expression cassette of the present disclosure does not include a promoter. 5' untranslated region (5'UTR)

[0148] In some embodiments, an expression cassette of the present disclosure comprises a 5' untranslated region (5'UTR).

[0149] In some embodiments, the 5'UTR comprises or consists of the fGlob 5'UTR. In some embodiments, the fGlob 5'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 52. In some embodiments, the fGlob 5'UTR comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 52.

[0150] In some embodiments, the 5'UTR comprises or consists of the hCaMKII 5'UTR. In some embodiments, the hCaMKII 5'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 53. In some embodiments, the hCaMKII 5'UTR comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 53.

[0151] In some embodiments, the 5'UTR comprises or consists of the hSyn 5'UTR. In some embodiments, the hSyn 5'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 54. In some embodiments, the hSyn 5'UTR comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 54.

[0152] In some embodiments, the 5'UTR comprises or consists of the mCaMKII 5'UTR. In some embodiments, the mCaMKII 5'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 55. In some embodiments, the mCaMKII 5'UTR comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 55.

[0153] In some embodiments, the 5'UTR comprises or consists of the mCaMKII-V2 5'UTR. In some embodiments, the mCaMKII-V2 5'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, 56 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:56. In some embodiments, the mCaMKII-V2 5'UTR comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:56.

[0154] In some embodiments, the expression cassette of the present disclosure does not include a 5'UTR. Introns

[0155] In some embodiments, the expression cassette of the present disclosure comprises an intron. In some embodiments, the intron is located between the promoter and the transgene. In some embodiments, the intron is located between the 5'UTR and the transgene. In some embodiments, the intron is located between the promoter and the 5'UTR.

[0156] In some embodiments, the intron comprises or consists of the cβAct+rGlob intron. In some embodiments, the cβAct+rGlob intron comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 57. In some embodiments, the cβAct+rGlob intron comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 57.

[0157] In some embodiments, the intron comprises or consists of the hCMV+rGlob intron. In some embodiments, the hCMV+rGlob intron comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 58. In some embodiments, the hCMV+rGlob intron comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 58.

[0158] In some embodiments, the intron comprises or consists of an hSyn intron. In some embodiments, the hSyn intron comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 59. In some embodiments, the hSyn intron comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 59.

[0159] In some embodiments, the intron comprises or consists of an hTPI intron. In some embodiments, the hTPI intron comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 60. In some embodiments, the hTPI intron comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 60.

[0160] In some embodiments, the intron comprises or consists of the TPL+eMLP intron. In some embodiments, the TPL+eMLP intron comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 61. In some embodiments, the TPL+eMLP intron comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 61.

[0161] In some embodiments, the expression cassette of the present disclosure does not contain an intron. 3' Enhancer

[0162] In some embodiments, the expression cassette of the present disclosure comprises a 3' enhancer.

[0163] In some embodiments, the 3' enhancer comprises or consists of the 511-810EES 3' enhancer. In some embodiments, the 511-810EES 3' enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 62. In some embodiments, the 511-810EES 3' enhancer comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 62.

[0164] In some embodiments, the 3' enhancer comprises or consists of a FullEES 3' enhancer. In some embodiments, the FullEES 3' enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 63. In some embodiments, the FullEES 3' enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 63.

[0165] In some embodiments, the 3' enhancer comprises or consists of the WPREx 3' enhancer. In some embodiments, the WPREx 3' enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 64. In some embodiments, the WPREx 3' enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 64.

[0166] In some embodiments, the 3' enhancer comprises or consists of the WPREx-V2 3' enhancer. In some embodiments, the WPREx-V2 3' enhancer comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 65. In some embodiments, the WPREx-V2 3' enhancer comprises or consists of a polynucleotide sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 65.

[0167] In some embodiments, the expression cassette of the present disclosure does not comprise a 3' enhancer. 3' untranslated region (3'UTR)

[0168] In some embodiments, an expression cassette of the present disclosure comprises a 3' untranslated region (3'UTR).

[0169] In some embodiments, the 3'UTR comprises or consists of an alpha globin 3'UTR. In some embodiments, the alpha globin 3'UTR comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 66. In some embodiments, the alpha globin 3'UTR comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 66.

[0170] In some embodiments, the expression cassette of the present disclosure does not include a 3'UTR. Polyadenylation sequence (polyA)

[0171] In some embodiments, an expression cassette of the present disclosure includes a polyadenylation sequence ("polyA", also known as a "poly A tail", "poly-A", or "poly-A tail").

[0172] In some embodiments, the polyA comprises or consists of a bGH polyA. In some embodiments, the bGH polyA comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 67. In some embodiments, the bGH polyA comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 67.

[0173] In some embodiments, the polyA comprises or consists of hGH polyA. In some embodiments, the hGH polyA comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 68. In some embodiments, the hGH polyA comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 68.

[0174] In some embodiments, the polyA comprises or consists of hGH-V2 polyA. In some embodiments, the hGH-V2 polyA comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 69. In some embodiments, the hGH-V2 polyA comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 69.

[0175] In some embodiments, the polyA comprises or consists of rβ globin polyA. In some embodiments, the rβ globin polyA comprises or consists of a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 70. In some embodiments, the rβ globin polyA comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to the sequence of SEQ ID NO: 70.

[0176] In some embodiments, the expression cassette of the present disclosure does not include polyA. Cassettes containing integrated elements

[0177] An exemplary expression cassette is illustrated in Figure 1. In some embodiments, the order of the regulatory elements, from 5' to 3', follows one of the examples provided in Table 2 below. Table 2. Non-limiting examples of expression cassettes [Table 2-1] [Table 2-2] "*": the NRSE element in Example No. 20 is embedded within the CMV promoter instead of being placed upstream of the CMV promoter in this cassette.

[0178] In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 71-93, excluding the transgene region (SEQ ID NO: 36). In some embodiments, the expression cassette comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to any one of SEQ ID NOs: 71-93, excluding the transgene region (SEQ ID NO: 36).

[0179] In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 121-124, excluding the transgene region (SEQ ID NO: 32). In some embodiments, the expression cassette comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to any one of SEQ ID NOs: 121-124, excluding the transgene region (SEQ ID NO: 32).

[0180] In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 71-93 and 121-124. In some embodiments, the expression cassette comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to any one of SEQ ID NOs: 71-93 and 121-124.

[0181] In some embodiments, the present disclosure provides a recombinant nucleic acid comprising an expression cassette of the present disclosure and an adeno-associated virus (AAV) inverted terminal repeat (ITR) flanking each end of the expression cassette. In some embodiments, the recombinant nucleic acid comprises a 5'ITR sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 94 or 119. In some embodiments, the recombinant nucleic acid comprises a 3'ITR sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 95 or 120.

[0182] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 96-118 and 125-128. In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide mutations (substitutions, deletions, and / or additions) compared to any one of SEQ ID NOs: 96-118 and 125-128.

[0183] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 96-118 excluding the transgene region (SEQ ID NO: 36). In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to any one of SEQ ID NOs: 96-118 excluding the transgene region (SEQ ID NO: 36).

[0184] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 125-128 excluding the transgene region (SEQ ID NO: 32). In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides mutated (substituted, deleted, and / or added) compared to any one of SEQ ID NOs: 125-128 excluding the transgene region (SEQ ID NO: 32).

[0185] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:48; (iii) an hSyn 5'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:54; (iv) an hTPI intron comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:60; (v) transgene; (vi) a FullEES 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:63; (vii) a rβ globin polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 70 Includes. In some embodiments, the transgene encodes a ligand-gated ion channel comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human glycine receptor. In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:73.

[0186] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hSyn promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:48; (iii) an hSyn 5'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:54; (iv) an hSyn intron comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:59; (v) transgene; (vi) a WPREx3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:64; (vii) an alpha globin 3'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:66; and (viii) an hGH polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:68; Includes. In some embodiments, the transgene encodes a ligand-gated ion channel comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human glycine receptor. In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:81.

[0187] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:37; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (iii) an hCaMKII 5'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:53; (iv) an hCMV+rGlob intron comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:58; (v) a transgene; (v) transgene; (vi) a WPREx3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:64; (vii) an alpha globin 3'UTR comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:66; and (viii) an hGH polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:68; Includes. In some embodiments, the transgene encodes a ligand-gated ion channel comprising a ligand binding domain derived from human α7-nAChR and an ion pore domain derived from human glycine receptor. In some embodiments, the expression cassette comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:83.

[0188] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:38; (ii) an hSyn-V2 promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:49; (iii) a transgene; (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 65; and (v) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69; Includes.

[0189] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:38; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (iii) a transgene; (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 65; and (v) an hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69; Includes.

[0190] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) an hSyn-V2 promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:49; (ii) a transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 65; and (iv) hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69. Includes.

[0191] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) an hCaMKIIa promoter comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:43; (ii) a transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 65; and (iv) hGH-V2 polyA comprising a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:69. Includes. vector

[0192] In some embodiments, the disclosure provides a vector comprising a recombinant nucleic acid, where the recombinant nucleic acid comprises an expression cassette of the disclosure.

[0193] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a bacmid.

[0194] In some embodiments, the vector is encapsulated in a particle, such as a lipid nanoparticle or an exosome.

[0195] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is derived from a replication-defective virus. In some embodiments, the viral vector is an adenovirus, a retrovirus (e.g., lentivirus), an adeno-associated virus (AAV), a poxvirus, an alphavirus, a vaccinia virus, or a herpes simplex virus (e.g., HSV-1). Adeno-associated virus (AAV) vectors

[0196] Adeno-associated virus (AAV), a member of the Parvoviridae family, is a small, non-enveloped, icosahedral virus with a single-stranded linear DNA genome of approximately 4.7 kilobases (kb). AAV was found as a contaminant in purified adenovirus stocks, and therefore the virus has been assigned to the Dependovirus genus (DM Knipe, PM Howley, Field's Virology, Lippincott Williams & Wilkins, Philadelphia, ed. Sixth, 2013). In its wild-type state, AAV is naturally replication-deficient, so AAV depends on a helper virus, typically an adenovirus, to provide the necessary protein factors for replication. The 4.7 kb genome of AAV is flanked by two inverted terminal repeats (ITRs) that fold into a hairpin shape that is important for replication. Being naturally replication-deficient and able to transduce nearly all cell types in the human body, AAV is an ideal vector for therapeutic use in gene therapy or vaccine delivery. In its wild type state, the life cycle of AAV includes a latent phase in which AAV genome is site-specifically integrated into host chromosome after infection, and an infectious phase in which the integrated genome is rescued, replicated, and packaged into infectious virus after infection with either adenovirus or herpes simplex virus.When vectorized, the viral Rep and Cap genes of AAV are removed and provided in trans during virus production, making ITR the only remaining viral DNA (A. Vasileva, R. Jessberger, Nature reviews. Microbiology, 3, 837-847 (2005)).Rep and Cap are then replaced with a range of possible transfer vector configurations to perform gene addition or gene targeting.These vectorized AAVs transduce both dividing and non-dividing cells and show robust and stable expression in stationary tissues.Various published U.S. applications describe AAV vectors and virions, including U.S. Publication Nos. 2015 / 0176027, 2015 / 0023924, 2014 / 0348794, 2014 / 0242031, and 2012 / 0164106, all of which are incorporated by reference in their entireties herein.

[0197] However, despite the excellent ability of AAV vectors to transduce various tissues and cell types, there is still a need in the art for AAV vectors that show improved transduction of specific neuronal cells, both in terms of transduction efficiency and desired tropism.In addition, administration method may also affect the location, amount and type of cells that are transduced.Since there is a physical limit to how much AAV can be delivered in a single injection, and the desired outcome (e.g., therapeutic outcome) is related to stimulating or suppressing the activity of the correct neuronal population in a subject, high levels of transduction and correct tropism are required for delivery to neurons.

[0198] The present disclosure provides AAV capsid polypeptides that demonstrate significantly improved transduction of subpopulations of neurons in a subject in vivo, in vitro and / or ex vivo.

[0199] The genomic organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) long. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences of nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, 2 and 3) form the capsid and contribute to the tropism of the virus. The terminal 145 nt ITRs are self-complementary and organized such that energetically stable intramolecular duplexes that form T-shaped hairpins can form. These hairpin structures serve as origins for viral DNA replication and as primers for cellular DNA polymerase complexes. After wild-type (wt) AAV infection in mammalian cells, the Rep genes are expressed and function in the replication of the viral genome.

[0200] In some embodiments, the outer protein "capsid" of the viral vector is naturally occurring, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the capsid is synthetically engineered (e.g., by directed evolution or rational design) to have certain unique features that do not occur in nature, such as altered tropism, increased transduction efficiency, and / or immune evasion. One example of a rationally designed capsid is the mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid polypeptide.

[0201] Non-limiting examples of AAV ITRs that can be used in an AAV vector include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16.

[0202] The AAV vector that contains two ITRs has a payload capacity of about 4.4 kB. Self-complementary AAV (scAAV) vector contains a third ITR and packages two strands of the recombinant part of the vector, leaving only about 2.1 kB for the polynucleotide contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0203] Expanded packaging capacity (about 9 kB), roughly double the packaging capacity of AAV, has been achieved using a dual AAV vector strategy. Dual vector strategies useful for producing AAV contemplated herein include, but are not limited to, splicing (trans-splicing), homologous recombination (overlap), or a combination of the two (hybrid). In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' end of the 5' half of the vector, and a splice acceptor (SA) signal is placed at the 5' end of the 3' half of the vector. Upon coinfection of the same cell with dual AAV vectors and inverted terminal repeat (ITR)-mediated head-to-tail concatenation of the two halves, trans-splicing leads to the production of mature mRNA and full-size protein (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the two halves of the large expression cassette contained in the dual AAV vector may contain homologous overlapping sequences (the 3' end of the 5' half vector and the 5' end of the 3' half vector, dual AAV overlap), which mediates the reconstitution of a single large genome by homologous recombination (Duan et al., 2001). This strategy relies on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region derived from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al., 2008, Ghosh et al., 2011) to the trans-splicing vector. The added region is located downstream of the SD signal of the 5' half vector and upstream of the SA signal of the 3' half vector to increase recombination between the dual AAVs.

[0204] "Hybrid AAV" refers to an AAV genome packaged in a capsid of a different AAV serotype (and preferably a different serotype derived from one or more AAV ITRs), and can be alternatively referred to as a pseudotype AAV. For example, a genome of AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 can be encapsidated in a capsid of AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a variant thereof, provided that the AAV capsid and genome (and preferably one or more AAV ITRs) are of different serotypes. In certain embodiments, pseudotyped AAV particles can be referred to as being of the "x / y" type, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid, e.g., a 2 / 5 AAV particle has ITRs derived from AAV2 and a capsid derived from AAV5.

[0205] "Host cells" include cells that are transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present disclosure. Host cells may include virus-producing cells and cells infected with viral vectors. In some embodiments, in vivo host cells are infected with viral vectors contemplated herein.

[0206] High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. Nos. 5,658,776; 6,566,118; 6,989,264; and 6,995,006; US2006 / 0188484; WO98 / 22607; WO2005 / 072364; and WO / 1999 / 011764; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003; Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; lnoue et al., (1998) J. Virol. 72, 7024. Methods for producing pseudotyped AAV vectors have also been reported (e.g., WO00 / 28004), and various modifications or formulations of AAV vectors have reduced their immunogenicity upon administration in vivo (see, e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562).

[0207] In some embodiments, an AAV vector comprising an AAV capsid polypeptide (or a variant of the present disclosure) contributes to targeted expression of an engineered receptor to a subpopulation of cells or neurons of a subject. In some embodiments, the neurons are nociceptors.

[0208] In some embodiments, the AAV comprises a self-complementary genome. As defined herein, an AAV comprising a "self-complementary" or "double-stranded" genome refers to an AAV whose coding region is engineered to be configured to form an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001), the entirety of which is incorporated by reference. The present disclosure contemplates the use of an AAV comprising a self-complementary genome, in some cases, because upon infection (such transduction), rather than waiting for cell-mediated synthesis of the second strand of the AAV genome, the two complementary halves of the scAAV combine to form one double-stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. It is understood that rather than the complete coding capacity found in AAV (4.7-6 kb), AAV containing self-complementary genomes can only retain about half that amount (about 2.4 kb).

[0209] In some embodiments, AAV comprises a single-stranded genome. As defined herein, "single-stranded" genome refers to a genome that is not self-complementary. In some embodiments, the present disclosure relates to a single-stranded AAV vector that can achieve efficient gene transfer to anterior segment in mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017), the entire contents of which are incorporated by reference. Capsid Polypeptide

[0210] In some embodiments, the present disclosure provides capsid polypeptides that allow corresponding encapsidated AAV vectors to achieve enhanced transduction and / or targeting to at least a subpopulation of neuronal cells. Exemplary capsid polypeptides of the present disclosure are listed in Table 3 below: Table 3. Exemplary Capsid Polypeptides [Table 3-1] [Table 3-2]

[0211] In some embodiments, the capsid polypeptide of the present disclosure is AAV2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:13.

[0212] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:14.

[0213] In some embodiments, the capsid polypeptide of the disclosure is AAV2.5-TV. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492V in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:3. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:3, and the capsid polypeptide comprises a valine at a position corresponding to V492 in SEQ ID NO:3. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 3, and the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 3. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 15.

[0214] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5-2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to Y705 and / or Y731 of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to Y705F and / or Y731F of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:4. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:4, and the capsid polypeptide comprises a phenylalanine at a position(s) corresponding to F705 and / or F731 of SEQ ID NO:4. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 4, and the capsid polypeptide comprises a phenylalanine at position(s) corresponding to F705 and / or F731 of SEQ ID NO: 4. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 16.

[0215] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5-TV2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492, Y705, and / or Y731 of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492V, Y705F, and / or Y731F of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:5. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:5, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO:5, and the capsid polypeptide comprises a phenylalanine at position(s) corresponding to F705 and / or F731 of SEQ ID NO:5. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 5, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 5, and wherein the capsid polypeptide comprises a phenylalanine at a position(s) corresponding to F705 and / or F731 of SEQ ID NO: 5. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 17.

[0216] In some embodiments, the capsid polypeptide of the present disclosure is AAV5. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:18.

[0217] In some embodiments, the capsid polypeptide of the present disclosure is AAV6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:7. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:7. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:7. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:19.

[0218] In some embodiments, the capsid polypeptide of the present disclosure is AAV9. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:20.

[0219] In some embodiments, the capsid polypeptide of the disclosure is AAV9-TV. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution corresponding to T492V in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:9. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:9, and the capsid polypeptide comprises a valine at a position corresponding to V492 in SEQ ID NO:9. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 9, and the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 9. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:21.

[0220] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to Y705 and / or Y731 of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to Y705F and / or Y731F of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:10. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:10, and the capsid polypeptide comprises a phenylalanine at a position(s) corresponding to F705 and / or F731 of SEQ ID NO:10. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 10, and the capsid polypeptide comprises a phenylalanine at position(s) corresponding to F705 and / or F731 of SEQ ID NO: 10. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:22.

[0221] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-TV2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492, Y705, and / or Y731 of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492V, Y705F, and / or Y731F of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:11, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO:11, and the capsid polypeptide comprises a phenylalanine at position(s) corresponding to F705 and / or F731 of SEQ ID NO:11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 11, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 11, and wherein the capsid polypeptide comprises a phenylalanine at a position(s) corresponding to F705 and / or F731 of SEQ ID NO: 11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:23.

[0222] In some embodiments, the AAV vector comprises an AAV capsid polypeptide comprising an amino acid mutation at one or more positions corresponding to T492, Y705, and / or Y731 of the AAV9 capsid polypeptide (SEQ ID NO: 8), which is of serotype AAV2, AAV2.5, AVV5, AAV6, AAV9, or another AAV serotype. In some embodiments, the one or more positions are two or more positions, two positions, or three positions. In some embodiments, the viral vector comprises an AAV capsid polypeptide comprising an AAV capsid polypeptide comprising one or more amino acid substitutions corresponding to T492V, Y705F, and / or Y731F of the AAV9 capsid polypeptide (SEQ ID NO: 8), or any combination thereof, which is of serotype AAV2, AAV2.5, AVV5, AAV6, AAV9, or another AAV serotype. In some embodiments, the one or more substitutions is two or more substitutions, two substitutions, or three substitutions.

[0223] As used herein, "T492, Y705 and / or Y731" refers to, for example, T492+Y705, T492+Y731, T492+Y705+Y731, or any other possible combination thereof. Similarly, "T492V, Y705F and / or Y731F" refers to, for example, T492V+Y705F, T492V+Y731F, T492V+Y705F+Y731F, or any other possible combination thereof.

[0224] In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.7% sequence identity to SEQ ID NO:8, and the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:8, and the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the substitution is T492V. In some embodiments, the substitution is T492I. In some embodiments, the substitution is T492L. In some embodiments, the T492 residue is substituted with a hydrophobic amino acid selected from valine (Val), leucine (Leu), and isoleucine (Ile). In some embodiments, the T492 residue is substituted with a hydrophobic amino acid selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). Neurons and Nerve Fibers

[0225] In some embodiments, the present disclosure provides methods of delivering a recombinant nucleic acid comprising an expression cassette to a target neuron located in the hippocampus, i.e., a hippocampal neuron.

[0226] In some embodiments, the hippocampal neuron is an excitatory neuron.In some embodiments, the expression cassette comprises a CAMKII family promoter (e.g., hCaMKIIa), causing high and / or selective expression of the encoded transgene in excitatory hippocampal neuron.In some embodiments, the hippocampal neuron is a Ca2+ / calmodulin-dependent protein kinase II (CaMKII) positive excitatory neuron.

[0227] In some embodiments, the hippocampal neurons are inhibitory neurons. In some embodiments, the hippocampal neurons are GABAergic neurons.

[0228] In some embodiments, the present disclosure provides a method for delivering a recombinant nucleic acid comprising an expression cassette to a target neuron located in the dorsal root ganglion (or spinal ganglion; also known as the posterior root ganglion). The dorsal root ganglion (DRG) is a cluster (ganglion) of neurons in the dorsal root of the spinal nerve. The cell bodies of sensory neurons, known as primary neurons, are located in the dorsal root ganglion. The axons of dorsal root ganglion neurons are known as afferent nerves. In the peripheral nervous system, afferent nerves refer to the axons that relay sensory information to the central nervous system (i.e., the brain and spinal cord).

[0229] In some embodiments, the present disclosure provides a method of delivering a recombinant nucleic acid comprising an expression cassette to a target neuron located in the trigeminal ganglion (or Gasserian ganglion, or semilunar ganglion, or Gasserian ganglion). The trigeminal ganglion (TGG) is a sensory ganglion of the trigeminal nerve (CNV) that occupies a cavity in the dura (Meckel's space), which overlies the trigeminal nerve impingement near the apex of the petrous part of the temporal bone.

[0230] Axons (also known as nerve fibers) are the long, thin projections of neuronal cells in vertebrates that typically conduct electrical impulses known as action potentials away from the nerve cell body. Each neuron has only one axon. Thus, neuronal cells can be classified by their axons (nerve fibers).

[0231] Three general classes of nerve fibers (groups A, B, and C) have been classified by Erlanger and Gasser: group A nerve fibers are heavily myelinated, group B nerve fibers are moderately myelinated, and group C nerve fibers are unmyelinated.

[0232] There are four subdivisions of group A nerve fibers (A fibers): alpha (α), beta (β), gamma (γ), and delta (δ). These subdivisions have different amounts of myelination and axon thickness and therefore transmit signals at different rates. Aα type fibers include the alternative classification system of type Ia and type Ib sensory fibers, which are fibers originating from muscle spindle endings and Golgi tendons, respectively. Aβ type fibers, and Aγ type fibers, are type II afferent fibers originating from stretch receptors. Many of the Aβ type fibers originating from the skin are specialized for touch. Aδ type fibers are afferent fibers of nociceptors. Aδ fibers carry information from peripheral mechanoreceptors and thermoreceptors to the dorsal horn of the spinal cord. Aδ fibers serve to receive and transmit information primarily associated with acute pain (sharp, immediate, and relatively short-lasting). This type of pain can result from several classes of stimuli: temperature-induced, mechanical, and chemical. This may be part of a withdrawal reflex initiated by Aδ fibers in the reflex arc that activate a withdrawal response. Aδ fibers carry cold pain, pressure pain, and acute pain signals; because they are thin (2-5 μm in diameter) and myelinated, they transmit impulses faster than unmyelinated C fibers, but more slowly than the other, thicker myelinated A group nerve fibers. Their conduction velocity is moderate.

[0233] Group B nerve fibers (B fibers) are axons that are moderately myelinated, meaning that they are less myelinated than group A nerve fibers and more myelinated than group C nerve fibers. They are usually general visceral afferent fibers and preganglionic nerve fibers of the autonomic nervous system.

[0234] Group C nerve fibers (C fibers) are unmyelinated and have small diameters and low conduction velocities. Group C fibers include postganglionic fibers in the autonomic nervous system (ANS) and dorsal root nerve fibers (IV fibers). These fibers carry sensory information. Damage or injury to these nerve fibers causes neuropathic pain.

[0235] The peripheral end of the mature nociceptor is where the stimulus is detected and converted into electrical energy. When the electrical energy reaches a threshold, an action potential is induced and driven toward the central nervous system (CNS). This results in a chain of events that allows for the subjective awareness of pain. The sensory specificity of the nociceptor is established solely by a high threshold for a particular feature of the stimulus. The nociceptor is triggered only when a high threshold is reached by either the chemical, thermal, or mechanical environment. Most nociceptors are classified by the environmental modalities to which they respond. Some nociceptors respond to more than one of these modalities and are consequently designated as multimodal. Other nociceptors do not respond to any of these modalities (but they can respond to stimuli under inflammatory conditions) and are referred to as inactive or silent.

[0236] Nociceptors have two different types of axons. The first are A-delta fiber axons. They are myelinated and can transmit action potentials towards the CNS at a speed of about 20 meters per second. The other type is the slower conducting C-fiber axon. These only conduct at a speed of about 2 meters per second. This is due to light myelination or non-myelination of the axon. As a result, pain comes in two phases. The first phase is mediated by the fast conducting A-delta fibers and the second part is due to the (polymodal) C-fibers. Pain associated with A-delta fibers can be associated with an initial extremely sharp pain. The second phase is a more prolonged and slightly less intense sensation of pain as a result of acute injury. When there is a large or prolonged input to the C-fibers, there is a progressive potentiation in the spinal dorsal horn; this phenomenon, similar to tetanus in muscles, is called wind-up. When wind-up occurs, there is a probability of increased sensitivity to pain.

[0237] Different classes of nerve fibers can be distinguished by biomarkers such as neurofilament 200 (NF200, also known as neurofilament heavy chain polypeptide), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4). Aα and Aβ fibers are typically NF200 positive but IB4 negative. Aδ fibers are typically positive for both NF200 and IB4. C fibers are typically IB4 positive but NF200 negative. Thus, many of the NF200+ neurons contain heavily myelinated Aα or moderately myelinated Aβ fibers. In some cases, these neurons mediate signals for light touch and proprioception. However, some NF200+ neurons may also mediate signals for nociception. Many of the IB4+ neurons contain unmyelinated C fibers or Aδ fibers for nociception. And many of the CGRP+ neurons contain unmyelinated C fibers or lightly myelinated Aδ fibers for nociception. Characteristics of the expression cassette

[0238] In some embodiments, the expression cassette of the present disclosure mediates a higher level of expression of the encoded transgene in one or more neurons compared to a control expression cassette (e.g., a control exhibiting tissue-specific expression). In some embodiments, the control expression cassette comprises all of the regulatory elements of expression cassette number 18 in Table 2, including the hSyn-V2 promoter for neuron-specific expression. In some embodiments, such a control expression cassette comprises the polynucleotide sequence of SEQ ID NO: 88, excluding the sequence of the transgene (SEQ ID NO: 36). In some embodiments, the expression cassette of the present disclosure mediates an equivalent or higher level of expression of the encoded transgene in one or more neurons compared to a control expression cassette exhibiting ubiquitous rather than tissue-specific transgene expression. In some embodiments, the control expression cassette comprises all of the regulatory elements of expression cassette number 17 in Table 2, including the CAG (CMV enhancer, cβ actin promoter, and rβ actin-rGlob intron) regulatory elements for strong and ubiquitous expression in eukaryotic cells. In some embodiments, such a control expression cassette comprises the polynucleotide sequence of SEQ ID NO: 87, excluding the sequence of the transgene (SEQ ID NO: 36). A person skilled in the art will readily recognize the appropriate control to use in each case, and typically the control expression cassette will have the same transgene polynucleotide sequence as that of the expression cassette to which it is being compared.

[0239] Expression levels of the transgene can be measured by techniques known in the art, including, for example, ELISA, ddPCR and / or immunofluorescence analysis, including those described in the Examples section of this disclosure, as well as other methods known in the art.

[0240] In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in one or more target neurons compared to a control expression cassette (e.g., a control with tissue-specific expression). In some embodiments, the transgene expression level is increased by about 5%, 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 100%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 7-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 70-fold, or about 100-fold (including all ranges and subranges therebetween) compared to that of the control expression cassette. In some embodiments, the transgene expression level is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, or at least 100-fold (including all ranges and subranges therebetween) compared to that of the control expression cassette. In some embodiments, the control expression cassette comprises a polynucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:88 (expression cassette number 18 in Table 2), excluding the sequence of the transgene.

[0241] In some embodiments, the expression cassette mediates equivalent or higher levels of expression of the encoded transgene in one or more target neurons compared to a ubiquitous control expression cassette, rather than tissue-specific transgene expression. In some embodiments, the transgene expression level is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, or at least 100-fold (including all ranges and subranges therebetween) of the transgene expression level of the control expression cassette. In some embodiments, the control expression cassette comprises a polynucleotide sequence having at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:87 (expression cassette number 17 in Table 2), excluding the sequence of the transgene.

[0242] In some embodiments, the transgene expression level refers to the mRNA expression level. In some embodiments, the transgene expression level refers to the protein expression level.

[0243] In some embodiments, the target neuron is a hippocampal neuron, hi some embodiments, the target neuron is an excitatory neuron.

[0244] In some embodiments, the target neuron is a DRG or TGG neuron.

[0245] In some embodiments, the target neuron comprises A fibers. In some embodiments, the target neuron comprises Aα or Aβ fibers. In some embodiments, the target neuron comprises Aδ fibers. In some embodiments, the target neuron comprises C fibers.

[0246] In some embodiments, the target neuron comprises nerve fibers that are NF200 positive and IB4 negative. In some embodiments, the target neuron comprises nerve fibers that are NF200 positive and IB4 positive. In some embodiments, the target neuron comprises nerve fibers that are NF200 negative and IB4 positive. In some embodiments, the target neuron comprises nerve fibers that are CGRP positive. Transgene

[0247] In some embodiments, the expression cassette of the present disclosure comprises a transgene. In some embodiments, the transgene encodes a protein, a peptide, miRNA, siRNA, or gRNA. In some embodiments, the transgene encodes two or more biomolecules (e.g., proteins) separated by an internal ribosome entry site, allowing the co-expression of two or more biomolecules.

[0248] In some embodiments, the expression cassette of the present disclosure comprises a transgene encoding a receptor. In some embodiments, the receptor is an engineered ligand-gated ion channel (eLGIC) or a chimeric version thereof. In some embodiments, the engineered LGIC can be activated by a small molecule ligand. In some embodiments, administration of an expression cassette encoding eLGIC delivers it to a subpopulation of neuronal cells in a subject, causing expression of eLGIC in the neurons. In some embodiments, expression of eLGIC can modulate the activity of these neurons by administration of a corresponding small molecule ligand.

[0249] In some embodiments, transgene is operably linked to one or more control / regulatory elements that direct its transcription or expression.Control elements (i.e., regulatory elements) can include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance the secretion of encoded products, if desired.Exemplary regulatory elements are described in this disclosure.

[0250] Such control elements may include control sequences normally associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, endogenous cellular promoters heterologous to the gene of interest, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMV-IE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Additionally, sequences derived from non-viral genes, such as the mouse metallothionein gene, can also be used.

[0251] In some embodiments, a cell type- or tissue-specific promoter is operably linked to the transgene to selectively or preferentially produce the corresponding gene product encoded by the transgene in a particular cell type or tissue, hi some embodiments, an inducible promoter is operably linked to the transgene.

[0252] In some embodiments, an expression cassette encoding a transgene can be delivered to neurons using non-viral methods (e.g., using non-viral vectors, synthetic nanoparticles, etc.). Engineered ligand-gated ion channels (eLGICs)

[0253] The present disclosure describes a receptor, its variants, and its method of use in treating focal epilepsy and / or neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia.In some embodiments, the receptor is an engineered ligand-gated ion channel (LGIC).In some embodiments, the receptor is a chimeric eLGIC.

[0254] In some embodiments, the receptor is an engineered receptor (e.g., eLGIC). The term "engineered receptor" as used herein refers to a receptor that has been experimentally altered to be physically and / or functionally different from its corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. The term "wild-type receptor" as used herein refers to a receptor that has a polypeptide sequence that is identical to the polypeptide sequence of a protein found in nature. Wild-type receptors include receptors that naturally occur in humans as well as orthologues that naturally occur in other eukaryotes, e.g., protists, fungi, plants or animals, e.g., yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates. In some embodiments, the parent receptor is a non-natural receptor; that is, it is a receptor that does not occur in nature, e.g., a receptor that has been engineered from a wild-type receptor. For example, the parent receptor may be an engineered receptor that includes one or more subunits from one wild-type receptor along with one or more subunits from a second wild-type receptor. The resulting protein thus includes subunits from two or more wild-type receptors. Thus, in some embodiments, the parent receptor is a chimeric receptor. Engineered receptors of the present disclosure include, for example, parent receptor mutants, and switch receptors.

[0255] In some embodiments, the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding parent receptor, for example, one or more mutations in one or more domains of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptor shares about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity (including all values ​​and subranges therebetween) with the corresponding parent receptor. In some embodiments, the parent receptor mutant has 85% or more sequence identity with the corresponding parent receptor, for example, 90% or more, or 95% or more, for example, about 96%, about 97%, about 98%, or about 99% identity (including all values ​​and subranges therebetween) with the corresponding parent receptor. In some embodiments, the engineered receptor (e.g., parent receptor mutant) is generated by error-prone PCR.

[0256] In some embodiments, the ligand-binding domain (LBD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ligand-binding domain of the parent receptor, e.g., one or more mutations in the ligand-binding domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand-binding domain of the engineered receptor has 85% or more sequence identity with the corresponding ligand-binding domain of the parent receptor, e.g., 90% or more, or 95% or more, e.g., about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity (including all values ​​and subranges therebetween) with the corresponding ligand-binding domain of the parent receptor. In some embodiments, the ligand-binding domain of the engineered receptor shares at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity sequence identity (including all values ​​and subranges therebetween) with the corresponding ligand-binding domain of the parent receptor.

[0257] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ion pore domain of the parent receptor, e.g., one or more mutations in the ion pore domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ion pore domain of the engineered receptor has 85% or more sequence identity with the corresponding ion pore domain of the parent receptor, e.g., 90% or more, or 95% or more, e.g., about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity (including all values ​​and subranges therebetween) with the corresponding ion pore domain of the parent receptor. In some embodiments, the ion pore domain of the engineered receptor shares a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity (including all values ​​and subranges therebetween) with the corresponding ion pore domain of the parent receptor.

[0258] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation compared to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, substantially reduce, or abolish the function of the engineered receptor compared to the parent receptor, for example, by reducing the binding of endogenous ligand to the engineered receptor compared to the binding of endogenous ligand to the parent receptor, or by reducing the activity of the downstream signaling pathway(s) of the engineered receptor that is typically activated in response to the binding of the ligand to the corresponding parent receptor. In some embodiments, the mutation is an amino acid substitution.

[0259] In some embodiments, the amino acid mutation is a gain-of-function amino acid mutation compared to the corresponding parent receptor. A "gain-of-function" amino acid mutation refers to one or more mutations that modify the function of the engineered receptor compared to the parent receptor, for example, by altering or enhancing the affinity of the engineered receptor for a ligand compared to the binding of the 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 the endogenous ligand to the corresponding parent receptor. In some embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for a ligand. In certain embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for an agonist ligand. In some embodiments, the gain-of-function mutation results in an antagonist ligand that acts as an agonist ligand upon binding to the engineered receptor (e.g., resulting in the activation of an agonist signaling pathway instead of an antagonist signaling pathway). In some embodiments, the gain-of-function mutation results in a modulator ligand that acts as an agonist ligand upon binding to the engineered receptor. In some embodiments, the mutation is an amino acid substitution.

[0260] In some embodiments, the engineered receptors of the present disclosure, or the ligand binding domains and / or ion pore domains thereof, comprise one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations compared to the corresponding parent receptor. In some embodiments, the mutations are amino acid substitutions.

[0261] In some embodiments, the loss-of-function mutation and the gain-of-function mutation are at the same residue, i.e., they are the same mutation. In other embodiments, the loss-of-function mutation and the gain-of-function mutation are mutations at different amino acid residues. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptor (or its ligand binding domain and / or ion pore domain) of interest that includes a loss-of-function mutation and / or a gain-of-function mutation shares about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or less sequence identity (including all ranges and subranges therebetween) with the corresponding parent receptor, e.g., a wild-type receptor or a non-natural receptor (or its ligand binding domain and / or ion pore domain). In some embodiments, a subject engineered receptor (or its ligand binding domain and / or ion pore domain) shares 85% or more sequence identity, e.g., 85%, 90%, or 95% or more sequence identity, and in some examples, 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity (including all values ​​and subranges therebetween), with a corresponding parent receptor (or its ligand binding domain and / or ion pore domain).

[0262] In some aspects, the engineered receptor of the present disclosure comprises a receptor resulting from the combination of one or more amino acid sequences, e.g., subunits, from one wild-type receptor with one or more amino acid sequences, e.g., subunits, from a second wild-type receptor. In other words, the engineered receptor comprises amino acid sequences that are heterologous to each other, where "heterologous" means that they do not exist together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, the chimeric receptor serves as a parent receptor from which the engineered receptor of the present disclosure is generated. In some embodiments, the chimeric receptor comprises a ligand binding domain from a first LGIC and an ion pore domain from a second LGIC.

[0263] In some embodiments, the parent receptor mutant exhibits increased affinity for agonist ligands, hi 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.

[0264] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGICs refer to a large group of transmembrane proteins that allow the passage of ions upon activation by a specific ligand. LGICs are composed of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Ligand binding to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding causes a dramatic change in the permeability of the channel to a specific ion or ions; when it is inactive or closed, ions cannot effectively pass through the channel, but upon ligand binding, up to 107 ions / sec can pass through. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and facilitate the influx of ions into the cytosol. In some embodiments, LGICs respond to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and facilitate the efflux of ions from the cytosol to the extracellular environment. Importantly, activation of LGIC results in the transport of ions (e.g., Ca2+, Na+, K+, Cl-, etc.) across the cell membrane and not the transport of the ligand itself.

[0265] LGIC receptors are composed of multiple subunits and may be either homomeric or heteromeric receptors. Homomeric receptors are composed of subunits that are all of the same type. Heteromeric receptors are composed of subunits where at least one subunit is different from at least one other subunit contained within the receptor. For example, glycine receptors are composed of five subunits where there are two types: α-subunits where there are four isoforms (α1-α4) and β-subunits where there is a single known isoform. An exemplary homomeric GlyR is a GlyR composed of five α1-GlyR subunits. Similarly, homomeric GABAA receptors may be composed of β3-GABAA subunits and nAchR receptors may be composed of α7-nAchR subunits. An exemplary heteromeric GlyR may be composed of one or more α-subunits and one or more β-subunits (e.g., α1β-GlyR). Exemplary LGIC receptor subunits are shown in Table 4. Table 4: LGIC receptors and subunits [Table 4-1] [Table 4-2]

[0266] Illustrative examples of families of LGICs suitable for use in certain embodiments include, but are not limited to, Cys-loop receptors such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), lambda-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchRs); as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaCs), ionotropic glutamate receptors, IP3 receptors, P2X receptors, ryanodine receptors, and zinc-activated channels (ZACs).

[0267] Specific non-limiting examples of LGICs suitable for use with the methods described herein include HTR3A, HTR3B, HTR3C, HTR3D, HTR3E, ASIC1, ASIC2, ASIC3, SCNN1A, SCNN1B, SCNN1D, SCNN1G, GABRA1, GABRA2, GABRA3, GABRA4, GABRA5, GABRA6, GABRB1, GABRB2, GABRB3, GABRG1, GABRG2, GABRG3, GABRD, GABRE, GABRQ, GABRP, GABRR1, GABRR2, GABRR3, GLRA1, GLRA2, GLRA3, GLRA4, GLRB, GRIA1, GRIA2, GRIA3, These include GRIA4, GRID1, GRID2, GRIK1, GRIK2, GRIK3, GRIK4, GRIK5, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, ITPR1, ITPR2, ITPR3, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG, CHRND, CHRNE, P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, RYR1, RYR2, RYR3, and ZACN.

[0268] Non-limiting examples of wild-type LGIC receptor sequences that are useful in generating engineered receptors of the present disclosure include the following, in which the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is underlined:

[0269] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor alpha 7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 25), encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5): [ka]

[0270] In some embodiments, the wild-type LGIC receptor is the human alpha 1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 26), encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1): [ka]

[0271] In some embodiments, the wild-type LGIC receptor is the human alpha 2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO: 27), encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1): [ka]

[0272] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO: 28), encoded by the GLRA3 gene (GenBank Accession No. NM_006529.3): [ka]

[0273] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO: 29), encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.3): [ka]

[0274] In some aspects, the engineered receptor is a chimeric LGIC receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence from at least a first LGIC and an ion pore conducting domain sequence 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 is derived. In some embodiments, the derived amino acid sequence may contain an alteration at at least one amino acid position compared to the corresponding region of the LGIC from which it is derived. In some embodiments, the amino acid sequence derived from the LGIC sequence differs from the corresponding region of the original amino acid sequence by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the derived amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity (including all ranges and subranges therebetween) to the corresponding region of the LGIC amino acid sequence.

[0275] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequence and ion pore domain sequence of Cys loop receptors are well known in the art and can be easily identified from the literature by using publicly available software such as PubMed, Genbank, Uniprot, etc. In some embodiments, the ligand binding domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ligand binding domain of the first LGIC. In some embodiments, the ion pore domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is shown in bold and the ion pore domain is shown underlined.

[0276] 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:26). In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 29-235 of GlyRα1, e.g., amino acids 29-235, amino acids 29-240, amino acids 29-246, amino acids 29-248, amino acids 29-250, or amino acids 29-252 of SEQ ID NO:26. In certain such embodiments, the ligand binding domain consists essentially of amino acids 29-235 of SEQ ID NO: 26, consists essentially of amino acids 29-240 of SEQ ID NO: 26, consists essentially of amino acids 29-246 of SEQ ID NO: 26, consists essentially of amino acids 29-248 of SEQ ID NO: 26, consists essentially of amino acids 29-250 of SEQ ID NO: 26, or consists essentially of amino acids 29-252 of SEQ ID NO: 26. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human GlyRα1.

[0277] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises around amino acids 23-220 of the human α7-nAChR (SEQ ID NO:25), for example, amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO:25. In some embodiments, the ligand binding domain consists essentially of amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 25. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0278] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 23-220 of human α7-nAChR (SEQ ID NO:25), such as amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of SEQ ID NO:25. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0279] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 245-457 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 240-457, amino acids 245-457, amino acids 248-457, amino acids 249-457, amino acids 250-457, amino acids 255-457, or amino acids 260-457 of SEQ ID NO:26. In some embodiments, the ion pore domain consists essentially of amino acids 245-457 of SEQ ID NO:26, consists essentially of amino acids 248-457 of SEQ ID NO:26, consists essentially of amino acids 249-457 of SEQ ID NO:26, or consists essentially of amino acids 250-457 of SEQ ID NO:26.

[0280] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO:27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO: 27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 258-452 of SEQ ID NO: 27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 260-452 of SEQ ID NO: 27.

[0281] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 253-464 of SEQ ID NO: 28. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 257-464 of SEQ ID NO: 28. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 259-464 of SEQ ID NO: 28.

[0282] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 253-449 of SEQ ID NO: 29. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 257-449 of SEQ ID NO: 29. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 259-449 of SEQ ID NO: 29.

[0283] In some embodiments, the ion pore domain is derived from an ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 230-502, e.g., amino acids 227-502, amino acids 230-502, amino acids 231-502, amino acids 232-502, or amino acids 235-502 of α7-nAChR (SEQ ID NO:25). In certain such embodiments, the ion pore domain consists essentially of amino acids 227-502 of SEQ ID NO:25, consists essentially of amino acids 230-502 of SEQ ID NO:25, consists essentially of amino acids 231-502 of SEQ ID NO:25, consists essentially of amino acids 232-502 of SEQ ID NO:25, or consists essentially of amino acids 235-502 of SEQ ID NO:25.

[0284] In some embodiments, the ion pore domain of the subject chimeric ligand-gated ion channel comprises an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ion pore domain. By "M2-M3 linker domain" or "M2-M3 linker" is meant a sequence within the ion pore domain of the LGIC that is flanked at the amino (N)-terminus by the C-terminus of the transmembrane domain 2 (M2) of the receptor and at the carboxy (C)-terminus by the N-terminus of the transmembrane domain 3 (M3) of the receptor. The M2-M3 linker of the LGIC can be easily determined in the art and / or by using any publicly available protein analysis tool, such as Expasy, uniProt, etc. In some embodiments, when the ion pore domain of the chimeric receptor comprises a heterologous M2-M3 linker, the M2-M3 linker is derived from the same receptor as the ligand binding domain of the chimeric receptor. For example, if a ligand-gated ion channel of interest comprises a ligand binding domain from AChR and an ion pore domain from GlyR, the ion pore domain sequence may comprise an M2-M3 linker sequence from AChR. In some embodiments, the ion pore domain is from GlyRα1 and the M2-M3 linker is from α7-nAChR. In some embodiments, the native M2-M3 linker sequence removed from the ion pore domain corresponds to around amino acids 293-313 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 304-310, 293-306, 298-310, 305-311, 302-313, etc. In some such embodiments, the inserted M2-M3 linker is derived from around amino acids 281-295 of the α7-nAChR (SEQ ID NO:25), e.g., amino acids 290-295, 281-290, 281-295, 283-295, 287-292, etc., or a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to amino acids 281-295 or 283-295 of the α7-nAChR (SEQ ID NO:25).

[0285] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a Cys loop domain sequence that is heterologous to the Cys loop sequence of the ligand-binding domain. "Cys loop domain sequence" or "Cys loop sequence" refers to a domain in the ligand-binding domain of the Cys loop LGIC that forms a loop structure flanked by cysteines at the N-terminus and C-terminus. Without wishing to be bound by theory, upon binding of a ligand to the ligand-binding domain, the Cys loop is structurally shifted to be close to the M2-M3 loop, and this shift is believed to mediate the biophysical conversion of ligand binding in the extracellular domain to signal transduction in the ion pore domain (reviewed in Miller and Smart, Trends in Pharmacological Sci 2009:31(4)). The replacement of endogenous Cys loop sequence with heterologous Cys loop sequence can increase the conductivity of LGIC by 1.5 times or more, for example, at least 2 times, 3 times or 4 times, in some cases at least 5 times or 6 times, and at least 7 times, 8 times, 9 times or 10 times at a certain dose. The Cys loop domain of Cys loop receptor can be easily determined by the art and / or by using any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand binding domain of chimeric receptor comprises heterologous Cys loop sequence, the Cys loop sequence is derived from the same receptor as the ion pore domain of chimeric receptor. For example, when the subject chimeric ligand-gated ion channel comprises a ligand binding domain derived from AChR and an ion pore domain derived from GlyR, the subject ligand-gated ion channel may comprise the ligand binding domain sequence derived from AChR, except for the sequence of the Cys loop domain derived from GlyR. In some embodiments, the ligand binding domain is derived from an α7-nAChR and the Cys loop sequence is derived from a GLyR.In some embodiments, the Cys loop sequence removed from the ligand binding domain corresponds to around amino acids 150-164 of α7-nAChR (SEQ ID NO:25), e.g., amino acids 150-157 of α7-nAChR. In some embodiments, the Cys loop sequence inserted is from around amino acids 166-180 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 166-172 of GlyRα1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 166-180 of GlyRα1. In some embodiments, the Cys loop sequence inserted is from around amino acids 172-186 of GlyRα2 (SEQ ID NO:27), e.g., amino acids 172-178 of GlyRα2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 172-186 of GlyRα2. In some embodiments, the inserted Cys loop sequence is derived from around amino acids 171-185 of GlyRα3 (sequence number 28 or 29), e.g., amino acids 171-177 of GlyRα3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 171-185 of GlyRα3.

[0286] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a β1-2 loop domain sequence that is heterologous to the β1-2 loop domain sequence of the ligand-binding domain. By "β1-2 loop domain sequence" or "β1-2 loop or β1-β2 loop" is meant a domain in the ligand-binding domain of the Cys loop LGIC that is flanked at its N-terminus by the C-terminus of the β1 sheet and at its C-terminus by the N-terminus of the β2 sheet. Without wishing to be bound by theory, it is believed that the β1-2 loop helps mediate the biophysical conversion of ligand binding in the extracellular domain to the ion pore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). Upon ligand binding, it is believed that the β1-2 loop, together with the Cys loop, comes close to the M2-M3 loop and mediates the biophysical conversion of ligand binding in the extracellular domain to signal transduction in the ion pore domain where the M2-M3 loop resides (reviewed in Miller and Smart, supra). Substitution of the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of LGIC by 1.5-fold or more, for example, at least 2-fold, 3-fold or 4-fold, in some cases at least 5-fold or 6-fold, and at least 7-fold, 8-fold, 9-fold or 10-fold at certain doses. The β1-2 loop of a Cys-loop receptor can be easily determined by using the art and / or any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand-binding domain of a chimeric receptor comprises a heterologous β1-2 loop sequence, the β1-2 loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the subject chimeric ligand-gated ion channel comprises a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the sequence of the β1-2 loop domain of the ligand-binding domain may be derived from GlyR. In some embodiments, the ligand-binding domain is derived from α7-nAChR.In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to around amino acids 64-72 or 67-70 of α7-nAChR (SEQ ID NO:25), e.g., amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the β1-2 loop sequence inserted is around amino acids 79-85 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 80-85, 81-84, 79-85, or 81-84 of GlyRα1, and includes at most three, at most two, at most one, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα2, and the β1-2 loop inserted corresponds to around amino acids 86-91 of GlyRα2 (SEQ ID NO:27), and includes at most three, at most two, at most one, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα3 and the β1-2 loop inserted corresponds to about amino acids 85-90 of GlyRα3 (SEQ ID NO: 28 or 29) and contains at most 3, at most 2, at most 1, or no amino acid mutations. In some embodiments, the mutations are amino acid substitutions.

[0287] In some embodiments, the present disclosure provides a chimeric LGIC receptor comprising a ligand binding domain derived from human α7-nAChR, the ligand binding domain comprising one or more amino acid substitutions of the present disclosure, and an ion pore domain derived from 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 human glycine receptor. In some embodiments, the ligand binding domain comprises a β1-2 loop domain derived from human glycine receptor.

[0288] Non-limiting examples of sequences of chimeric LGIC receptors of the present disclosure include the sequences disclosed herein as SEQ ID NOs: 30-31 and 33. In some embodiments, the chimeric LGIC receptor has 85% or more sequence identity to the sequences provided herein in SEQ ID NOs: 30-31 and 33, e.g., 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the sequences provided herein in SEQ ID NOs: 30-31 and 33. In the sequences, the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is shown underlined.

[0289] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα1 Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 33: [ka]

[0290] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that includes the human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) that includes the GlyRα1 β1-2 loop sequence (lowercase) and the Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0291] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 attachment point) that contains the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka] Amino acid mutations in ligand-gated ion channels

[0292] As discussed above, in some aspects, the engineered receptor of interest comprises at least one amino acid mutation that changes the potency of ligand to engineered receptor compared to its potency to non-mutated parent receptor.In other words, one or more amino acid mutations, for example, loss-of-function mutations or gain-of-function mutations, shift the potency of engineered receptor to ligand compared to the potency of non-mutated parent receptor.In some embodiments, the mutation is an amino acid substitution.In some embodiments, the one or more mutations are in the ligand-binding domain of engineered receptor. In some embodiments, when the ligand-binding domain of engineered receptor is a Cys-loop receptor protein, one or more amino acid mutations are substitutions with the residues corresponding to the residues of α7-nAChR (SEQ ID NO:25) selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213 and Y217.In some embodiments, one residue is substituted.In some embodiments, 2, 3, 4, or 5 or more residues are substituted, for example, 6, 7, 8, 9 or 10 residues are substituted. In certain embodiments, the residue corresponds to a residue of α7-nAChR (SEQ ID NO: 25) selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence.

[0293] In some embodiments, the one or more substitutions reduce the potency of the engineered receptor for acetylcholine and non-natural ligands, e.g., 2-fold or less, 3-fold or less, 4-fold or less, 5-fold or less, 10-fold or less, 20-fold or less, 30-fold or less, 50-fold or less, or 100-fold. In certain embodiments, the one or more substitutions correspond to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of α7-nAChR. In other examples, the one or more substitutions selectively reduce the potency of acetylcholine to the engineered receptor. In other words, the one or more substitutions reduce the potency of the engineered receptor for acetylcholine while essentially maintaining the potency for the non-natural ligand, or reduce the potency of the engineered receptor for acetylcholine by 2 or less, for example, 3, 4, 5 or less, and in some cases, 10, 20, 50, or 100 or less, than it would otherwise reduce the potency of the engineered receptor for the non-natural ligand. In some embodiments, the substitution corresponds to L131E, L131S, L131T, L131D, or S172D of α7-nAChR. In yet other embodiments, the one or more substitutions selectively reduce the potency of the non-natural ligand for the engineered receptor.In other words, the one or more substitutions reduce the potency of the engineered receptor against a non-natural ligand while essentially maintaining the potency against acetylcholine, or reduce the potency of the engineered receptor against a non-natural ligand by 2-fold or less, for example, 3-fold, 5-fold or less, and in some cases, 10-fold, 20-fold or 50-fold or less than it would otherwise reduce the potency of the engineered receptor against acetylcholine. In some embodiments, the substitution corresponds to W77M, Y115W, S172T, or S172C of α7-nAChR. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126, and facinicline / RG3487.

[0294] In other embodiments, the one or more substitutions increase the potency of the engineered receptor for acetylcholine and / or non-natural ligands, e.g., 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In some embodiments, the 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, the one or more substitutions increase the potency of both acetylcholine and non-natural ligand. 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, the one or more substitutions selectively increase the potency of acetylcholine for the engineered receptor. In other words, the one or more substitutions increase the potency of the engineered receptor for acetylcholine by 2-fold or more, for example, 3-fold, 4-fold, or 5-fold or more, and in some examples, 10-fold, 20-fold, 50-fold, or 100-fold, more than it increases the potency of the engineered receptor for non-natural ligand. In some embodiments, the substitution corresponds to L141W, S172T, S172C, S188P or S188W of α7-nAChR. In certain embodiments, the one or more substitutions are in the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC-5619, TC6987, ABT-126 and facinicline / RG3487. In yet other examples, the one or more substitutions selectively increase the potency of the non-natural ligand against the engineered receptor.In other words, the one or more substitutions increase the potency of the engineered receptor for a non-natural ligand by 2-fold or more, e.g., 3-fold, 5-fold or more, and in some cases, 10-fold, 20-fold or 50-fold or more, more than it increases the potency of the engineered receptor for acetylcholine.

[0295] In some embodiments, the amino acid residue mutated in the subject engineered receptor is not the amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of wild-type a7 nAChR (SEQ ID NO: 25). In some embodiments, the mutation is an amino acid substitution. In some embodiments, the amino acid residue mutated in the subject engineered receptor is the amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of wild-type a7 nAChR (SEQ ID NO: 25). In some embodiments, the substitution is not a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution corresponds to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in wild-type α7 nAChR. In some embodiments, when such a substitution is present in an engineered receptor, it is present in combination with one or more amino acid mutations described herein.

[0296] In some embodiments, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 25) mediate the binding of natural ligand acetylcholine. In some embodiments, mutations at these residues reduce the binding of acetylcholine and can 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 mediate the binding of non-natural ligand AZD0328 to this receptor, and mutations at these residues increase the affinity of AZD0328 and / or other ligands to this receptor and can therefore be considered gain-of-function mutations. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues of the ligand binding domain region of α7-nAChR (SEQ ID NO: 25) or the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217. In some embodiments, the mutation is an amino acid substitution. In certain embodiments, the mutation in one or more amino acid residues in the ligand binding domain region of α7-nAChR (sequence number 25) or the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of α7-nAChR is a substitution with one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217.

[0297] In some embodiments, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 25) mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues can reduce the binding of acetylcholine and / or nicotine. In some embodiments, R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR mediate the binding of non-natural ligand ABT126, and mutations at one or more of these residues can increase the affinity of ABT126 and / or other ligands to α7-nAChR. In some embodiments, the mutation is an amino acid substitution. In some embodiments, R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR mediate binding of the non-natural ligand TC6987, and mutation of one or more of these residues can increase the affinity of TC6987 and / or other ligands for the α7-nAChR. In some embodiments, R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR mediate binding of the non-natural ligand facinicline / RG3487, and mutation of one or more of these residues can increase the affinity of facinicline / RG3487 and / or other ligands for the α7-nAChR. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues in the ligand binding domain region of an α7-nAChR or a ligand binding domain of a chimeric receptor comprising the ligand binding domain region of an α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N120, N129, L131, L141, S170, W171, S172, S188, Y210, C212, C213 and Y217.In some embodiments, one or more amino acid residues alter the binding of acetylcholine and / or nicotine to α7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from C212 and S170. In some embodiments, the mutation in one or more amino acid residues alters the binding of ABT126 to α7-nAChR, wherein one or more amino acid residues are selected from the group consisting of R101, Y115, L131, L141, W171, S172, S188, Y210 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from R101, S188 and Y210. In some embodiments, the mutation in one or more amino acid residues alters the binding of TC6987 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210, and C213. In some embodiments, the mutation in one or more amino acid residues alters the binding of fasiniline / RG3487 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR. In certain such embodiments, the amino acids are selected from Y210, R101, and N129.

[0298] The present disclosure provides engineered receptors having two or more mutations, such as amino acid substitutions, compared to the parent receptor. In some embodiments, the parent receptor comprises a ligand binding domain derived from human α7 nicotinic acetylcholine receptor (α7-nAChR). In some embodiments, the parent receptor is a chimeric receptor. In some embodiments, the parent receptor comprises an ion pore domain derived from 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 comprises a Cys loop domain derived from human glycine receptor. In some embodiments, the parent receptor comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the engineered receptor comprises two amino acid substitutions compared to the parent receptor comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the ligand binding domain of the engineered receptor comprises a β1-2 loop domain derived from human glycine receptor α1 subunit.

[0299] In some embodiments, the ligand binding domain of the engineered receptor comprises amino acid substitutions at 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: 25).

[0300] In some embodiments, the two amino acid substitutions are at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution at S172D. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution at Y115D. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of L131S and S172D, L131T and S172D, L131D and S172D, Y115D and S170T, Y115D and L131Q, and Y115D and L131E. In some embodiments, the ligand binding domain comprises an amino acid substitution of L131E.

[0301] In some embodiments, the ligand binding domain comprises one or more amino acid substitutions at amino acid residues selected from the group consisting of Y140, R101, L131, Y115, and Y210, where the amino acid residues correspond to amino acid residues of α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid substitution of R101W and / or Y210V. In some embodiments, the ligand binding domain comprises two or more amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of R101 and L131, Y115 and Y210, and R101 and Y210. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of R101F and L131G, R101F and L131D, Y115E and Y210W, R101W and Y210V, R101F and Y210V, R101F and Y210F, R101M and L131A, and R101M and L131F. In some embodiments, the ligand binding domain comprises three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand binding domain comprises amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0302] In some embodiments, the ligand-binding domain comprises 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.

[0303] In some embodiments, the ligand-binding domain comprises a hydrophobic amino acid substitution at residue Y210 and an amino acid substitution of R101W or R101F. In some embodiments, the amino acid substitution at residue Y210 is Y210V, Y210F, or Y210W.

[0304] Those skilled in the art will easily recognize suitable control receptors for comparison with the engineered receptors of the present disclosure. In some embodiments, the control receptor is identical in sequence to the engineered receptor, except for one or more identifying amino acid mutations (e.g., substitutions). In all cases, reference to the control receptor is meant to indicate that the described change in properties (e.g., potency against ligands) is the result of the amino acid mutation(s) of the engineered receptor of the present disclosure.

[0305] The present disclosure provides an engineered receptor, which is a chimeric ligand-gated ion channel (LGIC) receptor, comprising: (a) a ligand-binding domain derived from human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprising a Cys loop domain derived from human glycine receptor α1 subunit; and (b) an ion pore domain derived from human glycine receptor α1 subunit. In some embodiments, the engineered receptor is derived from a parent engineered receptor that comprises or consists of the amino acid sequence of SEQ ID NO: 33, and further comprises one or more amino acid substitutions based on the parent engineered receptor.

[0306] In some embodiments, the potency of the engineered receptor for acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is at least 1.5 times lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine (e.g., less than about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)). In some embodiments, the potency of the engineered receptor for acetylcholine is evaluated by its EC50 for acetylcholine based on a cell reporter assay using YFP fluorescence quenching. 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 a higher expression level of the engineered receptor in or on the cell surface in the presence of physiological concentrations of acetylcholine without passing a significant amount of current through the cell.

[0307] In some embodiments, the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least 1.5 times higher (e.g., less than about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, determining potency involves determining the EC50 based on a cellular reporter assay using YFP fluorescence quenching as described in Example 2 of the present disclosure. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 15 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 150 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 the non-natural ligand is less than 10 nM. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 100 nM. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 1 μM.

[0308] In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least 1.5 times (e.g., less than about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, determining efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand.

[0309] In some aspects, the ligand-gated ion channel of interest comprises one or more non-desensitizing mutations. In some embodiments, the mutations are amino acid substitutions. When used in the context of a ligand-gated ion channel, "desensitization" refers to a progressive reduction in ion influx in the prolonged presence of an agonist. This results in a progressive loss of efficacy of the neuron to the ligand. By non-desensitizing mutation is meant an amino acid mutation that prevents the neuron from becoming less responsive or unresponsive to the ligand by preventing the LGIC from becoming desensitized to the ligand. Non-desensitizing mutations can be easily identified by introducing a LGIC carrying the mutation into a neuron and analyzing the current flux over time during prolonged exposure to the ligand. If the LGIC does not contain a non-desensitizing mutation, the current recovers from the peak to steady state during prolonged exposure, whereas if the LGIC contains a non-desensitizing mutation, the current remains at the peak flux for the duration of exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (post-processing of the proprotein to remove the signal peptide, V294L). LGIC desensitization, methods for measuring LGIC desensitization, and non-desensitizing mutations are well known in the art; see, for example, Gielen et al. Nat Commun 2015 Apr 20, 6:6829, and Keramidas et al. Cell Mol Life Sci. 2013 Apr;70(7):1241-53, the entire disclosures of which are incorporated herein by reference.

[0310] In some aspects, the subject ligand-gated ion channel comprises one or more converting mutations. In some embodiments, the mutation is an amino acid substitution. By converting mutation is meant a mutation that changes the permeability of the ion pore domain of the LGIC such that it becomes permissive to the conduction of non-native ions, i.e., ions that cannot be naturally passed. In some cases, a mutation, e.g., replacement of amino acid residues 260-281 in human α7-nAChR (CHRNA7) (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 34) or the corresponding amino acid in another cation-permeable LGIC with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 35), converts the permeability from cations to anions. In some cases, mutations such as substitution of amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC with glutamic acid (E) (which converts the LGIC from anion-permeable to calcium-permeable, such as A293E substitution in GLRA1), or deletion of amino acid residue 278 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC, substitution of amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC with glutamic acid (E), and substitution of amino acid residue 293 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC with valine (V) (which converts the LGIC from anion-permeable to cation-permeable, such as P278Δ, A279E, T293V in GLRA1).

[0311] Further engineered receptors beyond those described herein can be identified by in vitro screening and validation methods. In some embodiments, a library of parent receptor mutants is generated from a limited number of parent receptors. The parent receptors can be mutated using methods known in the art, including error-prone PCR. In some embodiments, the library of parent receptor mutants is then transfected into yeast or mammalian cells and screened with high efficiency to identify functional receptors (e.g., to identify parent receptor mutants that can signal in response to a ligand). In some embodiments, the functional parent receptor mutants identified in this primary screen are then expressed in mammalian cells and screened for potency against ligands, for example, by plate reader and / or electrophysiological assays described herein. Parent receptor mutants that show increased binding affinity to agonist ligands or allow the use of antagonist or modulator ligands as agonists in secondary screens can then be selected and further in vitro and / or in vivo validation and characterization assays can be performed.Such screening assays are known in the art, e.g., Armbruster, BN et al. (2007) PNAS, 104, 5163-5168; Nichols, CD and Roth, BL (2009) Front. Mol. Neurosci. 2, 16; Dong, S. et al. (2010) Nat. Protoc. 5, 561-573; Alexander, GM et al. (2009) Neuron 63, 27-39; Guettier, JM et al. (2009) PNAS 106, 19197-19202; Ellefson JW et al. (2014) Nat Biotechnol. 32 (1): 97-101; Maranhao AC and Ellington AD. (2017) ACS Synth Biol. 20; 6 (1): 108-119; Talwar S et al. (2013) PLoS One; 8 (3): e58479; Gilbert DF et al. (2009) Front Mol Neurosci. 30; 2: 17; Lynagh and Lynch, (2010), Biol Chem. 14: 285 (20), 14890-14897; Islam R. et al. (2016) ACS Chem Neurosci. 21; 7 (12): 1647-1657; and Myers et al. (2008) Neuron. 8: 58 (3): 362-373.

[0312] A summary of these exemplary engineered receptors is provided below in Table 5. Each of these receptors comprises or consists of the amino acid sequence of SEQ ID NO: 33, except for the indicated amino acid mutations. Table 5: Exemplary engineered receptors [Table 5-1] [Table 5-2] Ligand

[0313] In some embodiments, the ligand of the present disclosure refers to an exogenous drug or compound that has a known mechanism of action on mammalian cells (e.g., known to act as an agonist, antagonist, or modulator of a receptor). Such ligands can also be referred to as "binding agents." The ligands of the present disclosure may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the ligands include drugs or compounds approved by the U.S. Food and Drug Administration (FDA) for clinical use in treating a particular disease (e.g., a neurological disease). 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 research. In some embodiments, the ligands are analogs of one of the above ligands. In some embodiments, the ligand is selected from the group consisting of AZD0328, ABT-126, AQW-051, cannabidiol, cilansetron, PH-399733, facinicline / RG3487 / MEM-3454, TC-6987, and TC-5619 / AT-101. In some embodiments, the ligand is selected from the group consisting of ABT-126, AZD-0328, RG3487, TC-6987, TC-6683, varenicline, and TC-5619. In some embodiments, the ligand is TC-5619.

[0314] In certain embodiments, the ligand is an analog of cilansetron, for example, as described by one of the following compound formulas 2-7 in either its R or S enantiomer. [ka]

[0315] 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. The term antagonist is used herein to refer to a ligand that inhibits a signaling response.

[0316] In some embodiments, the ligand has the formula: [ka] The compound is AZD-0328, which is described in

[0317] In some embodiments, the ligand has the formula: [ka] The compound is TC-6987 described in

[0318] In some embodiments, the ligand has the formula: [ka] The compound is ABT-126, described in

[0319] In some embodiments, the ligand has the formula: [ka] and TC-5619 / Braddaniclin, as described in .

[0320] In some embodiments, the ligand has the formula: [ka] The compound is TC-6683 described in

[0321] In some embodiments, the ligand has the formula: [ka] and varenicline, as described in

[0322] In some embodiments, the ligand has the formula: [ka] and fascinicline / RG3487 as described in

[0323] Exemplary combinations of engineered receptors and non-natural ligands described herein are provided in Table 6 below. Each engineered receptor in Table 6 may exist as a protein, a polynucleotide encoding a protein, or a vector comprising a polynucleotide encoding a protein. In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR. In some embodiments, the engineered receptor comprises an ion pore domain derived from human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1. In some embodiments, the engineered receptor comprises 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 indicated mutations in Table 6. Table 6: Non-limiting examples of combinations of engineered receptors and non-natural ligands [Table 6] composition

[0324] The present disclosure also provides compositions (e.g., pharmaceutical compositions). In some embodiments, the composition or pharmaceutical composition comprises a recombinant nucleic acid comprising an expression cassette of the present disclosure. In some embodiments, the expression cassette comprises a transgene encoding an engineered receptor (e.g., a ligand-gated ion channel) of the present disclosure. In some embodiments, the transgene encodes SEQ ID NO: 33 or a variant thereof. In some embodiments, the composition or pharmaceutical composition comprises a pharma- ceutically acceptable carrier, diluent, excipient, and / or buffer. In some embodiments, the pharma- ceutically acceptable carrier, diluent, excipient, and / or buffer is suitable for use in humans.

[0325] Such excipients, carriers, diluents, and / or buffers (collectively referred to as "pharmaceutical vehicles") include any pharmaceutical agent that can be administered without undue toxicity. They may be approved by federal or state regulatory agencies for use in mammals, such as humans, or may be listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. Such pharmaceutical vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical vehicles may be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary, stabilizing, thickening, lubricating, and coloring agents may be used. When administered to a mammal, the compounds and compositions of the present disclosure and the pharmaceutical acceptable vehicles, excipients, or diluents may be sterile. In some instances, aqueous media such as water, saline solution, and aqueous dextrose and glycerol solutions are used as vehicles when a compound of the present disclosure is administered intravenously.

[0326] The pharmaceutical composition may take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained release formulations thereof, or any other form suitable for administration to a mammal. In some cases, the pharmaceutical composition is formulated for administration by routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for their formulation are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, which are incorporated herein by reference.

[0327] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may be present in such vehicles. A wide variety of pharma-ceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are fully described in a variety of publications, including, for example, A. Gennaro, (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[0328] In some embodiments, the pharmaceutical composition comprises a liquid that includes the recombinant nucleic acid in a solution, suspension, or both. As used herein, a liquid composition comprises a gel. In some embodiments, the liquid composition is aqueous. In some embodiments, the composition is an in situ gellable aqueous composition, e.g., an in situ gellable aqueous solution.

[0329] Non-limiting examples of compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption enhancers or retarders that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharma-ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) can also be incorporated into the composition.

[0330] In some embodiments, pharmaceutical compositions are formulated to be compatible with a particular route of administration or delivery. Thus, pharmaceutical compositions contain carriers, diluents, and / or excipients suitable for administration by various routes.

[0331] The choice of excipient will be determined in part by the particular vector, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present disclosure.

[0332] For example, the vectors can be formulated into preparations for injection by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents such as vegetable oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, together with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers and preservatives, if desired.

[0333] Suitable compositions for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of active compounds. Preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting examples include water, saline, dextrose, fructose, ethanol, animal oils, vegetable oils or synthetic oils.

[0334] Cosolvents and adjuvants may be added to the formulation.Non-limiting examples of cosolvents include alcohols with hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0335] In some embodiments, the pharmaceutical compositions of the present disclosure are suitable for parenteral administration, for example, as aqueous and non-aqueous, isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulations isotonic with the blood of the intended recipient, and as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient, such as water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described.

[0336] As another example, the compound can be formulated into preparations suitable for oral administration, including: (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as a solid or granules; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings, and pharmacologically compatible excipients. Lozenge forms may include flavors, usually pastilles comprising the active ingredient in sucrose and acacia or tragacanth, and an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like, containing, in addition to the active ingredient, such excipients as described herein.

[0337] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of the compound of the present disclosure calculated in an amount sufficient to produce a desired effect, in association with a pharma- ceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. method

[0338] In some aspects of the present disclosure, the compositions and methods disclosed herein can be used to treat neurological disease or disorder in a subject in need of such treatment.In some aspects, the compositions and methods disclosed herein can be used to express transgenes in neurons.In some aspects, the compositions and methods disclosed herein can be used in assays.In some aspects, the compositions and methods disclosed herein can be used as research tools.In some aspects, the compositions and methods disclosed herein can be used as nucleic acid delivery tools.

[0339] In some aspects of the present disclosure, a method of treating a neurological disease or disorder in a subject is provided, comprising administering to the subject an effective amount of a recombinant nucleic acid comprising an expression cassette of the present disclosure. In some embodiments, the recombinant nucleic acid is delivered to a hippocampal neuron. In some embodiments, the recombinant nucleic acid is delivered to a dorsal root ganglion neuron or a trigeminal ganglion neuron. In some embodiments, the expression cassette comprises a transgene. In some embodiments, the transgene encodes an engineered LGIC. In some embodiments, the method further comprises activating the engineered LGIC and then administering a small molecule ligand / drug / agonist that modulates the activity of the target neuron, thereby treating the neurological disease or disorder (e.g., pain or epilepsy) in the subject.

[0340] In some aspects of the present disclosure, a method for treating focal epilepsy in a subject is provided, comprising administering to the subject an effective amount of a recombinant nucleic acid comprising an expression cassette, wherein the expression cassette comprises a transgene encoding the engineered LGIC receptor of the present disclosure.In some embodiments, the recombinant nucleic acid is delivered into hippocampal neurons.In some embodiments, the method further comprises activating the engineered LGIC, and then administering a small molecule ligand that modulates the activity of target neurons, thereby treating focal epilepsy in the subject.

[0341] In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used in the manufacture of medicaments for treating neurological diseases or disorders. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used in the manufacture of medicaments for treating neurological diseases or disorders associated with hippocampal neuronal dysregulation, such as focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, and fragile X syndrome. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat focal epilepsy. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat schizophrenia. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat autism spectrum disorder. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat Alzheimer's disease. In some embodiments, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat Rett syndrome. In some aspects, a vector, recombinant nucleic acid, or composition disclosed herein is used to treat Fragile X Syndrome.

[0342] The present disclosure, in part, contemplates compositions and methods for controlling, managing, preventing or treating epilepsy in a subject.In some embodiments, epilepsy is focal epilepsy.In some embodiments, focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0343] In some embodiments, the compositions and methods herein can be used to improve the level of epileptic seizures in subjects.In some embodiments, the compositions and methods herein can be used to prevent or control the level of epileptic seizures in subjects.Epileptic seizures can be classified as tonic-clonic seizures, tonic seizures, clonic seizures, myoclonic seizures, absence seizures or atonic seizures.

[0344] In some embodiments, the compositions and methods herein can reduce the number or frequency of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% (including all ranges and subranges therebetween). In some embodiments, the compositions and methods herein can reduce the number or frequency of epileptic seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% (including all ranges and subranges therebetween).

[0345] In some embodiments, the compositions and methods herein can reduce the level and / or duration of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% (including all ranges and subranges therebetween). In some embodiments, the compositions and methods herein can reduce the level and / or duration of epileptic seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% (including all ranges and subranges therebetween).

[0346] In various embodiments, a method for controlling, managing, preventing, or treating epilepsy (e.g., focal epilepsy) in a subject comprises administering to the subject an effective amount of a recombinant nucleic acid comprising an expression cassette of the present disclosure. Without wishing to be bound by any particular theory, the present disclosure contemplates using an expression cassette comprising a transgene encoding an engineered LGIC as disclosed herein to modulate neuronal activity to alleviate epilepsy in a subject.

[0347] In various embodiments, vectors and / or recombinant nucleic acids containing expression cassettes encoding engineered receptors that activate or depolarize neuronal cells are administered to (or introduced into) one or more neuronal cells that control epilepsy. In the presence of a ligand, neuronal cells expressing the engineered receptor are activated, reducing susceptibility to epilepsy.

[0348] In some embodiments, the epilepsy is focal epilepsy (focal epileptic seizures). Focal epilepsy is a neurological condition whose primary symptom is recurrent seizures affecting one hemisphere (half) of the brain.

[0349] Focal epilepsy falls into four categories: (a) focal awareness-preserving seizures, where the subject is aware of what is happening during the seizure; (b) focal awareness-impaired seizures, where the subject is confused or does not know or remember what is happening during the seizure; (c) focal motor seizures, where the subject moves to some degree - anything from convulsions to spasms to rubbing their hands to walking around; and (d) focal non-motor seizures, where the subject does not convulse or make any other movements during the seizure. Instead, it causes a change in the way the subject feels or thinks (e.g., feeling intense emotions, strange sensations, or symptoms such as heart racing, goosebumps, or feeling hot or cold).

[0350] Focal epilepsy is characterized by seizures originating in a specific part (lobe) of the brain. Focal epilepsy includes idiopathic localization-related epilepsy (ILRE), frontal lobe epilepsy, temporal lobe epilepsy, parietal lobe epilepsy, and occipital lobe epilepsy.

[0351] Idiopathic localization-related epilepsy (ILRE) is caused by unknown factors.

[0352] Frontal lobe epilepsy is a term for recurrent seizures that begin in the frontal lobe, the area of ​​the brain behind the forehead.

[0353] Temporal lobe epilepsy is a term for recurrent seizures that originate in the temporal lobe, a part of the brain located on the side of the head behind the temples and cheekbones. The temporal lobe is the area of ​​the brain that most commonly produces seizures. The mesial (middle) parts of both temporal lobes are very important in epilepsy - it is the frequent origin of seizures and prone to damage or scarring. Mesial temporal lobe epilepsy (mTLE) is the most common form of human epilepsy. Often its pathophysiological substrate is hippocampal sclerosis. Thus, one strategy to treat focal epilepsy such as mTLE is by targeting hippocampal neurons.

[0354] Parietal Lobe Epilepsy. The parietal lobe is the part of the brain that lies on the top and sides of the head. Known as the "association cortex," the parietal lobe is responsible for attaching meaning to brain functions. This is where the brain creates visual images, sounds are recognized as words, and touch is associated with specific objects. In a sense, the parietal lobe is where perceptions are matched up with physical reality.

[0355] Occipital lobe epilepsy. Occipital lobe epilepsy is the term for recurrent seizures that begin in the occipital lobe, the part of the brain at the back of the head that is primarily responsible for vision.

[0356] In some aspects of the present disclosure, a method for treating peripheral neuropathy and neuropathic pain such as trigeminal neuralgia in a subject is provided, comprising administering an effective amount of recombinant nucleic acid to the subject, wherein the recombinant nucleic acid comprises an expression cassette comprising a transgene encoding the engineered LGIC receptor of the present disclosure.In some embodiments, the recombinant nucleic acid is delivered into DRG or TGG neuron.In some embodiments, the method further comprises activating engineered LGIC, and then administering a small molecule ligand that modulates the activity of target neuron, thereby treating peripheral neuropathy and neuropathic pain such as trigeminal neuralgia in a subject.

[0357] In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder associated with DRG / TGG neuron dysregulation, such as neuropathic pain. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a spinal cord-related disease, such as spasticity, spinal cord injury, and avulsion injury. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat spasticity. In some embodiments, the treatment of spasticity comprises transducing neurons in the spinal column. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat spinal cord injury. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat avulsion injury. In some embodiments, the treatment of spinal cord injury or avulsion injury comprises transducing neurons in the dorsal horn.

[0358] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating pain in a subject. In some embodiments, the pain is neuropathic pain. "Pain" refers to discomfort and / or strange sensations in the subject's body. The sensation of pain can range from mild and occasional to severe and constant. Pain can be classified as acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain, or psychogenic pain. In some cases, pain is caused or associated with disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, Parkinson's disease, pain associated with epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and pain associated with central nervous system trauma, strain / sprain, and burn; nociceptive pain, including myocardial infarction, acute pancreatitis, post-operative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0359] The compositions and methods herein can be used to improve the level of pain in a subject. In some cases, the level of pain in a subject is improved by at least at least 5%, at least at least 10%, at least at least 15%, at least at least 20%, at least at least 25%, at least at least 30%, at least at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or about 100% (including all ranges and subranges therebetween). The level of pain in a subject can be evaluated by various methods. In some cases, the level of pain is evaluated by self-report (i.e., the human subject expresses a verbal report of the level of pain he / she is experiencing). In some cases, the level of pain is evaluated by behavioral indicators of pain, such as facial expressions, limb movements, vocalization, anxiety and guarding. These types of assessments can be useful, for example, when a subject is unable to self-report (e.g., young children, unconscious subjects, non-human subjects). Pain levels can be assessed after treatment with a composition of the present disclosure, compared to the level of pain the subject experienced prior to treatment with the composition.

[0360] In various embodiments, a method for controlling, managing, preventing, or treating pain in a subject comprises administering to the subject an effective amount of a recombinant nucleic acid comprising an expression cassette encoding an engineered LGIC of the present disclosure. Without wishing to be bound by any particular theory, the present disclosure contemplates using vectors and / or recombinant nucleic acids encoding the engineered LGICs disclosed herein to modulate neuronal activity to reduce pain in a subject.

[0361] In various embodiments, a recombinant nucleic acid comprising an expression cassette encoding an engineered receptor that activates or depolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells, e.g., inhibitory interneurons, in which pain sensation is to be reduced. In the presence of a ligand, the neuronal cells expressing the engineered receptor are activated, reducing their sensitivity to pain and enhancing the analgesic effect of stimulating these neuronal cells.

[0362] In various embodiments, a recombinant nucleic acid comprising an expression cassette encoding an engineered receptor that inactivates or hyperpolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells that are to have increased nociception or sensitivity to pain, such as nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc. In the presence of the ligand, the neuronal cells expressing the engineered receptor are inactivated, decreasing sensitivity to pain and enhancing the analgesic effect.

[0363] In some embodiments, the compositions and methods of the present disclosure are effective in reducing pain.The examples of pain suitable for treatment by the compositions and methods of the present disclosure 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, burn, back pain, eye pain, visceral pain, cancer pain (e.g. bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, post-herpetic neuralgia, post-operative pain, post-stroke pain and menstrual pain.

[0364] Pain can be classified as acute or chronic. "Acute pain" refers to pain that begins suddenly and is usually sharp in quality. Acute pain may be mild and last only a moment, or it may be severe and last for weeks or months. In many cases, acute pain does not last longer than three months, and it disappears when the underlying cause of the pain is treated or cured. However, unrelieved acute pain can result in chronic pain. "Chronic pain" refers to ongoing or recurrent pain that lasts beyond the normal course of an acute illness or injury, or that lasts longer than three to six months and adversely affects the well-being of an individual. In some embodiments, the term "chronic pain" refers to pain that continues when it should not. Chronic pain can be nociceptive pain or neuropathic pain.

[0365] In some embodiments, the pain is predicted or expected to occur in association with or as a result of an injury, infection, or medical intervention. In some embodiments, the infection causes nerve damage. In some embodiments, the medical intervention is surgery, such as surgery to the central core of the body. In some embodiments, the medical intervention is surgery to remove part or all of one or more tissues, tumors, or organs of the body. In some embodiments, the medical intervention is an amputation. In some embodiments, the compositions and methods of the present disclosure are effective in reducing acute pain. In some embodiments, the compositions and methods of the present disclosure are effective in reducing chronic pain.

[0366] Clinical pain exists when discomfort and abnormal sensitivity appear among the patient's symptoms. Different individuals may present with different pain symptoms. These include: 1) spontaneous pain, which may be dull, causalgia, or stabbing; 2) exaggerated pain response to noxious stimuli (hyperalgesia); and 3) pain caused by normally innocuous stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Patients suffering from various forms of acute and chronic pain may have similar symptoms, but the underlying mechanisms may be different and therefore may require different treatment strategies. Thus, pain can also be divided into several different subtypes according to different pathophysiology, such as nociceptive pain, inflammatory pain, and neuropathic pain.

[0367] In some embodiments, the compositions and methods of the present disclosure are effective in reducing nociceptive pain. In some embodiments, the compositions and methods of the present disclosure are effective in reducing inflammatory pain. In some embodiments, the compositions and methods of the present disclosure are effective in reducing neuropathic pain.

[0368] Nociceptive pain is induced by tissue injury or by strong stimuli that may cause injury. Moderate to severe acute nociceptive pain is the main feature of pain from central nervous system trauma, contusion / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumor-related pain (e.g., bone pain, headache, facial pain or visceral pain) or pain associated with cancer treatment (e.g., post-chemotherapy syndrome, chronic postoperative pain syndrome or post-radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiation therapy. Back pain may be due to herniated or ruptured intervertebral disc or abnormality of lumbar facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament. Back pain may resolve naturally, but in some patients where it lasts for more than 12 weeks, it can become a chronic condition and be particularly debilitating.

[0369] Neuropathic pain can be defined as the pain that is initiated or caused by primary lesion or dysfunction in nervous system.The etiology of neuropathic pain includes, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, and the pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0370] Neuropathic pain can be associated with pain disorders, a term that refers to diseases, disorders or conditions associated with or caused by pain. Examples of pain disorders include arthritis, allodynia, typical trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia, causlagia, sciatica disorders, osteoarthritis, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis or pain associated with cancer.

[0371] In some embodiments, the neuropathic pain is peripheral neuropathy. Peripheral neuropathy refers to a condition that occurs when the nerves that carry messages from the rest of the body to the brain and spinal cord and from the brain and spinal cord to the rest of the body are damaged or diseased. The various types of peripheral neuropathy range from carpal tunnel syndrome (a common traumatic injury after chronic repetitive use of the hand and wrist, such as with computer use) to nerve damage associated with diabetes. In general, peripheral neuropathy can be classified into mononeuropathy and polyneuropathy. Mononeuropathy includes carpal tunnel syndrome, ulnar nerve palsy, radial nerve palsy, and peroneal nerve palsy. Polyneuropathy occurs when multiple peripheral nerves in the body malfunction at the same time. Polyneuropathy can have a wide variety of causes, including exposure to certain toxins, such as from alcohol abuse, nutritional deficiencies (especially vitamin B deficiency), and complications from diseases such as cancer or kidney failure. One of the most common forms of chronic polyneuropathy is diabetic neuropathy, a condition that occurs in people with diabetes. It is more severe in people whose blood sugar levels are poorly controlled. Less commonly, diabetes can also cause mononeuropathy. One of the most severe polyneuropathy is Guillain-Barre syndrome, a rare disease that strikes suddenly when the body's immune system attacks the nerves inside the body as they leave the spinal cord. Symptoms tend to appear quickly and get worse quickly, and can result in paralysis. Early symptoms include weakness and tingling, which can eventually spread up the arms. In more severe cases, blood pressure problems, heart rate problems, and difficulty breathing can occur. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic form of Guillain-Barre where symptoms continue for months or even years. Early diagnosis and treatment are important for CIDP patients, who have a 30% risk of eventually becoming wheelchair bound.

[0372] In some embodiments, the neuropathic pain is trigeminal neuralgia. Trigeminal neuralgia (TN), also called painful tics, is a chronic pain condition that affects the trigeminal nerve or the fifth cranial nerve, one of the most widely distributed nerves in the head. The trigeminal nerve is a set of cranial nerves in the head. It is the nerve responsible for providing sensation to the face. One trigeminal nerve runs on the right side of the head, while the other runs on the left side. Each of these nerves has three different branches. After the trigeminal nerve leaves the brain and travels inside the skull, it splits into three smaller branches that control sensation throughout the face: Ophthalmic nerve (V1): The first branch controls sensation in a person's eyes, upper eyelids, and forehead. Maxillary nerve (V2): The second branch controls sensation in the lower eyelid, cheek, nostrils, upper lip, and upper gums. Mandibular nerve (V3): The third branch controls sensation in the jaw, lower lip, lower gums, and some of the muscles used for chewing.

[0373] TN is a form of neuropathic pain. The typical or "classic" form of the disorder (called "Type 1" or TN1) causes extreme, scattered, sudden, burning, or shock-like facial pain lasting anywhere from a few seconds to as long as two minutes per episode. These attacks occur in rapid succession and can last up to two hours in rapid succession. The "atypical" form of the disorder (called "Type 2" or TN2) is characterized by constant aching, burning, stabbing pain of somewhat lower intensity than Type 1. Both forms of pain can occur in the same person, sometimes at the same time. The intensity of the pain can be physically and mentally incapacitating. TN is associated with a variety of conditions. TN can be caused by blood vessels compressing the trigeminal nerve as it leaves the brainstem. This compression causes wear or damage to the protective coating (myelin sheath) around the nerve. Symptoms of TN can also occur in people with multiple sclerosis, a disease that causes deterioration of the myelin sheath of the trigeminal nerve. Rarely, TN symptoms can be caused by nerve compression from a tumor or a tangle of arteries and veins called an arteriovenous malformation. Injury to the trigeminal nerve (perhaps the result of sinus surgery, oral surgery, stroke, or facial trauma) can also result in neuropathic facial pain.

[0374] The inflammatory process is a complex series of biochemical and cellular events that are activated in response to tissue injury or the presence of foreign substances, resulting in swelling and pain. Joint pain is a common form of inflammatory pain.

[0375] Other types of pain amenable to treatment with the compositions and methods of the present disclosure include, but are not limited to, pain resulting from musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis, and pyomyositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma, and skeletal muscle ischemia; headaches, such as migraine (including migraine with aura and migraine without aura), cluster headaches, tension-type headaches, mixed headaches, and headaches associated with vascular disorders; and orofacial pain, including toothache, earache, burning mouth syndrome, and temporomandibular myofascial pain.

[0376] The effective amount of the compositions and methods of the present disclosure for reducing the amount of pain experienced by human subjects can be determined using various pain scales. Patient self-report can be used to evaluate whether pain is reduced; see, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231. Alternatively, observational pain scales can be used. LANSS pain scale can be used to evaluate whether pain is reduced; see, for example, Bennett (2001) Pain 92:147. Visual analog pain scale can be used; see, for example, Schmader (2002) Clin. J. Pain 18:350. Likert pain scale can be used; for example, 0 is no pain, 5 is moderate pain, and 10 is the worst pain possible. Self-report pain scales for children include, for example, facial pain scale; Wong-Baker facial pain rating scale; and color analog scale. Self-report pain scales for adults include, for example, visual analog scales; verbal numerical rating scales; verbal descriptor scales; and the Brief Pain Questionnaire.Examples of pain measurement scales include the Alder Hey Triage pain score (Stewart et al. (2004) Arch. Dis. Child. 89:625); behavioral pain scales (Payen et al. (2001) Critical Care Medicine 29:2258); the Brief Pain Questionnaire (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23: 129); the Nonverbal Pain Indicator Checklist (Feldt (2000) Pain Manag. Nurs. 1 : 13); the Critical Care Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); the COMFORT scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); the Dallas Pain Questionnaire (Ozguler et al. (2002) Spine 27:1783); the Dolorimeter Pain Index (Hardy et al. (2002) Spine 27:1783); al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); the Face Pain Scale-Revised (Hicks et al. (2001) Pain 93:173); the Face Legs Activity Cry Consolability scale; the McGill Pain Questionnaire (Melzack (1975) Pain 1 :277); the Descriptor Discrimination Scale (Gracely and Kwilosz (1988) Pain 35:279); the Numeric 11-point Box (Jensen et al. (1989) Clin. J. Pain 5: 153); the Numeric Rating Scale (Hartrick et al. (2003) Pain Pract. 3:310); the Wong-Baker FACES pain rating scale; and the Visual Analog Scale (Huskisson (1982) J. Rheumatol. 9:768).

[0377] In some embodiments, a method of alleviating pain in a subject is provided, comprising administering a recombinant nucleic acid comprising an expression cassette of the present disclosure, which introduces an engineered LGIC encoded by a transgene into a neuronal cell, and controlling the activity of the cell by providing an effective amount of a ligand that activates the engineered receptor, thereby alleviating pain in the subject. The method provides significant analgesia for pain without off-target effects such as general central nervous system depression. In certain embodiments, the method provides at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including all ranges and subranges therebetween) reduction in neuropathic pain in the subject compared to untreated subjects. In some embodiments, the method includes measuring pain in the subject before and after administration of the ligand, and pain in the subject is reduced by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including all ranges and subranges therebetween). In such examples, the measurement may be made 4 hours or more after administration of the ligand, for example, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, or 4 days or more after administration of the ligand.

[0378] In some cases, the compositions and methods are used to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumors), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that can be treated by the compositions and methods herein include apraxia, agraphia, alcoholism, dyslexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, ataxia telangiectasia, attention deficit hyperactivity disorder, auditory processing disorder, autism spectrum disorder, bipolar disorder, Bell's palsy, brachial plexus injury, brain tumors, and neurodegenerative disorders. Injury, brain injury, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, head injury, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, co Finn-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital facial diplegia, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cyclothymic disorder, cytomegalic inclusion body disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, onset Coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, speech disorder, autonomic neuropathy, dyscalculia, dysgraphia, dyskinesia, reading disorder, dystonia, empty sella syndrome, encephalitis, cerebral herniation, trigeminal nerve region hemangiomatosis, fecal incontinence, enuresis, epilepsy, intellectual disability in women, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry disease, Fahr's syndrome, syncope, familial convulsive paralysis, febrile seizures,Fisher syndrome, Friedreich's ataxia, fibromyalgia, Foville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy febrile seizures plus, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, heterotopic grey matter, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury , headache, hemifacial spasms, hereditary spastic paraplegia, hereditary polyneuropathy ataxia, otic shingles, herpes zoster, Hirayama syndrome, Hirschsprung's disease, Holmes-Addie syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hyperadrenocorticism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile Refsum's disease, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, isodicentric 15, Joubert syndrome, Karak syndrome, Carr Sayre syndrome, Kinsbone syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral medullary (Wallenberg) syndrome, learning disabilities, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, white matter atrophy, leukoencephalopathy with vanishing white matter, dementia with Lewy bodies, gliosis, locked-in syndrome, intervertebral disc disease, lumbar spinal stenosis, Lafora disease, Im's disease - neurological sequelae, Machado-Joseph disease (Spinocerebellar ataxia type 3), Megaencephaly, Macropsia, Mal de Débarquement, Macrocephalic leukoencephalopathy with subcortical cysts, Megacephaly, Merkelson-Rosenthal syndrome, Meniere's disease, Meningitis, Menkes disease, Metachromatic leukodystrophy, Microcephaly, Micropsia, Migraine, Miller-Fisher syndrome, Mini-stroke (transient ischemic attack), Sound aversion, Mitochondrial myopathy, Moebius syndrome syndrome), unilateral muscular atrophy, motor disability, moyamoya disease, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinolytic diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, myotonia congenita, narcolepsy, neuro-Behçet's disease, neurofibromatosis, neuroleptic malignant syndrome,Neurological signs of AIDS, neurological sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neuropathy, Nyman-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, sequelae of latent spinal dysraphism, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, repetitive vision, paresthesia, Parkinson's disease, congenital paramyotonia, paraneoplastic disorders, seizures (Paroxysmal attack), Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, pervasive developmental disorder, photophotic sneeze reflex, phytanic acid storage disease, Pick's disease, compressed nerve, pituitary tumor, PMG, polyneuropathy, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, post-herpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemifacial atrophy, progressive multifocal Leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrant blindness, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen's encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg's syndrome, chorea (Saint Vitus dance), Sandhoff disease, Schilder's disease, schizencephaly, sensory processing disorder, septo-optic dysplasia, shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal tumor, spinal muscular atrophy, spinal-bulbar muscular atrophy, spinocerebellar ataxia, split encephalopathy, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, stroke, Sturge-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, Sydenham's chorea, fainting, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, tardive dysphrenia,Tarlov cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered spinal cord syndrome, Thomsen's disease, thoracic outlet syndrome, painful tics, Todd's palsy, Tourette's syndrome, toxic encephalopathy, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, von Hippel-Lindau disease (VHL), Biliusk encephalomyelitis (VE), Wallenberg syndrome, West syndrome, whiplash injury, William's syndrome, Wilson's disease, or Zellweger syndrome.

[0379] The subject treated by the methods and compositions disclosed herein may be a human or a non-human animal. In some embodiments, the subject is a human. In some embodiments, the human is an adult (18 years of age or older). Non-limiting examples of non-human animals include non-human primates, livestock animals, household pets, and laboratory animals. For example, the non-human animal may be an ape (e.g., chimpanzee, baboon, gorilla, or orangutan), an Old World monkey (e.g., rhesus monkey), a New World monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a llama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal. The compositions and methods described herein are suitable for the treatment of veterinary animals. Veterinary animals may include, but are not limited to, dogs, cats, horses, cows, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some aspects, contacting a tissue or cell population with a composition comprises administering the composition to the cell population or subject. In some embodiments, administration is performed in vitro, for example, by adding the composition to a cell culture system. In some aspects, administration is performed in vivo, for example, by administration by a specific route. When administering more than one composition, the compositions may be administered by the same route at the same time (e.g., on the same day) or by the same route at different times. Alternatively, the compositions may be administered by different routes at the same time (e.g., on the same day) or by different routes at different times. Administration

[0380] The present disclosure provides a dosing regimen for administering the recombinant nucleic acid comprising the expression cassette of the present disclosure or a pharmaceutical composition thereof. In some embodiments, the recombinant nucleic acid is incorporated into a vector (e.g., an AAV vector) for administration.

[0381] In some embodiments, the recombinant nucleic acid is administered or introduced into one or more neuronal cells. The neuronal cells may be of the same type or a mixed population of neuronal cells of different types.

[0382] In some embodiments, the neuronal cell is a hippocampal neuron. In some embodiments, the neuronal cell is an excitatory neuron. In some embodiments, the recombinant nucleic acid is administered or introduced into one or more hippocampal neuronal cells.

[0383] In some embodiments, the neuronal cell is a nociceptor neuron or a peripheral sensory neuron. Examples of sensory neurons include, but are not limited to, dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In some embodiments, the neuronal cell is an inhibitory interneuron involved in neuronal pain circuits. In some embodiments, the recombinant nucleic acid of the present disclosure is administered or introduced into one or more DRG neuronal cells. In some embodiments, the recombinant nucleic acid of the present disclosure is administered or introduced into one or more TGG neuronal cells.

[0384] Non-limiting examples of administration methods include subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, infusion, intracranial, intrathecal, intranasal, intraganglionic, intraspinal, cisternal and intraneuronal administration. In some embodiments, administration may include injection of a liquid formulation of the recombinant nucleic acid or vector containing the recombinant nucleic acid. In some embodiments, administration may include oral delivery of a solid formulation of the vector or recombinant nucleic acid. In some embodiments, oral formulations can be administered with food. In some embodiments, the recombinant nucleic acid of the present disclosure is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneuronally, intraganglionally, intraspinal or intracerebroventricularly to a subject to introduce the recombinant nucleic acid into one or more neuronal cells.

[0385] In some embodiments, recombinant nucleic acids of the disclosure are administered to neurons intracranially, intrathecally (IT), intracerebrally, intracerebroventricularly, or by direct injection into the epileptic focus of the hippocampus.

[0386] In some embodiments, a recombinant nucleic acid of the disclosure is administered to a sensory neuron or nociceptor, such as a DRG neuron, a TGG neuron, etc., by intrathecal (IT) or intraganglionic (IG) administration.

[0387] Intrathecal (IT) administration includes delivery via the spine or cisterna magna. The IT route delivers recombinant nucleic acid to the cerebrospinal fluid (CSF). This administration route 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 has been achieved by inserting an IT catheter via the cisterna magna and advancing it caudally to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a routine bedside procedure that shows an excellent safety profile.

[0388] In some embodiments, the recombinant nucleic acid of the present disclosure is administered to a subject by intraganglionic administration. Intraganglionic administration may include direct injection into one or more ganglia. The IG route can deliver the recombinant nucleic acid directly to the DRG or TGG parenchyma. In some embodiments, IG administration to the DRG is performed by open neurosurgical procedure. In some embodiments, open neurosurgical procedure is invasive and undesirable in humans. In some embodiments, minimally invasive, CT imaging guided techniques to safely target the DRG can be used, for example, for human subjects. The recombinant nucleic acid can be delivered into the DRG parenchyma using a custom-made needle assembly for convection-enhanced delivery (CED). In a non-limiting example, the recombinant nucleic acid of the present disclosure can be delivered to one or more dorsal root ganglia and / or trigeminal ganglia for the treatment of chronic pain. In another non-limiting example, the recombinant nucleic acid of the present disclosure can be delivered to the nodose ganglion (vagus nerve) for the treatment of epilepsy.

[0389] In some embodiments, the recombinant nucleic acid of the present disclosure is administered to the subject by intracranial administration (i.e., directly into the brain). In a non-limiting example of intracranial administration, the recombinant nucleic acid of the present disclosure can be delivered to the cortex of the brain, for example, to treat epileptic seizure focus, to the paraventricular hypothalamus, for example, to treat satiety disorder, or to the central amygdala, for example, to treat satiety disorder. In some embodiments, the recombinant nucleic acid can be administered to the subject by intraneural injection (i.e., directly into the nerve). The nerve can be selected based on the indication to be treated, for example, injection into the sciatic nerve to treat chronic pain, or injection into the vagus nerve to treat epilepsy or satiety disorder. In yet another particular case, the recombinant nucleic acid can be administered to the subject by injection into the sensory nerve terminal, for example, to treat chronic pain. In some embodiments, the recombinant nucleic acid of the present disclosure is administered to the subject by direct injection into the epileptic focus of the hippocampus.

[0390] Dosages may vary and may depend on whether the treatment is preventive or therapeutic, the type, onset, progression, severity, frequency, duration, or probability of disease treatment for the desired clinical endpoint, previous or concurrent treatment, the general health, age, sex, race, or immunocompetence of the subject, and other factors understood by those skilled in the art. Dose amount, number, frequency, or duration may be increased or decreased proportionately as indicated by adverse side effects of the treatment or therapy, complications or other risk factors, and the condition of the subject. Those skilled in the art will appreciate the factors that may affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0391] In some embodiments, the recombinant nucleic acid is incorporated into a vector, such as a viral vector. In some embodiments, the vector dose can be expressed as the number of vector genome units delivered to a subject. The size of an individual vector genome generally depends on the type of viral vector used. The vector genome of the present disclosure can be about 1.0 kilobase, 1.5 kilobase, 2.0 kilobase, 2.5 kilobase, 3.0 kilobase, 3.5 kilobase, 4.0 kilobase, 4.5 kilobase to 5.0 kilobase, or more than 5.0 kilobase (including all ranges and subranges therebetween).

[0392] In some embodiments, the vector dose administered is about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , about 9×10 6 , about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 , about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , about 1×10 9 , about 2×10 9 , about 3×10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9, about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 6×10 14 , about 7×10 14 , about 8×10 14 , about 9×10 14 , about 1×10 15 , about 2×10 15 , about 3×10 15 , about 4×10 15 , about 5×10 15 , about 6×10 15 , about 7×1015 , about 8×10 15 , about 9×10 15 , about 1×10 16 , about 2×10 16 , about 3×10 16 , about 4×10 16 , about 5×10 16 , about 6×10 16 , about 7×10 16 , about 8×10 16 , about 9×10 16 , about 1×10 17 or more vector genome units (including all ranges and subranges therebetween).

[0393] In some embodiments, the vector dose administered is at least 1×10 6 , at least 2 × 10 6 , at least 3 × 10 6 , at least 4 × 10 6 , at least 5 × 10 6 , at least 6 × 10 6 , at least 7 × 10 6 , at least 8 × 10 6 , at least 9×10 6 , at least 1 × 10 7 , at least 2 × 10 7 , at least 3 × 10 7 , at least 4 × 10 7 , at least 5 × 10 7 , at least 6 × 10 7 , at least 7 × 10 7 , at least 8 × 10 7 , at least 9×10 7 , at least 1 × 10 8 , at least 2 × 10 8 , at least 3 × 10 8 , at least 4 × 10 8 , at least 5 × 10 8 , at least 6 × 10 8 , at least 7 × 10 8 , at least 8 × 10 8 , at least 9×10 8 , at least 1 × 10 9 , at least 2 × 109 , at least 3 × 10 9 , at least 4 × 10 9 , at least 5 × 10 9 , at least 6 × 10 9 , at least 7 × 10 9 , at least 8 × 10 9 , at least 9×10 9 , at least 1 × 10 10 , at least 2 × 10 10 , at least 3 × 10 10 , at least 4 × 10 10 , at least 5 × 10 10 , at least 6 × 10 10 , at least 7 × 10 10 , at least 8 × 10 10 , at least 9×10 10 , at least 1 × 10 11 , at least 2 × 10 11 , at least 3 × 10 11 , at least 4 × 10 11 , at least 5 × 10 11 , at least 6 × 10 11 , at least 7 × 10 11 , at least 8 × 10 11 , at least 9×10 11 , at least 1 × 10 12 , at least 2 × 10 12 , at least 3 × 10 12 , at least 4 × 10 12 , at least 5 × 10 12 , at least 6 × 10 12 , at least 7 × 10 12 , at least 8 × 10 12 , at least 9×10 12 , at least 1 × 10 13 , at least 2 × 10 13 , at least 3 × 10 13 , at least 4 × 10 13 , at least 5 × 10 13 , at least 6 × 10 13 , at least 7 × 10 13 , at least 8 × 10 13 , at least 9×10 13, at least 1 × 10 14 , at least 2 × 10 14 , at least 3 × 10 14 , at least 4 × 10 14 , at least 5 × 10 14 , at least 6 × 10 14 , at least 7 × 10 14 , at least 8 × 10 14 , at least 9×10 14 , at least 1 × 10 15 , at least 2 × 10 15 , at least 3 × 10 15 , at least 4 × 10 15 , at least 5 × 10 15 , at least 6 × 10 15 , at least 7 × 10 15 , at least 8 × 10 15 , at least 9×10 15 , at least 1 × 10 16 , at least 2 × 10 16 , at least 3 × 10 16 , at least 4 × 10 16 , at least 5 × 10 16 , at least 6 × 10 16 , at least 7 × 10 16 , at least 8 × 10 16 , at least 9×10 16 , or at least 1 × 10 17 of vector genome units (including all ranges and subranges therebetween).

[0394] In some embodiments, the vector dose administered is 1×10 6 Below, 2×10 6 Below, 3 x 10 6 Below, 4×10 6 Below, 5 x 10 6 Below, 6 x 10 6 Below, 7 x 10 6 Below, 8 x 10 6 Below, 9 x 10 6 Below, 1×10 7 Below, 2×10 7 Below, 3 x 10 7 Below, 4×10 7Below, 5×10 7 Below, 6×10 7 Below, 7×10 7 Below, 8×10 7 Below, 9×10 7 Below, 1×10 8 Below, 2×10 8 Below, 3×10 8 Below, 4×10 8 Below, 5×10 8 Below, 6×10 8 Below, 7×10 8 Below, 8×10 8 Below, 9×10 8 Below, 1×10 9 Below, 2×10 9 Below, 3×10 9 Below, 4×10 9 Below, 5×10 9 Below, 6×10 9 Below, 7×10 9 Below, 8×10 9 Below, 9×10 9 Below, 1×10 10 Below, 2×10 10 Below, 3×10 10 Below, 4×10 10 Below, 5×10 10 Below, 6×10 10 Below, 7×10 10 Below, 8×10 10 Below, 9×10 10 Below, 1×10 11 Below, 2×10 11 Below, 3×10 11 Below, 4×10 11 Below, 5×10 11 Below, 6×10 11 Below, 7×10 11 Below, 8×10 11 Below, 9×10 11 Below, 1×10 12 Below, 2×10 12 Below, 3×10 12 Below, 4×10 12 Below, 5×10 12 Below, 6×10 12 Below, 7×10 12 Below, 8×10 12 Below, 9×10 12Below, 1×10 13 Below, 2×10 13 Below, 3 x 10 13 Below, 4×10 13 Below, 5 x 10 13 Below, 6 x 10 13 Below, 7 x 10 13 Below, 8 x 10 13 Below, 9 x 10 13 Below, 1×10 14 Below, 2×10 14 Below, 3 x 10 14 Below, 4×10 14 Below, 5 x 10 14 Below, 6 x 10 14 Below, 7 x 10 14 Below, 8 x 10 14 Below, 9 x 10 14 Below, 1×10 15 Below, 2×10 15 Below, 3 x 10 15 Below, 4×10 15 Below, 5 x 10 15 Below, 6 x 10 15 Below, 7 x 10 15 Below, 8 x 10 15 Below, 9 x 10 15 Below, 1×10 16 Below, 2×10 16 Below, 3 x 10 16 Below, 4×10 16 Below, 5 x 10 16 Below, 6 x 10 16 Below, 7 x 10 16 Below, 8 x 10 16 Below, 9 x 10 16 or less, or 1×10 17 The vector genome units include the following (including all ranges and subranges therebetween):

[0395] In some embodiments, the vector dose is expressed as vector genome units per kilogram of subject body weight (vg / kg). In some embodiments, the vector dose numbers above are based on a subject weighing 50 kg, and the doses can be converted accordingly to vg / kg and applied to different subjects based on the subject's body weight. For example, about 5×10 14The vector genome unit dose is approximately 1 × 10 per kilogram. 13 It can be converted to vector genome units (vg / kg).

[0396] In some embodiments, the vector dose is expressed according to the concentration or potency of the vector administered to the subject. In some embodiments, the vector dose can be expressed as units per volume (e.g., genome units / volume) x volume.

[0397] In some embodiments, the vector of the present disclosure is administered in a volume of fluid. In some embodiments, the vector is administered in a volume of about 0.01 mL, about 0.02 mL, about 0.03 mL, about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.1 mL, about 0.15 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 2.0 mL, about 3. 0 mL, about 4.0 mL, about 5.0 mL, about 6.0 mL, about 7.0 mL, about 8.0 mL, about 9.0 mL, about 10.0 mL, about 11.0 mL, about 12.0 mL, about 13.0 mL, about 14.0 mL, about 15.0 mL, about 16.0 mL, about 17.0 mL, about 18.0 mL, about 19.0 mL, about 20.0 mL, about 25.0 mL, or greater than 25.0 mL (including all ranges and subranges therebetween). In some embodiments, the vector is at least 0.01 mL, at least 0.02 mL, at least 0.03 mL, at least 0.04 mL, at least 0.05 mL, at least 0.06 mL, at least 0.07 mL, at least 0.08 mL, at least 0.09 mL, at least 0.1 mL, at least 0.15 mL, at least 0.2 mL, at least 0.25 mL, at least 0.3 mL, at least 0.4 mL, at least 0.5 mL, at least 0.6 mL, at least 0.7 mL, at least 0.8 mL, at least 0.9 mL, at least 1.0 mL, at least and / or at least 2.0 mL, at least 3.0 mL, at least 4.0 mL, at least 5.0 mL, at least 6.0 mL, at least 7.0 mL, at least 8.0 mL, at least 9.0 mL, at least 10.0 mL, at least 11.0 mL, at least 12.0 mL, at least 13.0 mL, at least 14.0 mL, at least 15.0 mL, at least 16.0 mL, at least 17.0 mL, at least 18.0 mL, at least 19.0 mL, at least 20.0 mL, or at least 25.0 mL (including all ranges and subranges therebetween).In some embodiments, the vector is 0.01 mL or less, 0.02 mL or less, 0.03 mL or less, 0.04 mL or less, 0.05 mL or less, 0.06 mL or less, 0.07 mL or less, 0.08 mL or less, 0.09 mL or less, 0.1 mL or less, 0.15 mL or less, 0.2 mL or less, 0.25 mL or less, 0.3 mL or less, 0.4 mL or less, 0.5 mL or less, 0.6 mL or less, 0.7 mL or less, 0.8 mL or less, 0.9 mL or less, 1.0 mL or less, 2.0 mL or less 0.0mL or less, 3.0mL or less, 4.0mL or less, 5.0mL or less, 6.0mL or less, 7.0mL or less, 8.0mL or less, 9.0mL or less, 10.0mL or less, 11.0mL or less, 12.0mL or less, 13.0mL or less, 14.0mL or less, 15.0mL or less, 16.0mL or less, 17.0mL or less, 18.0mL or less, 19.0mL or less, 20.0mL or less, or 25.0mL or less (including all ranges and subranges therebetween).

[0398] In some embodiments, the vectors contemplated herein are at least 1×10 9 Genomic units / mL, at least 1 × 10 10 Genomic units / mL, at least 5 × 10 10 Genomic units / mL, at least 1 × 10 11 Genomic units / mL, at least 5 × 10 11 Genomic units / mL, at least 1 × 10 12 Genomic units / mL, at least 5 × 10 12 Genomic units / mL, at least 6 × 10 12 Genomic units / mL, at least 7 × 10 12 Genomic units / mL, at least 8 × 10 12 Genomic units / mL, at least 9 × 10 12 Genomic units / mL, at least 10 × 10 12 Genomic units / mL, at least 15 × 10 12 Genomic units / mL, at least 20 × 10 12 Genomic units / mL, at least 25 × 10 12 Genomic units / mL, at least 50 × 10 12 Genomic units / mL, or at least 100 × 10 12In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of about 1×10 genome units / mL, including all ranges and subranges therebetween. 9 Genome units / mL, approximately 1 x 10 10 Genomic units / mL, approximately 5 × 10 10 Genome units / mL, approximately 1 x 10 11 Genomic units / mL, approximately 5 × 10 11 Genome units / mL, approximately 1 x 10 12 Genomic units / mL, approximately 5 × 10 12 Genomic units / mL, approximately 6 x 10 12 Genomic units / mL, approximately 7 x 10 12 Genomic units / mL, approximately 8 x 10 12 Genomic units / mL, approximately 9 × 10 12 Genomic units / mL, approximately 10 x 10 12 Genomic units / mL, approximately 15 x 10 12 Genomic units / mL, approximately 20 x 10 12 Genomic units / mL, approximately 25 x 10 12 Genomic units / mL, approximately 50 x 10 12 Genomic units / mL, or approximately 100 x 10 12 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of 1×10 genome units / mL, including all ranges and subranges therebetween. 9 Genomic units / mL or less, 1 x 10 10 Genomic units / mL or less, 5 x 10 10 Genomic units / mL or less, 1 x 10 11 Genomic units / mL or less, 5 x 10 11 Genomic units / mL or less, 1 x 10 12 Genomic units / mL or less, 5 x 10 12 Genomic units / mL or less, 6 x 10 12 Genomic units / mL or less, 7 x 10 12 Genomic units / mL or less, 8 x 10 12 Genomic units / mL or less, 9 x 10 12 Genomic units / mL or less, 10 x 10 12 Genomic units / mL or less, 15 x 10 12 Genomic units / mL or less, 20 x 10 12 Genomic units / mL or less, 25 x 10 12Genomic units / mL or less, 50 x 10 12 Genomic units / mL or less, or 100 x 10 12 It is administered to subjects at a titer of genomic units / mL or less (including all ranges and subranges therebetween).

[0399] In some embodiments, the vectors contemplated herein are about 5×10 9 Infectious units / mL, approximately 6 × 10 9 Infectious units / mL, approximately 7 × 10 9 Infectious units / mL, approximately 8 × 10 9 Infectious units / mL, approximately 9 × 10 9 Infectious units / mL, approximately 1 × 10 10 Infectious units / mL, approximately 1.5 × 10 10 Infectious units / mL, approximately 2 × 10 10 Infectious units / mL, approximately 2.5 × 10 10 Infectious units / mL, approximately 5 × 10 10 Infectious units / mL, approximately 1 × 10 11 Infectious units / mL, approximately 2.5 × 10 11 Infectious units / mL, approximately 5 × 10 11 Infectious units / mL, approximately 1 × 10 12 Infectious units / mL, approximately 2.5 × 10 12 Infectious units / mL, approximately 5 × 10 12 Infectious units / mL, approximately 1 × 10 13 Infectious units / mL, approximately 5 × 10 13 infectious units / mL, or approximately 1 × 10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of at least 5×10 infectious units / mL (including all ranges and subranges therebetween). 9 Infectious units / mL, at least 6 × 10 9 Infectious units / mL, at least 7 × 10 9 Infectious units / mL, at least 8 × 10 9 Infectious units / mL, at least 9 × 10 9 Infectious units / mL, at least 1 × 10 10 Infectious units / mL, at least 1.5 × 10 10 Infectious units / mL, at least 2 × 10 10 Infectious units / mL, at least 2.5 × 10 10Infectious units / mL, at least 5 × 10 10 Infectious units / mL, at least 1 × 10 11 Infectious units / mL, at least 2.5 × 10 11 Infectious units / mL, at least 5 × 10 11 Infectious units / mL, at least 1 × 10 12 Infectious units / mL, at least 2.5 × 10 12 Infectious units / mL, at least 5 × 10 12 Infectious units / mL, at least 1 × 10 13 Infectious units / mL, at least 5 × 10 13 infectious units / mL, or at least 1 × 10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of 5×10 infectious units / mL, including all ranges and subranges therebetween. 9 Infectious units / mL or less, 6 x 10 9 Infectious units / mL or less, 7 x 10 9 Infectious units / mL or less, 8 x 10 9 Infectious units / mL or less, 9 x 10 9 Infectious units / mL or less, 1 x 10 10 Infectious units / mL or less, 1.5 x 10 10 Infectious units / mL or less, 2 x 10 10 Infectious units / mL or less, 2.5 x 10 10 Infectious units / mL or less, 5 x 10 10 Infectious units / mL or less, 1 x 10 11 Infectious units / mL or less, 2.5 x 10 11 Infectious units / mL or less, 5 x 10 11 Infectious units / mL or less, 1 x 10 12 Infectious units / mL or less, 2.5 x 10 12 Infectious units / mL or less, 5 x 10 12 Infectious units / mL or less, 1 x 10 13 Infectious units / mL or less, 5 x 10 13 Infectious units / mL or less, or 1 x 10 14 It is administered to subjects at a titer of infectious units / mL or less (including all ranges and subranges therebetween).

[0400] In some embodiments, the vector of the present disclosure comprises about 5×1010 Transducing units / mL, approximately 1 x 10 11 Transducing units / mL, approximately 2.5 x 10 11 Transducing units / mL, approximately 5 x 10 11 Transducing units / mL, approximately 1 x 10 12 Transducing units / mL, approximately 2.5 x 10 12 Transducing units / mL, approximately 5 x 10 12 Transducing units / mL, approximately 1 x 10 13 Transducing units / mL, approximately 5 x 10 13 transducing units / mL, or approximately 1 x 10 14 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of at least 5×10 transducing units / mL, including all ranges and subranges therebetween. 10 Transducing units / mL, at least 1 x 10 11 Transducing units / mL, at least 2.5 x 10 11 Transducing units / mL, at least 5 x 10 11 Transducing units / mL, at least 1 x 10 12 Transducing units / mL, at least 2.5 x 10 12 Transducing units / mL, at least 5 x 10 12 Transducing units / mL, at least 1 x 10 13 Transducing units / mL, at least 5 x 10 13 Transducing units / mL, or at least 1 x 10 14 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of 5×10 transducing units / mL, including all ranges and subranges therebetween. 10 Transducing units / mL or less, 1 x 10 11 Transducing units / mL or less, 2.5 x 10 11 Transducing units / mL or less, 5 x 10 11 Transducing units / mL or less, 1 x 10 12 Transducing units / mL or less, 2.5 x 10 12 Transducing units / mL or less, 5 x 10 12 Transducing units / mL or less, 1 x 10 13 Transducing units / mL or less, 5 x 10 13 Transducing units / mL or less, or 1 x 10 14It is administered to subjects at a titer of no more than 100 transducing units / mL (including all ranges and subranges therebetween).

[0401] In some embodiments, the vector dose is determined by the route of administration. In some embodiments, intraganglionic injections are administered at about 1×10 in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intrathecal injection contains about 1×10 vector genome in a volume of about 1.0 mL to about 12.0 mL. 10 ~Approx. 1×10 15 In some embodiments, the intracranial injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intraneural injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intraspinal injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the cisternal infusion contains about 5×10 vector genomes in a volume of about 0.5 mL to about 5.0 mL. 9 ~Approx. 5×10 13 In some embodiments, the subcutaneous injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 It may include a vector genome.

[0402] In some embodiments, the vector dose is the total dose of a si...

Claims

1. In the order from 5' to 3', i. a 5' enhancer, ii. a promoter; iii. 5' untranslated region (UTR); iv. a. a ligand-binding domain comprising an amino acid sequence having at least 85% sequence identity to amino acids 23-220 of the α7 nicotinic acetylcholine receptor (α7-nAChR; SEQ ID NO: 25); b. a Cys loop domain in the ligand-binding domain derived from the human glycine alpha receptor; and c. An ion pore domain comprising an amino acid sequence having at least 85% sequence identity with amino acids 255-457 of human glycine receptor α1 (GlyRα1; SEQ ID NO:26). a transgene encoding a ligand-gated ion channel comprising v. a 3' enhancer, and vi. Polyadenylation sequence (poly A) wherein said transgene is operably linked to said promoter.

2. The recombinant nucleic acid described in claim 1, wherein the Cys loop domain in the ligand binding domain comprises amino acids 166 to 172 or 166 to 180 of SEQ ID NO: 26 (GlyRα1).

3. The recombinant nucleic acid of claim 1, wherein the ligand binding domain comprises a β1-2 loop domain derived from the human glycine α1 receptor, and optionally, the β1-2 loop domain comprises amino acids 81 to 84 of SEQ ID NO: 26 (GlyRα1).

4. The recombinant nucleic acid described in claim 1, wherein the introduced gene encodes a ligand-gated ion channel comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

33.

5. The recombinant nucleic acid of claim 1, wherein the ligand-binding domain comprises one or more amino acid substitutions in amino acid residues corresponding to Q79, R101, Y115, T128, L131, Q139, S170, S172, Y210 and / or Y217 of SEQ ID NO:

25.

6. The recombinant nucleic acid described in claim 1, wherein the 5' enhancer comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 37 to 39.

7. The recombinant nucleic acid of claim 1, wherein the promoter comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 41 to 51, and optionally, the promoter is a neuron-specific promoter.

8. The expression cassette a. intron and said transgene in 5' to 3' order and after said 5' UTR; or b. in 5' to 3' order and after the promoter, an intron and the 5'UTR and optionally, wherein the intron comprises a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 57-61. The recombinant nucleic acid of claim 1.

9. The recombinant nucleic acid of claim 1, wherein the 5'UTR comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 52 to 56.

10. The recombinant nucleic acid of claim 1, wherein the 3' enhancer comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 62 to 65.

11. The recombinant nucleic acid described in claim 1, wherein the polyA comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 67 to 70.

12. 2. The recombinant nucleic acid of claim 1, wherein the expression cassette further comprises a non-neuronal silencing element embedded in the promoter, wherein the non-neuronal silencing element comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

40.

13. 2. The recombinant nucleic acid of claim 1, wherein the expression cassette further comprises, in 5' to 3' order and after the 5' enhancer, a non-neuronal silencing element and the promoter, and optionally, the non-neuronal silencing element comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

40.

14. 2. The recombinant nucleic acid of claim 1, wherein the expression cassette further comprises, in 5' to 3' order and after the 3' enhancer, a 3' UTR and the polyA, and optionally the 3' UTR comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

66.

15. The recombinant nucleic acid of claim 1, wherein the ion pore domain comprises amino acids 255 to 457 of human GlyRα1 (sequence number 26), and the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to sequence number 33.

16. 2. The recombinant nucleic acid of claim 1, wherein the transgene comprises or consists of a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 32, optionally wherein the transgene is codon-optimized for expression in a human cell, and optionally wherein the human cell is a neuron.

17. The expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 37; (ii) an hSyn promoter comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 48; (iii) an hSyn 5′UTR comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 54; (iv) an hSyn intron comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 59; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 64; (vii) an alpha globin 3′UTR comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 66; and (viii) an hGH polyA comprising a polynucleotide sequence having at least 90% identity to SEQ ID NO: 68 The recombinant nucleic acid of claim 1, comprising:

18. 2. The recombinant nucleic acid of claim 1, comprising adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking each end of the expression cassette, optionally comprising a 5' ITR sequence having at least 90% identity to SEQ ID NO: 94 or 119 and a 3' ITR sequence having at least 90% identity to SEQ ID NO: 95 or 120.

19. 10. An AAV vector comprising the recombinant nucleic acid of claim 1, optionally wherein the AAV vector is an AAV5, AAV6, or AAV9 serotype.

20. 20. The AAV vector of claim 19, comprising a capsid protein having at least 95% identity to the AAV9 capsid protein (SEQ ID NO: 8), and optionally, the capsid protein further comprises one or more amino acid substitutions at amino acid residues corresponding to T492, Y705 and / or Y731 of SEQ ID NO:

8.

21. 21. A host cell comprising a nucleic acid according to any one of claims 1 to 18 or a vector according to claim 19 or 20.

22. A method for producing an AAV vector according to claim 19 or 20.

23. 21. A kit comprising the recombinant nucleic acid of any one of claims 1 to 18 or the AAV vector of claim 19 or 20, optionally further comprising a non-natural ligand of the ligand-gated ion channel, optionally wherein the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487.

24. A composition for use in a method for expressing an introduced gene in a cell, comprising a recombinant nucleic acid described in any one of claims 1 to 18 or an AAV vector described in claim 19 or 20, the method comprising delivering the recombinant nucleic acid or the AAV vector to the cell.

25. A composition for use in a method of transducing a cell, comprising an AAV vector according to claim 19 or 20, the method comprising contacting the cell with the AAV vector, and optionally the cell being a neuron.

26. the neurons are hippocampal neurons, and optionally i. the neuron is an excitatory neuron, and optionally the neuron is a CAMK2-positive neuron; ii. the neuron is an inhibitory neuron, and optionally the neuron is a GABAergic neuron; or iii. The neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron, and optionally the neuron comprises isolectin B4 (IB4)-positive nerve fibers, NF200-positive nerve fibers, CGRP-positive nerve fibers, C fibers, and / or A-delta fibers; 26. The composition of claim 25.

27. 26. The composition of claim 25, wherein the cells are ex vivo cells or the cells are in vivo cells of a subject, optionally wherein the subject is a human.

28. 26. The composition of claim 25, wherein the cells comprising the expression cassette have a higher expression level of the transgene compared to corresponding cells comprising a control expression cassette, and optionally the higher expression is at least 5% higher than the transgene expression level of the control expression cassette comprising an expression cassette regulatory element of SEQ ID NO: 88 (Expression Cassette No. 18 in Table 2) or an expression cassette regulatory element of SEQ ID NO: 87 (Expression Cassette No. 17 in Table 2).

29. A composition for treating a neurological disease or disorder in a subject in need thereof, the composition comprising a recombinant nucleic acid described in any one of claims 1 to 18 or an AAV vector described in claim 19 or 20.

30. the neurological disease or disorder is epilepsy, optionally wherein the epilepsy is focal epilepsy or mesial temporal lobe epilepsy (mTLE), and optionally the recombinant nucleic acid or the AAV vector is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cisternal) administration, intracerebral administration, intraventricular administration, or direct injection into the epileptic focus of the hippocampus; optionally, reducing the duration, intensity, and / or frequency of epilepsy in said subject by at least 10%; 30. The composition of claim 29.

31. The composition described in claim 29, wherein the neurological disease or disorder is spasticity, spinal cord injury, or avulsion injury.

32. the neurological disease or disorder is neuropathic pain, and optionally (a) the neuropathic pain is peripheral neuropathy or trigeminal neuralgia, and optionally the recombinant nucleic acid or the AAV vector is administered by intrathecal (IT) or intraganglionic (IG) administration, optionally wherein the IG administration is direct IG administration to the dorsal root ganglion or the trigeminal ganglion, and optionally, pain in the subject is reduced by at least 10%; or (b) the neurological pain is sciatica, and optionally, the recombinant nucleic acid or the AAV vector is administered to the sciatic nerve, and optionally, pain in the subject is reduced by at least 10%.

30. The composition of claim 29.

33. 30. The composition of claim 29, wherein the recombinant nucleic acid or the AAV vector is administered by systemic, parenteral, intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputamenal, intrahippocampal, intrastriatal, or intracerebroventricular injection.

34. 1 x 10 ９ ~1 x 10 １４ 30. The composition of claim 29, wherein a copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

35. The method comprises administering a non-natural ligand of the ligand-gated ion channel encoded by the transgene, and optionally: The non-natural ligand is administered simultaneously with or after administration of the recombinant nucleic acid or AAV vector, and optionally: the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487; 30. The composition of claim 29.

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