Expression cassette for treating epilepsy and neuropathic pain

Abstract

The present disclosure provides expression cassettes, vectors and methods for expressing a transgene in a cell, such as a neuron. Also provided are methods of achieving desired expression levels of transgenes in neuronal cells, e.g., in combination with small molecule ligands to treat focal epilepsy or neuropathic pain.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 304,960, filed on January 31, 2022, and U.S. Provisional Application No. 63 / 312,480, filed on February 22, 2022, the entire contents of each of which are incorporated herein by reference.

[0003] References to Electronic Sequence Listings

[0004] 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 Art

[0005] Intractable neurological diseases are often associated with abnormally functioning neurons. Attempts to develop therapies for treating these conditions have been hampered by the lack of tractable target proteins associated with the disease.

[0006] Focal epilepsy is a chronic and debilitating neurological disorder that affects more than 2 million people in the United States and is characterized by unpredictable seizures that start from a specific location in the brain. Recurrent seizures can lead to cognitive and emotional deficits, and current interventions offer limited efficacy as well as multiple side effects. Focal seizures originate from abnormal discharges of a subset of neurons, but most anti-epileptic drugs rely on systemic compound administration and alter whole-brain activity. Therefore, patients with epilepsy suffer significant side effects due to the unconscious modulation of neurons involved in normal cognition and undesirable off-target changes in other biological systems. Medial temporal lobe epilepsy (mTLE) is the most common form of focal epilepsy, and it is often associated with hippocampal sclerosis as a histopathological abnormality.

[0007] Neuropathic pain includes peripheral neuropathy and trigeminal neuralgia, among others. The former is a common neurological disorder caused by damage or functional disorders of the peripheral nervous system. It is characterized by numbness, tingling, and pain, usually starting in the hands and feet, but may also affect other areas of the body. Trigeminal neuralgia, also known as suicide disorder, is caused by damage to the myelin sheath that protects the trigeminal cranial nerve. It causes extreme and disseminated shock-like facial pain that may last from a few seconds to a few minutes. Current pharmacological and surgical methods provide little relief and have the potential for addiction, as well as significant side effects.

[0008] The most commonly used treatments for chronic pain are opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can be addictive and may cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive disorders, hallucinations, and other systemic side effects. Despite the widespread use of drugs, their success rate in effectively relieving pain is surprisingly low. More invasive options for treating pain include nerve blocks and electrical stimulation. The most invasive and least popular method for managing pain is to completely remove the nerve or part of it that causes the pain through surgery.

[0009] An ideal treatment for pain and focal epilepsy would alter the activity of only the specific neurons that cause pain and / or seizure generation. Therefore, a new strategy for treating pain or focal epilepsy is to deliver heterologous proteins encoded by recombinant nucleic acids to a subset of neurons to control abnormal activity.

[0010] One consideration for this strategy is the choice of heterologous proteins used to control neuronal activity and their expression in target cells. Recently, designer receptors activated specifically by designer drugs (DREADDs) to alter seizure activity have produced promising results in animal models of epilepsy. However, inhibitory DREADDs rely on G protein-coupled receptors that indirectly activate ion channels through second messengers and may not consistently alter neuronal potentials.

[0011] An alternative therapeutic approach is to transduce target neurons with transgenes encoding engineered ligand-gated ion channels (LGICs) that can subsequently respond to exogenous ligands. Oral administration of such small molecule ligands, designed to interact only with engineered LGICs, can be finely tuned to control abnormal neuronal activity and suppress epileptic seizures without side effects.

[0012] There is a need in the art to design optimal expression cassettes that allow robust expression of transgenes in target cells to control neuronal activity. Such expression cassettes can be used to treat neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia, or focal epilepsy, such as mesial temporal lobe epilepsy. The present disclosure provides such expression cassettes, vectors, methods, and the like. Summary of the invention

[0013] In one aspect, the present invention 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.

[0014] 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.

[0015] In some embodiments, the expression cassette does not comprise a 5' enhancer.

[0016] 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.

[0017] 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 at least 90% identical to any one of SEQ ID NOs: 57-61.

[0018] In some embodiments, the expression cassette does not comprise an intron.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In some embodiments, the expression cassette comprises a non-neuronal silencing element embedded in a promoter, and wherein the non-neuronal silencing element comprises a polynucleotide sequence at least 90% identical to SEQ ID NO: 40. In some embodiments, the expression cassette comprises a non-neuronal silencing element located between the 5' enhancer and the promoter, and wherein the non-neuronal silencing element comprises a polynucleotide sequence at least 90% identical to SEQ ID NO: 40.

[0023] In some embodiments, the expression cassette comprises a 3'UTR located between the 3' enhancer and the polyA, and wherein the 3'UTR comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:66.

[0024] In some embodiments, the transgene encodes a ligand-gated ion channel (LGIC). In some embodiments, the ligand-gated ion channel includes a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR). In some embodiments, the ligand binding domain includes an amino acid sequence having at least 85% identity with amino acid residues 23-220 of SEQ ID NO:25. In some embodiments, the ligand binding domain includes one or more amino acid mutations listed in Table 5. In some embodiments, the ligand-gated ion channel includes an ion pore domain derived from a human glycine receptor. In some embodiments, the ion pore domain includes an amino acid sequence having at least 85% identity with amino acids 255-457 of SEQ ID NO:26, amino acids 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 includes a Cys loop domain derived from a human glycine receptor. In some embodiments, the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 25-31 and 33. In some embodiments, the human glycine receptor is human glycine receptor α1, and wherein the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity with 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 with SEQ ID NO: 32. In some embodiments, the transgene is codon-optimized for expression in human cells. In some embodiments, the human cell is a neuron.

[0025] In some embodiments, the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence 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:37; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence 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:43; (iii) an hCaMKII 5'UTR comprising a polynucleotide sequence 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:53; (iv) an hCMV+rGlob intron comprising a polynucleotide sequence 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:54; NO:58 having a polynucleotide sequence 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; (v) the transgene; (vi) WPREx 3' enhancer, which comprises a polynucleotide sequence 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:64; (vii) alpha globulin 3'UTR, which comprises a polynucleotide sequence 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:66; and (viii) hGH polyA, which comprises a polynucleotide sequence 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:67. NO:68 has a polynucleotide sequence that is 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.

[0026] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 173-1877 of SEQ ID NO: 108, the transgene, and a sequence that is 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 nucleotides 3237-4315 of SEQ ID NO: 108.

[0027] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) a CMV 5′ enhancer comprising a polynucleotide sequence 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: 37;

[0028] (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48;

[0029] (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54;

[0030] (iv) an hSyn intron comprising a polynucleotide sequence that is 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:59;

[0031] (v) the transgene;

[0032] (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64;

[0033] (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and

[0034] (viii) hGH polyA comprising a polynucleotide sequence that is 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:68.

[0035] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 173-1159 of SEQ ID NO: 106, the transgene, and a sequence that is 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 nucleotides 2519-3597 of SEQ ID NO: 106.

[0036] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) a CMV-V2 5′ enhancer comprising a polynucleotide sequence 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: 38;

[0037] (ii) an hSyn-V2 promoter comprising a polynucleotide sequence that is 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:49;

[0038] (iii) the transgene;

[0039] (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0040] (v) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0041] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 149-946 of SEQ ID NO: 125, the transgene, and a sequence that is 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 nucleotides 2285-3416 of SEQ ID NO: 125.

[0042] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) a CMV-V2 5′ enhancer comprising a polynucleotide sequence 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: 38;

[0043] (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43;

[0044] (iii) the transgene;

[0045] (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0046] (v) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0047] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 149-1527 of SEQ ID NO: 126, the transgene, and a sequence that is 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 nucleotides 2848-3979 of SEQ ID NO: 126.

[0048] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) an hSyn-V2 enhancer comprising a polynucleotide sequence 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:49;

[0049] (ii) the transgene;

[0050] (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0051] (iv) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0052] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 153-661 of SEQ ID NO: 127, the transgene, and a sequence that is 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 nucleotides 1982-3113 of SEQ ID NO: 127.

[0053] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) an hCaMKIIa promoter comprising a polynucleotide sequence 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:43;

[0054] (ii) the transgene;

[0055] (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0056] (iv) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0057] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 153-1224 of SEQ ID NO: 128, the transgene, and a sequence that is 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 nucleotides 2545-3676 of SEQ ID NO: 128.

[0058] In some embodiments, the expression cassette comprises, in 5′ to 3′ order: (i) a CMV 5′ enhancer comprising a polynucleotide sequence 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: 37;

[0059] (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48;

[0060] (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54;

[0061] (iv) an hTPI intron comprising a polynucleotide sequence that is 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:60;

[0062] (v) the transgene;

[0063] (vi) a FullEES 3' enhancer comprising a polynucleotide sequence that is 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:63; and

[0064] (vii) rβ globulin polyA, comprising a polynucleotide sequence that is 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:70.

[0065] In some embodiments, the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 173-1188 of SEQ ID NO:98, the transgene, and a sequence that is 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 nucleotides 2548-3824 of SEQ ID NO:98.

[0066] In some embodiments, the expression cassette comprises a polynucleotide sequence that is at least 90% identical 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 that is at least 90% identical 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 that is at least 90% identical to any one of SEQ ID NOs:71-93 and 121-124.

[0067] 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 at least 90% identical to SEQ ID NO: 94 or 119 and a 3'ITR sequence at least 90% identical to SEQ ID NO: 95 or 120.

[0068] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 96-118 and 125-128. In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 96-118 excluding the sequence of the transgene (SEQ ID NO: 36), and in some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 125-128 excluding the sequence of the transgene (SEQ ID NO: 32).

[0069] On the one hand, the present disclosure provides a vector comprising a 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 an AAV vector genome or consists of an AAV vector genome.

[0070] In one aspect, the present disclosure provides an AAV comprising a 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 having at least 95% identity to an AAV9 capsid protein (SEQ ID NO: 8) or an AAV9-TV capsid protein (SEQ ID NO: 9).

[0071] In one aspect, the present disclosure provides a host cell comprising a nucleic acid of the present disclosure or a vector of the present disclosure.

[0072] In one aspect, the present disclosure provides methods of producing the AAV of the present disclosure.

[0073] 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.

[0074] In one aspect, the present disclosure provides a method for expressing a transgene in a cell, comprising delivering a recombinant nucleic acid of the present disclosure or a vector of the present disclosure to the cell.

[0075] On the one hand, the present disclosure provides a method for transducing cells, comprising contacting cells with the AAV of the present disclosure. In some embodiments, the cells are neurons. In some embodiments, the neurons are hippocampal neurons. In some embodiments, the neurons are excitatory neurons. In some embodiments, the neurons are CAMK2-positive neurons. In some embodiments, the neurons are inhibitory neurons. In some embodiments, the neurons are GABAergic neurons. In some embodiments, the neurons are dorsal root ganglion neurons or trigeminal ganglion neurons. In some embodiments, the neurons include isolectin B4 (IB4)-positive nerve fibers. In some embodiments, the neurons include NF200-positive nerve fibers. In some embodiments, the neurons include CGRP-positive nerve fibers. In some embodiments, the neurons include C fibers. In some embodiments, the neurons include Aδ fibers. In some embodiments, the cells are ex vivo cells. In some embodiments, the cells are in vivo cells of a subject, optionally, wherein the subject is a person. In some embodiments, a cell comprising the expression cassette has a higher level of transgene expression compared to a corresponding cell comprising a control expression cassette, optionally wherein the higher expression 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 two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-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, a cell comprising the expression cassette has a comparable or higher transgene expression level as compared to a corresponding cell 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 two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-fold the transgene expression level of a control expression cassette comprising the polynucleotide sequence of SEQ ID NO: 87 excluding the transgene sequence.

[0076] On the one hand, the present disclosure provides a method for treating a disease or condition 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 condition is epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome. In some embodiments, epilepsy is focal epilepsy. In some embodiments, epilepsy is mesial temporal lobe epilepsy (mTLE). In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cerebellomedullary cisterna magna) administration, intracerebral administration, intraventricular administration, or direct injection into the epileptic lesion of the hippocampus. 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 (SEQ ID NO: 8). In some embodiments, the method reduces the duration, intensity and / or frequency of epilepsy by at least 10%.

[0077] On the one hand, the present disclosure provides a method for treating a disease or condition 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 condition is neuropathic pain, spasticity, spinal cord injury, or avulsion. In some embodiments, the disease or condition 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 directly into the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration. 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 pain levels by at least 10%.

[0078] In some embodiments, the recombinant nucleic acid, vector, or AAV is administered by systemic, parenteral, intravenous, brain, cerebrospinal, intrathecal, intracisternal, intraputaminal, intrahippocampal, intrastriatal, or intracerebroventricular injection. 9 -1×10 14A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

[0079] In some embodiments, the method comprises administering a ligand of a 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The patent or application file contains at least one drawing executed in color.

[0081] Figure 1 The designs of 27 expression cassettes are shown. For each cassette, the element on the left is located upstream (5') of the element on the right. The "*" symbol in cassette #20 indicates that the NRSE element is embedded in the CMV promoter in this cassette, rather than placed upstream of it.

[0082] Figure 2 Shown are ddPCR results of the CODA receptor mRNA ratio compared to the β-actin housekeeping gene in SK-N-AS cells and HeLa cells transfected with the indicated cassettes.

[0083] Figure 3 Shown are the results of FLAGELISA of CODA receptor protein in SK-N-AS cells and HeLa cells transfected with the indicated cassettes.

[0084] Figure 4 ddPCR results showing the ratio of CODA receptor mRNA expression compared to the β-actin housekeeping gene for nine expression cassettes packaged into AAV9 vectors and transduced in neonatal rat hippocampal mixed cultures.

[0085] Figure 5 ddPCR results showing the ratio of CODA receptor mRNA compared to the β-actin housekeeping gene in the hippocampus of rats injected directly in vivo with AAV9 carrying six expression cassettes.

[0086] Figure 6 Shown are immunofluorescence localization of FLAG-tagged CODA receptors from six expression cassettes tested in vivo in rat hippocampus.

[0087] Figure 7 Shown are seizure frequencies in the KA focal epilepsy mouse model injected with AAV containing the indicated expression cassettes followed by repeated administration of TC-5619.

[0088] Figure 8Shown is the design of an efficacy study using the KA focal epilepsy mouse model injected with AAV containing the indicated expression cassettes.

[0089] Fig. 9 ddPCR results showing the ratio of CODA receptor mRNA expression compared to the β-actin housekeeping gene for ten expression cassettes packaged as AAV6 vectors and transduced in human iPSC-derived sensory neuron progenitor cells.

[0090] Fig.10 ddPCR results showing the ratio of CODA receptor mRNA compared to the β-actin housekeeping gene in L3 and L4 DRG of rats injected directly in vivo with AAV6 carrying four expression cassettes.

[0091] Fig.11 Shown are immunofluorescence localization of FLAG-tagged CODA receptors from four expression cassettes tested in vivo in rat L5 DRG. DETAILED DESCRIPTION OF THE INVENTION

[0093] The details of the present disclosure are set forth in the attached specification below. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects and advantages of the present disclosure will be clear from the specification and claims. In the specification and the appended claims, unless the context clearly provides otherwise, the singular form also includes the plural. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0094] Will be described more fully according to the embodiment of the present disclosure below.However, the various aspects of the present disclosure can be implemented in different forms, and should not be construed as being limited to the embodiments set forth herein.On the contrary, these embodiments are provided so that the present disclosure will be comprehensive and complete, and will fully convey the scope of the present disclosure to those skilled in the art.Unless otherwise clearly indicated, all specified embodiments, features and terms are intended to include described embodiments, features or terms and their biological equivalents.The terms used in this specification are only used for the purpose of describing specific embodiments, and are not intended to be limited.

[0095] Before describing the methods and compositions of the present invention, it should be understood that the present disclosure is not limited to the specific methods or compositions described, as such methods and compositions may of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the present disclosure will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0096] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the present disclosure. Any method recited may be performed in the order of events recited or in any other order that is logically possible.

[0097] definition

[0098] Throughout this disclosure, the singular forms "a", "an", and "the" include plural referents unless the content clearly indicates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a peptide" includes reference to one or more peptides and equivalents thereof (e.g., polypeptides) known to those skilled in the art, and so forth.

[0099] Throughout this specification, the term "and / or" is used in this disclosure to mean "and" or "or" unless stated otherwise.

[0100] Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" means the inclusion of the stated elements or integers or groups of elements or integers but not the exclusion of any other elements or integers or groups of elements or integers. In addition, the recitation of numerical ranges throughout this specification expressly includes all integers and decimal points therebetween.

[0101] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting essentially of" means that the scope of the described composition, method, or kit is limited to specified materials or steps that do not materially affect one or more of the basic and novel characteristics of the subject disclosure. For example, a polypeptide "consisting essentially of a disclosed sequence" has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence, e.g., about 5 residues, 4 residues, 3 residues, 2 residues, or about 1 residue less than the recited boundary amino acid residues, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited boundary amino acid residues.

[0102] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting of" means that any element, step or ingredient not specified in the claims is not included in the composition, method or kit. For example, a polypeptide "consisting of a disclosed sequence" consists only of the disclosed amino acid sequence.

[0103] The terms "about" and "approximately" are used as equivalents. Any numerical value used in this application with or without about / approximately is meant to cover any normal fluctuations understood by a person of ordinary skill in the relevant art. In certain embodiments, unless otherwise stated or the context clearly implies otherwise, the terms "about" or "approximately" refer to a range of values ​​that fall within 10% in either direction (greater than or less than) of the stated reference value (except where such numbers exceed 100% of possible values).

[0104] The term "isolated" means material that is substantially or essentially free from components that normally accompany it as it is found in its native state. In some embodiments, the term "obtained" is used synonymously with isolated.

[0105] The terms "subject", "individual" and "patient" are used interchangeably to refer to a vertebrate, such as a mammal. The mammal can be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee) or human. Tissues, cells or derivatives thereof of a subject obtained in vivo or cultured in vitro are also contemplated. A human subject can be an adult, adolescent, child (2 to 14 years old), infant (1 to 24 months) or newborn (maximum 1 month). In some embodiments, an adult is an elderly person who is about 65 years old or older or about 60 years old or older.

[0106] The term "sample" refers to a certain volume and / or mass of biological material that is subjected to analysis. In some embodiments, sample includes tissue sample, cell sample, fluid sample, etc. In some embodiments, sample is taken from subject (for example, human subject) or provided by it. In some embodiments, sample includes a part of tissue taken from any internal organ, cancerous, precancerous or noncancerous tumor, brain, skin, hair (including hair root), eye, muscle, bone marrow, cartilage, white adipose tissue and / or brown adipose tissue. In some embodiments, fluid sample includes oral swab, blood, cord blood, saliva, semen, urine, ascites, pleural fluid, spinal fluid, lung lavage fluid, tears, sweat, etc. It will be understood by those of ordinary skill in the art that in some embodiments, "sample" is "primary sample" because it is obtained directly from source (for example, subject). In some embodiments, "sample" is the result of processing primary sample, for example, removing some potential contaminating components, separating some components and / or purifying some components of interest. In some embodiments, sample is cell or cell group (for example, neuronal cell). The cell sample can be directly derived from a subject (e.g., a primary sample) or can be a cell line. The cell line can include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., immortalized cell lines).

[0107] The term "treatment" as used herein and its grammatical equivalents generally refer to the use of a composition or method to reduce, eliminate or prevent the symptoms of a disease, and include achieving a therapeutic benefit and / or a preventive benefit. The therapeutic benefit means slowing down the progression of the symptoms of the disorder or condition being treated, stopping its progression, reversing its progression, or eradicating or alleviating the symptoms. The preventive benefit of treatment includes reducing the risk of the condition, delaying the progression of the condition, or reducing the likelihood of the condition occurring. In some embodiments, treatment refers to delivering the composition to a subject and / or cell population to affect physiological results. In some embodiments, treatment results in an improvement (e.g., reduction, alleviation or remedy) of one or more disease symptoms. Improvement may be an observable or measurable improvement, or may be an improvement in the general sense of the subject's health. The treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to reducing the severity of disease symptoms to a level comparable to that before the onset of the disease. In some embodiments, treatment may refer to a short-term (e.g., temporary or acute) and / or long-term (e.g., continuous or chronic) reduction of disease symptoms. In some embodiments, treatment may refer to the alleviation of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of a subject at risk of developing a particular disease in order to prevent the disease from developing. Preventing the development of a disease may refer to preventing disease symptoms entirely, delaying the onset of a disease, lessening the severity of symptoms of a subsequently developed disease, or reducing the likelihood of a disease developing.

[0108] The terms "manage" or "control" refer 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.

[0109] The term "effective amount" refers to an amount that can achieve the indicated result (e.g., transduction of target neurons). In some embodiments, an effective amount for in vivo administration refers to a dosage that can achieve the desired result, such as, for example, transducing a sufficient number of target neurons for therapeutic purposes.

[0110] A "therapeutically effective amount" is an amount of a composition that can achieve a desired therapeutic outcome. A therapeutically effective amount can vary depending on factors such as, but not limited to, the disease state and the age, sex, and weight of the subject. Generally speaking, a therapeutically effective amount is also an amount in which the therapeutic benefits of the composition outweigh any toxic or deleterious effects thereof. A "therapeutically effective amount" includes an amount of a composition that is effective for treating a subject.

[0111] "Increase" refers to an increase of at least 5% of the value compared to a reference or control level (e.g., an increased transduction efficiency). For example, an increase can include an increase of 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 500%, 1000% or more. An increase also refers to an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) higher than a reference or control level.

[0112] "Decrease", "reduce", "diminish" or their synonyms refer to a decrease of at least 5% of a value compared to a reference or control level (e.g., a decreased transduction efficiency). For example, a decrease can include a decrease of 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 500%, 1000% or more. A decrease also means a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) lower than a reference or control level.

[0113] The terms "reference" or "control" levels are used interchangeably throughout this disclosure and refer to the value of a specific physiological and / or therapeutic effect in a subject or sample that has not been treated with a composition of the present disclosure or in a subject or sample that has been treated with a control. In some embodiments, the control is a vehicle control. In some embodiments, a reference level refers to the value of a specific physiological and / or therapeutic effect as a measure in a subject or sample prior to administering a composition of the present disclosure (e.g., a baseline level).

[0114] 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 changes the function of the receptor, i.e., activates or inhibits its function. In some embodiments, the binding of a ligand to a receptor (such as a ligand-gated ion channel (LGIC)) causes 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., λ-aminobutyric acid (GABA), acetylcholine, serotonin, etc.) and a signaling intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., a synthetic or non-naturally occurring ligand (e.g., a binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by a variety of methods known in the art (e.g., detecting the 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.

[0115] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a receptor and a single binding site of a ligand. Unless otherwise indicated, as used throughout this disclosure, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described in this disclosure.

[0116] The term "wild-type" or "native" is a term of art understood by the skilled artisan and means the typical form of an organism, strain, gene, protein or feature found in nature, as opposed to a mutant or variant form. For example, a wild-type protein is the typical form of the protein found in nature.

[0117] The terms "non-natural," "variant," and "mutant" are used interchangeably throughout the specification and claims to refer to a mutant of a native or wild-type composition, such as a variant polypeptide having less than 100% sequence identity to a native or wild-type sequence.

[0118] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids. The term also encompasses modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, methylation, carboxylation, deamidation, acetylation, or conjugation with a labeling component.

[0119] The amino acid modification can be an amino acid substitution, an amino acid deletion and / or an amino acid insertion. The amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. Conservative substitutions (also referred to as conservative mutations, conservative substitutions or conservative variations) are amino acid substitutions in proteins that change a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used throughout this disclosure, "conservative variation" refers to replacing an amino acid residue with another biologically similar residue. Examples of conservative variations include: a hydrophobic residue (such as isoleucine, valine, leucine, or methionine) replaces another hydrophobic residue; or a polar residue replaces another polar residue, such as arginine replaces lysine, glutamic acid replaces aspartic acid, or glutamine replaces asparagine, etc. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; 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.

[0120] The term "engineered" is used throughout the specification and claims to refer to a non-naturally occurring composition or protein that has different properties than the parent composition or protein from which it is derived.

[0121] Typically, "sequence identity" or "sequence homology" refers to the nucleotide-to-nucleotide correspondence or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, the technique for determining sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded therefrom, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". The percent identity of two sequences (nucleotides or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. The percent identity can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including version 2.2.9) available from the National Institute of Health. The BLAST program is based on Karlin and Altschul, Proc. Natl. Acad. Sci.; and Altschul et al., Nucleic Acids Res. 25: The alignment method discussed in 3389-3402 (1997). In short, the BLAST program defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The program can be used to determine the percent identity over the full length of the compared protein. Default parameters are provided to optimize searches with short query sequences, such as in the blast program. The program also allows the use of SEG filters to mask segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The range of the desired degree of sequence identity is about 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

[0122] As used throughout this disclosure, "substantially identical" refers to sequences having 85% or more, such as 90% or more, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity, wherein the activity of the composition is not altered by modifications in the sequence that result in differences in sequence identity.

[0123] As used throughout this disclosure, "amino acid mutation" refers to any difference in an amino acid sequence relative to a corresponding parent sequence, for example, an amino acid substitution, deletion, and / or insertion.

[0124] As used throughout this disclosure, the term "virus vector" or "viral vector" refers to a viral particle that acts as a vehicle for nucleic acid delivery and comprises a nucleic acid (e.g., an AAV expression cassette) packaged within the viral particle. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0125] "AAV virion" or "AAV virus" or "AAV virus particle" or "AAV vector particle" or "AAV vector" refers to a virus particle that includes at least one AAV capsid polypeptide and an encapsidated polynucleotide (e.g., an AAV vector genome comprising an expression cassette). The polynucleotide may include a heterologous nucleic acid (i.e., a polynucleotide other than a wild-type AAV genome, such as an expression cassette to be delivered to a mammalian cell). AAV vectors are generally named based on the name (serotype) of the capsid polypeptide. For example, an AAV5 vector indicates that the AAV vector comprises an AAV5 capsid polypeptide.

[0126] The terms "vector genome unit", "genomic unit", "genomic particle (gp)" or "genomic copy (gc)" as used in reference to viral titer refer to the number of vector genomes encapsidated in a virion and have no bearing on infectivity or function. The number of genomic particles in a particular vector preparation can be measured by methods well understood in the art, e.g., quantitative PCR of genomic DNA, or, for example, in Clark et al. (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: 272-278:.

[0127] The terms "infectious unit (iu)", "infectious particles" or "replication units" as used with reference to viral titer refer to the number of recombinant AAV vector particles that are competent for infection and replication as measured by the infectious center assay (also called the replication center assay), e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0128] "Infectious" virions, viruses or viral particles are virions, viruses or viral particles that include a polynucleotide component that can be delivered to cells that are tropic for the viral species. The term does not necessarily mean that the virus has any ability to replicate. As used herein, an "infectious" virus or viral particle is a virus or viral particle that can infect a target cell after approaching the target cell and can express a heterologous nucleic acid in the target cell. Thus, "infectivity" refers to the ability of a viral particle to approach a target cell, enter a target cell and express a heterologous nucleic acid in the target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Viral infectivity can be expressed as a ratio of infectious viral particles to total viral particles.

[0129] The term "transduction unit" (tu) as 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 described, e.g., in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or in Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0130] The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as "transduction." The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a variety of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus includes a nucleotide sequence encoding GFP), where GFP is produced in cells infected with the viral particle and detected and / or measured; or measurement of protein produced, such as by enzyme-linked immunosorbent assay (ELISA) or fluorescence activated cell sorting (FACS).

[0131] The term "tropism" refers to the ability of an AAV vector to infect one or more specified cell types, but can also encompass how the vector functions to transduce cells in one or more specified cell types; that is, tropism refers to the preferential entry of the AAV virion into one or more certain cell or tissue types and / or preferential interaction with the cell surface to facilitate entry into certain cells or tissue types, optionally and preferably followed by expression (e.g., transcription and optionally translation) of sequences carried by the AAV virion in the cell, such as expression of heterologous nucleotide sequences in the case of recombinant viruses.

[0132] The term "tropism profile" refers to the pattern of transduction of one or more target cells, tissues, and / or organs by an AAV vector described herein.

[0133] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to an appropriate control (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more of the transduction or tropism of the control, respectively). The appropriate control will depend on a variety of factors, including the desired tropism profile. Similarly, whether a capsid and / or virus "does not efficiently transduce" a target tissue or "does not have efficient tropism" or similar terms for a target tissue can be determined by reference to an appropriate control.

[0134] As used throughout this disclosure, "neuronal activity," "neuronal activity," "neuronal discharges," and variants and synonyms thereof refer to electrical activity caused by stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch clamp techniques. In some embodiments, determining the activity of a neuron includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the activity level of a neuron depends on, or is affected by, an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential.

[0135] 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.

[0136] As used throughout this disclosure, "symptoms" of a disease refer to physical or mental characteristics that are considered indicative of a disease condition, particularly such characteristics that are noticeable 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 an epileptic seizure.

[0137] As used throughout this disclosure with respect to the position of amino acids, the term "corresponding to or corresponds to" means that when a first polypeptide sequence and a reference polypeptide sequence are compared, an amino acid in the first polypeptide sequence is aligned with a given amino acid in the reference polypeptide sequence. Those skilled in the art use software designed for this purpose, such as Clustal Omega version 1.2.4 and the default parameters of that version for comparison.

[0138] As used throughout this disclosure, the term "expression cassette" refers to a DNA segment containing 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 an appropriate host cell, the expression cassette is particularly capable of directing the cell's machinery to transcribe the transgene into RNA.

[0139] As used throughout this disclosure, the term "promoter" refers to one or more nucleic acid control sequences that instruct the transcription of an operably connected nucleic acid. A promoter may include a nucleic acid sequence near the transcription start site, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. A promoter may be inducible or repressive. An "inducible" promoter responds to certain changes in external conditions (such as temperature, concentration of a specific molecule, or oxygen or nutrient levels) to start increasing the transcription level of an operably connected nucleic acid. In some embodiments, a promoter is "tissue-specific", which causes the operably connected nucleic acid to be expressed at a significantly higher level in a specific cell type or tissue than in a control cell type or tissue. In some embodiments, a promoter may be neuron-specific and confer operably connected nucleic acids in neurons with significantly higher expression (e.g., 2 times, 5 times, or 10 times) than in non-neurons.

[0140] As used throughout this disclosure, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the second nucleic acid sequence.

[0141] As used throughout this disclosure, the term "enhancer" refers to a polynucleotide sequence that generally acts on the activity of a promoter in a cis position and thus stimulates the transcription of a gene operably linked to the promoter. Typically, the effect of an enhancer is quite independent of position and orientation, and therefore it can be located in front of or behind a promoter, within an intron, or even within a gene coding region. An enhancer can be located in the immediate vicinity of a gene coding region or a promoter, or can be quite far from a promoter. It may also have physical and / or functional overlap with a promoter.

[0142] Overview

[0143] The present disclosure provides expression cassettes 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.

[0144] In some embodiments, the expression cassettes of the present disclosure are optimized for one or more of the following properties:

[0145] Preferentially expressed in neurons;

[0146] Promote sustained gene expression;

[0147] Expressed at sufficient levels to be effective without causing serious cell health issues;

[0148] Easily packaged into AAV vectors.

[0149] In some embodiments, the expression cassette is further optimized to balance expression level, efficacy, and toxicity. In some embodiments, the expression cassette of the present disclosure allows the transgene to be expressed at medium to low levels in target cells, but enables a wider in vivo therapeutic window (e.g., the corresponding small molecule ligand of the transgene can be administered over a wider range without causing toxicity) compared to a control expression cassette that allows the transgene to be expressed at a higher level.

[0150] In some embodiments, the expression cassettes of the present disclosure mediate high levels of expression of the encoded transgene in dorsal root ganglia (DRG) and / or trigeminal ganglia (TGG) neurons. In some embodiments, the expression cassettes of the present disclosure enhance expression of the encoded transgene compared to a control expression cassette. In some embodiments, the expression cassettes of the present disclosure are delivered to DRG and / or TGG neurons to treat neuropathic pain. In some embodiments, the expression cassettes comprise polynucleotides encoding an engineered ligand-gated ion channel (eLGIC) receptor or a chimeric form thereof.

[0151] In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in vivo in human dorsal root ganglion and / or trigeminal ganglion neurons compared to a control expression cassette. In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in vitro in human dorsal root ganglion and / or trigeminal ganglion neurons compared to a control expression cassette. In some embodiments, the expression cassette mediates a higher level of expression of the encoded transgene in vitro in human dorsal root ganglion and / or trigeminal ganglion neurons compared to a control expression cassette.

[0152] In some embodiments, the expression cassette of the present disclosure mediates high levels of expression of the encoded transgene in hippocampal neurons. In some embodiments, the expression cassette mediates high levels of 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 the 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 form thereof.

[0153] In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in hippocampal neurons compared to a control expression cassette. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in hippocampal neurons compared to a control expression cassette.

[0154] 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 vitro compared to a control expression cassette in human hippocampal neurons. In some embodiments, the expression cassette mediates higher levels of expression of the encoded transgene in vitro compared to a control expression cassette in human hippocampal neurons.

[0155] In some embodiments, the expression cassette is contained in a vector (e.g., a viral vector or a non-viral vector). In some embodiments, the expression cassette is contained 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.

[0156] The present disclosure provides methods of using the expression cassettes or corresponding vectors of the present disclosure in the development of therapeutic regimens, vaccines, or research tools.

[0157] The present disclosure also provides methods for reducing the amount of total nucleic acid administered to a subject using the expression cassettes of the present disclosure. The methods include administering to the subject a lesser total amount of nucleic acid than the amount of nucleic acid administered to the subject when the nucleic acid includes a control expression cassette.

[0158] The present disclosure also provides methods of treating neuropathic pain (e.g., 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 form thereof.

[0159] The present disclosure also provides methods of treating focal epilepsy in a subject in need thereof, the methods 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 form thereof.

[0160] Expression cassette

[0161] In some embodiments, the expression cassette comprises one or more regulatory elements provided in Table 1 below. Figure 1 An exemplary arrangement of various elements is provided in .

[0162] Table 1: Non-limiting examples of regulatory elements

[0163]

[0164]

[0165] 5' enhancer

[0166] In some embodiments, the expression cassettes of the present disclosure comprise a 5' enhancer.

[0167] 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 that is 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% identical to SEQ ID NO: 37. In some embodiments, the CMV enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 37.

[0168] 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 that is 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% identical to SEQ ID NO: 38. In some embodiments, the CMV-V2 enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 38.

[0169] In some embodiments, the 5' enhancer comprises or consists of a CMV-V3 enhancer. In some embodiments, the CMV-V3 enhancer comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 39. In some embodiments, the CMV-V3 enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 39.

[0170] In some embodiments, the expression cassettes of the present disclosure do not comprise a 5' enhancer.

[0171] Non-neuronal silencing element

[0172] In some embodiments, the expression cassettes of the present disclosure comprise a non-neuronal silencing element, also referred to as a neuronal-restricted silencing element (NRSE).

[0173] NRSE has been identified in multiple neuronal genes and confers neuronal specificity by silencing transcription in non-neuronal cells. For example, neuron-restricted silencing elements are present in the promoter of the neuronal nicotinic acetylcholine receptor β2 subunit gene that determines its neuron-specific expression in the nervous system. In some embodiments, such NRSEs can silence or enhance transcription depending on the cellular environment within the nervous system. Studies have shown that certain NRSEs may activate transcription of synthetic promoters when located downstream of the 5' untranslated region or upstream of the TATA box less than 50 bp away, but will convert to silencers when located further upstream. For more discussion of NRSE, see BBessis et al., Proc Natl Acad Sci USA. 1997 May 27; 94 (11): 5906-5911, the contents of which are incorporated by reference as a whole for all purposes.

[0174] In some embodiments, the NRSE of the present disclosure comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 40. In some embodiments, the NRSE comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) nucleotides compared to the sequence of SEQ ID NO: 40.

[0175] In some embodiments, the NRSE is embedded in a promoter of the present disclosure. In some embodiments, the NRSE is placed between 30 and 50 base pairs after the TATA box in the expression cassette.

[0176] In some embodiments, the expression cassettes of the present disclosure do not comprise a neuron-restricted silencing element (NRSE).

[0177] Promoter

[0178] 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.

[0179] 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 that is 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% identical to SEQ ID NO: 41. In some embodiments, the cβ actin promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 41.

[0180] In some embodiments, the promoter comprises a CMV promoter or consists of a CMV promoter. In some embodiments, the CMV promoter comprises or consists of the following polynucleotide sequence: the polynucleotide sequence has 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 with SEQ ID NO: 42. In some embodiments, the CMV promoter comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 42.

[0181] In some embodiments, the promoter comprises or consists of a hCaMKIIa promoter. In some embodiments, the hCaMKIIa promoter comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 43. In some embodiments, the hCaMKIIa promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 43.

[0182] In some embodiments, the promoter comprises or consists of an hsCaMKIIa promoter. In some embodiments, the hsCaMKIIa promoter comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 44. In some embodiments, the hsCaMKIIa promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 44.

[0183] In some embodiments, the promoter comprises or consists of an hγ enolase promoter. In some embodiments, the hγ enolase promoter comprises or consists of the following polynucleotide sequence: the polynucleotide sequence has 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 with SEQ ID NO: 45. In some embodiments, the hγ enolase promoter comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 45.

[0184] In some embodiments, the promoter comprises or consists of a hMecp2 promoter. In some embodiments, the hMecp2 promoter comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 46. In some embodiments, the hMecp2 promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 46.

[0185] In some embodiments, the promoter comprises or consists of an hPDGF promoter. In some embodiments, the hPDGF promoter comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 47. In some embodiments, the hPDGF promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 47.

[0186] 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 that is 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% identical to SEQ ID NO: 48. In some embodiments, the hSyn promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 48.

[0187] In some embodiments, the promoter comprises or consists of an hSyn-V2 promoter. In some embodiments, the hSyn-V2 promoter comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 49. In some embodiments, the hSyn-V2 promoter comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 49.

[0188] In some embodiments, the promoter includes or consists of an mMecp2 promoter. In some embodiments, the mMecp2 promoter includes or consists of the following polynucleotide sequence: the polynucleotide sequence is 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% identical to SEQ ID NO: 50. In some embodiments, the mMecp2 promoter includes or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 50.

[0189] In some embodiments, the promoter comprises or consists of the msCaMKIIa promoter. In some embodiments, the msCaMKIIa promoter comprises or consists of the following polynucleotide sequence: the polynucleotide sequence has 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 with SEQ ID NO: 51. In some embodiments, the msCaMKIIa promoter comprises or consists of a polynucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 51.

[0190] In some embodiments, the expression cassettes of the present disclosure do not comprise a promoter.

[0191] 5' untranslated region (5'UTR)

[0192] In some embodiments, the expression cassettes of the present disclosure comprise a 5' untranslated region (5'UTR).

[0193] In some embodiments, the 5'UTR comprises or consists of a fGlob 5'UTR. In some embodiments, the fGlob 5'UTR comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 52. In some embodiments, the fGlob 5'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 52.

[0194] 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 that is 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% identical to SEQ ID NO: 53. In some embodiments, the hCaMKII 5'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 53.

[0195] 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 that is 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% identical to SEQ ID NO: 54. In some embodiments, the hSyn 5'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 54.

[0196] 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 that is 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% identical to SEQ ID NO: 55. In some embodiments, the mCaMKII 5'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 55.

[0197] 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 that is 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% identical to SEQ ID NO: 56. In some embodiments, the mCaMKII-V2 5'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 56.

[0198] In some embodiments, the expression cassettes of the present disclosure do not comprise a 5'UTR.

[0199] Intron

[0200] 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.

[0201] In some embodiments, the intron comprises or consists of a cβAct+rGlob intron. In some embodiments, the cβAct+rGlob intron comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 57. In some embodiments, the cβAct+rGlob intron comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 57.

[0202] In some embodiments, the intron comprises or consists of an hCMV+rGlob intron. In some embodiments, the hCMV+rGlob intron comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 58. In some embodiments, the hCMV+rGlob intron comprises or consists of a polynucleotide sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated (substituted, deleted and / or added) nucleotides compared to the sequence of SEQ ID NO: 58.

[0203] 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 that is 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% identical to SEQ ID NO: 59. In some embodiments, the hSyn intron comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 59.

[0204] 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 that is 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% identical to SEQ ID NO: 60. In some embodiments, the hTPI intron comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 60.

[0205] In some embodiments, the intron comprises or consists of a TPL+eMLP intron. In some embodiments, the TPL+eMLP intron comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 61. In some embodiments, the TPL+eMLP intron comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 61.

[0206] In some embodiments, the expression cassettes of the present disclosure do not comprise introns.

[0207] 3' enhancer

[0208] In some embodiments, the expression cassettes of the present disclosure comprise a 3' enhancer.

[0209] In some embodiments, the 3' enhancer comprises or consists of a 511-810EES 3' enhancer. In some embodiments, the 511-810EES 3' enhancer comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 62. In some embodiments, the 511-810EES 3' enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 62.

[0210] In some embodiments, the 3' enhancer comprises or consists of the FullEES 3' enhancer. In some embodiments, the FullEES 3' enhancer comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 63. In some embodiments, the FullEES 3' enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 63.

[0211] In some embodiments, the 3' enhancer comprises or consists of a WPREx 3' enhancer. In some embodiments, the WPREx 3' enhancer comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 64. In some embodiments, the WPREx 3' enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) nucleotides compared to the sequence of SEQ ID NO: 64.

[0212] In some embodiments, the 3' enhancer comprises or consists of a WPREx-V2 3' enhancer. In some embodiments, the WPREx-V2 3' enhancer comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 65. In some embodiments, the WPREx-V2 3' enhancer comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 65.

[0213] In some embodiments, the expression cassettes of the present disclosure do not comprise a 3' enhancer.

[0214] 3' untranslated region (3'UTR)

[0215] In some embodiments, the expression cassettes of the present disclosure comprise a 3' untranslated region (3'UTR).

[0216] 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 that is 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% identical to SEQ ID NO: 66. In some embodiments, the alpha globin 3'UTR comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 66.

[0217] In some embodiments, the expression cassettes of the present disclosure do not comprise a 3'UTR.

[0218] Polyadenylation sequence (PolyA)

[0219] In some embodiments, an expression cassette of the present disclosure comprises a polyadenylation sequence ("polyA", also referred to as a "polyA tail", "poly-A" or "poly-A tail").

[0220] In some embodiments, polyA comprises or consists of bGH polyA. In some embodiments, bGH polyA comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 67. In some embodiments, bGH polyA comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 67.

[0221] In some embodiments, the polyA comprises or consists of hGH polyA. In some embodiments, the hGH polyA comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 68. In some embodiments, the hGH polyA comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 68.

[0222] In some embodiments, polyA comprises or consists of hGH-V2 polyA. In some embodiments, hGH-V2 polyA comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 69. In some embodiments, hGH-V2 polyA comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 69.

[0223] In some embodiments, polyA comprises or consists of rβ globulin polyA. In some embodiments, rβ globulin polyA comprises or consists of a polynucleotide sequence that is 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% identical to SEQ ID NO: 70. In some embodiments, rβ globulin polyA comprises or consists of a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to the sequence of SEQ ID NO: 70.

[0224] In some embodiments, the expression cassettes of the present disclosure do not comprise polyA.

[0225] Box with integrated components

[0226] An exemplary expression cassette is Figure 1 In some embodiments, the order of the regulatory elements (5' to 3' order) follows one of the examples provided in Table 2 below.

[0227]

[0228]

[0229] In some embodiments, the expression cassette comprises a polynucleotide sequence that is 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% identical to any one of SEQ ID NOs: 71-93 excluding the transgenic region (SEQ ID NO: 36). In some embodiments, the expression cassette comprises a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 71-93 excluding the transgenic region (SEQ ID NO: 36).

[0230] In some embodiments, the expression cassette comprises a polynucleotide sequence that is 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% identical to any one of SEQ ID NOs: 121-124 excluding the transgenic region (SEQ ID NO: 32). In some embodiments, the expression cassette comprises a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 121-124 excluding the transgenic region (SEQ ID NO: 32).

[0231] 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 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 71-93 and 121-124.

[0232] In some embodiments, the disclosure provides a recombinant nucleic acid comprising an expression cassette of the 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 that is 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% identical to SEQ ID NO: 94 or 119. In some embodiments, the recombinant nucleic acid comprises a 3'ITR sequence that is 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% identical to SEQ ID NO: 95 or 120.

[0233] 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 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 96-118 and 125-128.

[0234] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence that is 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% identical to any one of SEQ ID NOs: 96-118 excluding the transgenic region (SEQ ID NO: 36). In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 96-118 excluding the transgenic region (SEQ ID NO: 36).

[0235] In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence that is 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% identical to any one of SEQ ID NOs: 125-128 excluding the transgenic region (SEQ ID NO: 32). In some embodiments, the recombinant nucleic acid comprises a polynucleotide sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of nucleotides compared to any one of SEQ ID NOs: 125-128 excluding the transgenic region (SEQ ID NO: 32).

[0236] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0237] (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37;

[0238] (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48;

[0239] (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54;

[0240] (iv) an hTPI intron comprising a polynucleotide sequence that is 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:60;

[0241] (v) genetically modified organisms;

[0242] (vi) a FullEES 3' enhancer comprising a polynucleotide sequence that is 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:63;

[0243] (vii) rβ globulin polyA, comprising a polynucleotide sequence that is 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:70.

[0244] 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 a 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.

[0245] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0246] (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37;

[0247] (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48;

[0248] (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54;

[0249] (iv) an hSyn intron comprising a polynucleotide sequence that is 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:59;

[0250] (v) genetically modified organisms;

[0251] (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64;

[0252] (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and

[0253] (viii) hGH polyA comprising a polynucleotide sequence that is 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:68.

[0254] 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 a 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.

[0255] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0256] (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37;

[0257] (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43;

[0258] (iii) an hCaMKII 5'UTR comprising a polynucleotide sequence that is 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:53;

[0259] (iv) an hCMV+rGlob intron comprising a polynucleotide sequence that is 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:58;

[0260] (v) genetically modified organisms;

[0261] (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64;

[0262] (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and

[0263] (viii) hGH polyA comprising a polynucleotide sequence that is 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:68.

[0264] 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 a 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.

[0265] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0266] (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence that is 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:38;

[0267] (ii) an hSyn-V2 promoter comprising a polynucleotide sequence that is 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:49;

[0268] (iii) the transgene;

[0269] (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0270] (v) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0271] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0272] (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence that is 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:38;

[0273] (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43;

[0274] (iii) the transgene;

[0275] (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0276] (v) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0277] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0278] (i) an hSyn-V2 5' enhancer comprising a polynucleotide sequence that is 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:49;

[0279] (ii) the transgene;

[0280] (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0281] (iv) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0282] In some embodiments, the expression cassette comprises, in 5' to 3' order:

[0283] (i) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43;

[0284] (ii) the transgene;

[0285] (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and

[0286] (iv) hGH-V2 polyA comprising a polynucleotide sequence that is 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:69.

[0287] Carrier

[0288] In some embodiments, the present disclosure provides a vector comprising a recombinant nucleic acid, wherein the recombinant nucleic acid comprises an expression cassette of the present disclosure.

[0289] 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.

[0290] In some embodiments, the vector is encapsulated in a particle, such as a lipid nanoparticle or an exosome.

[0291] 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., a lentivirus), an adeno-associated virus (AAV), a poxvirus, an alphavirus, a vaccinia virus, or a herpes simplex virus (e.g., HSV-1).

[0292] Adeno-associated virus (AAV) vector

[0293] Adeno-associated virus (AAV) is a member of the Parvoviridae family and is a small, non-enveloped icosahedral virus with a single-stranded, linear DNA genome of about 4.7 kilobases (kb). AAV is assigned to the genus Dependovirus because the virus was found to be a contaminant in purified adenovirus stocks (DM Knipe, PM Howley, Field's Virology, Lippincott Williams & Wilkins, Philadelphia, ed. Sixth edition, 2013). In the wild-type state, AAV relies on a helper virus (usually adenovirus) to provide the protein factors necessary for replication because AAV is naturally replication-defective. The 4.7kb genome of AAV is flanked by two reverse terminal repeats (ITRs) folded into a hairpin shape important for replication. AAV has a natural replication defect and is able to transduce almost all cell types in the human body, so it is an ideal vector for therapeutic use or vaccine delivery in gene therapy. In the wild-type state, the life cycle of AAV includes a latent period and an infectious period. During the latent period, the AAV genome is site-specifically integrated into the host chromosome after infection. During the infectious period, after infection with adenovirus or herpes simplex virus, the integrated genome is subsequently rescued, replicated and packaged into infectious virus. When vectorized, the viral Rep and Cap genes of AAV are removed during the virus production process and provided in trans, 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 series of possible transfer vector configurations to perform gene addition or gene targeting. These vectorized AAVs transduce dividing and non-dividing cells and show strong stable expression in static tissues. AAV vectors and virions are described in multiple published US applications, including US Publication Nos. 2015 / 0176027, 015 / 0023924, 2014 / 0348794, 2014 / 0242031, and 2012 / 0164106; all of which are incorporated herein by reference in their entirety.

[0294] However, despite the impressive ability of AAV vectors to transduce various tissues and cell types, there is still a need in the art for AAV vectors that have improved transduction efficiency and desired tropism for specific neuronal cells. In addition, the method of administration can also affect the location, amount, and type of cells transduced. Delivery to neurons requires high levels of transduction and correct tropism because there are physical limitations on how much AAV can be delivered in a single injection, and the desired outcome (e.g., therapeutic outcome) is related to stimulating or inhibiting the activity of the correct set of neurons in a subject.

[0295] The present disclosure provides AAV capsid polypeptides that exhibit significantly improved transduction of a subpopulation of neurons in a subject in vivo, in vitro, and / or ex vivo.

[0296] The genome structure of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Reverse terminal repeats (ITRs) are located on both sides of the unique coding nucleotide sequence of non-structural replication (Rep) proteins and structural (VP) proteins. VP proteins (VP1, VP2 and VP3) form capsids and contribute to the tropism of the virus. The terminal 145nt ITR is self-complementary and organized so that an energy-stable intramolecular duplex can be formed, which forms a T-shaped hairpin. These hairpin structures act as the origin of replication of viral DNA, thereby serving as primers for cellular DNA polymerase complexes. After wild-type (wt) AAV infection in mammalian cells, the Rep gene is expressed and plays a role in the replication of the viral genome.

[0297] In some embodiments, the outer protein "capsid" of the viral vector exists in nature, for example, 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 properties that do not exist in nature, such as altered tropism, increased transduction efficiency and / or immune escape. An example of a rationally designed capsid is a mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T) and lysine (K) residues on the VP3 viral capsid polypeptide.

[0298] Non-limiting examples of AAV ITRs that can be used in AAV vectors include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16.

[0299] AAV vectors containing two ITRs have a payload capacity of about 4.4 kB. Self-complementary AAV (scAAV) vectors contain a third ITR and package both strands of the recombinant portion of the vector, leaving only about 2.1 kB for the polynucleotides contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0300] The double AAV vector strategy has been used to achieve an expanded packaging capacity (about 9 kB) that is roughly twice the packaging capacity of AAV. The double vector strategy that can be used to produce the AAV considered herein includes, but is not limited to, splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybridization). In the double AAV trans-splicing strategy, the splicing donor (SD) signal is placed at the 3' end of the 5' half vector, and the splicing acceptor (SA) signal is placed at the 5' end of the 3' half vector. When the same cell is co-infected with two AAV vectors and the head-to-tail concatemerization of the two halves mediated by inverted terminal repeats (ITRs), trans-splicing results in the production of mature mRNA and full-length 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 can contain homologous overlapping sequences (the dual AAV overlaps at the 3' end of the 5' half vector and at the 5' end of the 3' half vector) that will mediate the reconstruction of a single large genome by homologous recombination (Duan et al., 2001). This strategy relies on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on the addition of a high recombination region from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al., 2008; Ghosh et al., 2011)) to the trans-splicing vector. To increase recombination between the dual AAVs, the added region is placed downstream of the SD signal in the 5' half vector and upstream of the SA signal in the 3' half vector.

[0301] "Hybrid AAV" refers to an AAV genome packaged with a capsid of a different AAV serotype (and preferably, a serotype different from one or more AAV ITRs), and may otherwise be referred to as pseudotyped AAV. For example, an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 or AAV16 genome can be encapsidated into an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 or AAV16 capsid or a variant thereof, provided that the AAV capsid and the genome (and preferably, one or more AAV ITRs) are of different serotypes. In certain embodiments, a pseudotyped AAV particle can be referred to as being of type "x / y," 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 from AAV2 and a capsid from AAV5.

[0302] "Host cells" include cells transfected, infected or transduced in vivo, ex vivo or in vitro with the recombinant vectors or polynucleotides disclosed herein. Host cells can include cells that produce viruses and cells infected with viral vectors. In some embodiments, host cells are infected in vivo with the viral vectors contemplated herein.

[0303] High-titer AAV preparations can be produced using techniques known in the art, for example, as described in U.S. Pat. Nos. 5,658,776; 6,566,118; 6,989,264; and 6,995,006; U.S. 2006 / 0188484; WO 98 / 22607; WO 2005 / 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), as well as various modifications or formulations of AAV vectors to reduce their immunogenicity following in vivo administration (see, e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562).

[0304] In some embodiments, an AAV vector comprising an AAV capsid polypeptide (or a mutant of the disclosure) facilitates targeted expression of an engineered receptor to a cell or neuronal subpopulation in a subject. In some embodiments, the neuron is a nociceptor.

[0305] In some embodiments, AAV includes a self-complementary genome. As defined herein, an AAV including a "self-complementary" or "double-stranded" genome refers to an AAV that has been engineered so that the coding region of AAV is configured to form an intramolecular double-stranded DNA template, such as 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) described, the entire contents of which are incorporated by reference. In some cases, the present disclosure contemplates the use of AAVs including self-complementary genomes because upon infection (such transduction), the two complementary halves of scAAV will associate to form a double-stranded DNA (dsDNA) unit in preparation for immediate replication and transcription rather than waiting for cell-mediated synthesis of the second strand of the AAV genome. It should be understood that AAVs including self-complementary genomes can only accommodate about half (about 2.4 kb) of the total coding capacity (4.7-6 kb) found in AAV, rather than the entire coding capacity.

[0306] In some embodiments, the AAV comprises a single-stranded genome. As defined herein, a "single-stranded" genome refers to a non-self-complementary genome. In some embodiments, the disclosure relates to a single-stranded AAV vector capable of achieving efficient gene transfer to the anterior segment of the 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 contents of which are incorporated by reference in their entirety.

[0307] Capsid polypeptide

[0308] In some embodiments, the present disclosure provides capsid polypeptides that enable the corresponding encapsidated AAV vector to achieve enhanced transduction and / or tropism for at least one neuronal cell subpopulation. Illustrative capsid polypeptides of the present disclosure are listed in Table 3 below:

[0309] Table 3: Illustrative Capsid Polypeptides

[0310]

[0311]

[0312] 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 a sequence according 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 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 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 mutations (substitutions, deletions, and / or additions) of amino acids 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.

[0313] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5. In some embodiments, the capsid polypeptide of the present disclosure comprises a sequence according to SEQ ID NO: 2, is essentially composed of a sequence according to SEQ ID NO: 2, or is composed of a sequence according to SEQ ID NO: 2. In some embodiments, the capsid polypeptide of the present disclosure comprises, is essentially composed of, or is composed of the following amino acid sequence: the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with SEQ ID NO: 2. In some embodiments, the capsid polypeptide of the present disclosure comprises, is essentially composed of, or is composed of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations (substitutions, deletions and / or additions) of amino acids 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.

[0314] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5-TV. In some embodiments, the capsid polypeptide of the present disclosure includes an amino acid substitution at a position corresponding to T492 of SEQ ID NO: 2. In some embodiments, the capsid polypeptide of the present disclosure includes an amino acid substitution corresponding to T492V of SEQ ID NO: 2. In some embodiments, the capsid polypeptide of the present disclosure includes a sequence according to SEQ ID NO: 3, is essentially composed of a sequence according to SEQ ID NO: 3, or is composed of a sequence according to SEQ ID NO: 3. In some embodiments, the capsid polypeptide of the present disclosure includes, is essentially composed of, or is composed of the following amino acid sequence: the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 3, wherein the capsid polypeptide includes a valine at a position corresponding to V492 of SEQ ID NO: 3. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) amino acids compared to the sequence of SEQ ID NO: 3, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 3. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist 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.

[0315] 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 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 according to SEQ ID NO: 4. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist 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, wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 / F731 of SEQ ID NO: 4. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of amino acids compared to the sequence of SEQ ID NO: 4, wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of 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 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.

[0316] 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 amino acid substitutions at positions corresponding to T492, Y705 and / or Y731 of SEQ ID NO: 2. In some embodiments, the capsid polypeptide of the present disclosure comprises amino acid substitutions 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 a sequence according to SEQ ID NO: 5. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to 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 corresponding to F705 and / or F731 of SEQ ID NO:5. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) amino acids compared to the sequence of SEQ ID NO: 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 corresponding to F705 and / or F731 of SEQ ID NO: 5. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist 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.

[0317] 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 a sequence according 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 that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of amino acids 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.

[0318] 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 a sequence according 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 at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 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 mutations (substitutions, deletions, and / or additions) of amino acids compared to the sequence of 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 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.

[0319] 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 a sequence according 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 that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence that has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of amino acids 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.

[0320] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-TV. In some embodiments, the capsid polypeptide of the present disclosure includes an amino acid substitution at a position corresponding to T492 of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes an amino acid substitution corresponding to T492V of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes a sequence according to SEQ ID NO: 9, is essentially composed of a sequence according to SEQ ID NO: 9, or is composed of a sequence according to SEQ ID NO: 9. In some embodiments, the capsid polypeptide of the present disclosure includes, is essentially composed of, or is composed of an amino acid sequence according to SEQ ID NO: 9: the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO: 9, wherein the capsid polypeptide includes a valine at a position corresponding to V492 of SEQ ID NO: 9. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) amino acids compared to the sequence of SEQ ID NO: 9, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 9. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist 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.

[0321] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-2YF. In some embodiments, the capsid polypeptide of the present disclosure includes amino acid substitutions at positions corresponding to Y705 and / or Y731 of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes amino acid substitutions corresponding to Y705F and / or Y731F of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes, consists essentially of, or consists of a sequence according to SEQ ID NO: 10. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist 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, wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 / F731 of SEQ ID NO: 10. In some embodiments, the capsid polypeptides of the present disclosure comprise, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of amino acids compared to the sequence of SEQ ID NO: 10, wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of 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 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.

[0322] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-TV2YF. In some embodiments, the capsid polypeptide of the present disclosure includes amino acid substitutions at positions corresponding to T492, Y705 and / or Y731 of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes amino acid substitutions corresponding to T492V, Y705F and / or Y731F of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure includes, consists essentially of, or consists of a sequence according to SEQ ID NO: 11. 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: 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 corresponding to F705 and / or F731 of SEQ ID NO: 11. In some embodiments, the capsid polypeptides of the present disclosure include, consist essentially of, or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutated (substituted, deleted, and / or added) amino acids compared to the sequence of SEQ ID NO: 11, wherein the capsid polypeptide includes a valine at a position corresponding to V492 of SEQ ID NO: 11, and wherein the capsid polypeptide includes a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO: 11. In some embodiments, the capsid polypeptides of the present disclosure include, consist essentially of, or consist 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.

[0323] In some embodiments, the AAV vector includes an AAV capsid polypeptide, the AAV capsid polypeptide includes an amino acid mutation at one or more positions corresponding to T492, Y705 and / or Y731 of an AAV9 capsid polypeptide (SEQ ID NO: 8), wherein the AAV capsid polypeptide is serotype AAV2, AAV2.5, AVV5, AAV6, AAV9 or another AAV serotype. In some embodiments, one or more positions are two or more positions, two positions or three positions. In some embodiments, the viral vector includes an AAV capsid polypeptide, the AAV capsid polypeptide includes one or more amino acid substitutions corresponding to T492V, Y705F and / or Y731F of an AAV9 capsid polypeptide (SEQ ID NO: 8) or any combination thereof, wherein the AAV capsid polypeptide polypeptide is serotype AAV2, AAV2.5, AVV5, AAV6, AAV9 or another AAV serotype. In some embodiments, the one or more substitutions are two or more substitutions, two substitutions or three substitutions.

[0324] 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.

[0325] 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 99.7% sequence identity to SEQ ID NO: 8, wherein the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 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 having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (substitutions, deletions, and / or additions) of amino acids compared to the sequence of SEQ ID NO: 8, wherein the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 of 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 by a hydrophobic amino acid selected from valine (Val), leucine (Leu) and isoleucine (Ile). In some embodiments, the T492 residue is substituted by a hydrophobic amino acid selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met) and tryptophan (Trp).

[0326] Neurons and nerve fibers

[0327] In some embodiments, the present disclosure provides methods of delivering a recombinant nucleic acid comprising an expression cassette to target neurons located in the hippocampus (ie, hippocampal neurons).

[0328] In some embodiments, hippocampal neurons are excitatory neurons. In some embodiments, the expression cassette comprises a CAMKII family promoter (e.g., hCaMKIIa) and causes the encoded transgene to be highly expressed and / or selectively expressed in excitatory hippocampal neurons. In some embodiments, hippocampal neurons are Ca2+ / calmodulin-dependent protein kinase II (CaMKII) positive excitatory neurons.

[0329] In some embodiments, the hippocampal neurons are inhibitory neurons. In some embodiments, the hippocampal neurons are GABAergic neurons.

[0330] 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 a dorsal root ganglion (or spinal ganglion; also referred to as a dorsal root ganglion). The dorsal root ganglion (DRG) is a collection of neurons (ganglion) in the dorsal root of a spinal nerve. The cell bodies of sensory neurons, known as primary neurons, are located in the dorsal root ganglion. The axons of the dorsal root ganglion neurons are called afferents. In the peripheral nervous system, afferents refer to axons that transmit sensory information to the central nervous system (i.e., the brain and spinal cord).

[0331] 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 trigeminal ganglion (or Gasserian ganglion, or semilunarganglia, or Gasser's ganglion). The trigeminal ganglion (TGG) is a sensory ganglion of the trigeminal nerve (CNV) that occupies a cavity (McCalder's cavity) in the dura mater, covering the trigeminal nerve impression near the petrous apex of the temporal bone.

[0332] An axon (also called a nerve fiber) is a long, thin protrusion of a vertebrate neuron cell that normally conducts electrical impulses (called action potentials) away from the nerve cell body. Each neuron has only one axon. Therefore, neuronal cells can be classified based on their axons (nerve fibers).

[0333] Erlanger and Gasser divided nerve fibers into three general categories (Groups A, B, and C). Group A nerve fibers are highly myelinated, Group B nerve fibers are moderately myelinated, and Group C nerve fibers are unmyelinated.

[0334] 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 speeds. Type Aα fibers include type Ia and type Ib sensory fibers of the alternative classification system, and are fibers from muscle spindle terminals and Golgi tendons, respectively. Type Aβ fibers and type Aγ fibers are type II afferent fibers from stretch receptors. Type Aβ fibers from the skin are mainly used for touch. Type Aδ fibers are afferent fibers of nociceptors. Aδ fibers transmit information from peripheral mechanoreceptors and temperature receptors to the dorsal horn of the spinal cord. Aδ fibers are used to receive and transmit information mainly related to acute pain (sharp, immediate, and relatively short-lasting). This type of pain may be caused by several stimuli: temperature-induced, mechanical, and chemical. This may be part of the retraction reflex triggered by the AS fibers in the reflex arc that activates the retraction reaction. Aδ fibers carry cold, pressure, and acute pain signals; because they are thin (2-5 μm in diameter) and myelinated, they fire faster than unmyelinated C fibers but slower than other, thicker, myelinated group A fibers. Aδ fibers have a moderate conduction velocity.

[0335] Group B nerve fibers (B fibers) are axons that are moderately myelinated, meaning they are less myelinated than group A nerve fibers and more myelinated than group C nerve fibers. Group B nerve fibers are typically general visceral afferent fibers and preganglionic nerve fibers of the autonomic nervous system.

[0336] Group C nerve fibers (C fibers) are unmyelinated and have a small diameter and slow conduction velocity. Group C fibers include postganglionic fibers in the autonomic nervous system (ANS) and nerve fibers at the dorsal root (IV fibers). These fibers transmit sensory information. Damage or damage to these nerve fibers can cause neuropathic pain.

[0337] The peripheral endings of mature nociceptors are where stimuli are detected and converted into electrical energy. When the electrical energy reaches a threshold, an action potential is induced and driven to the central nervous system (CNS). This leads to a chain of events that allows people to consciously realize the pain. Nociceptors are determined by their sensory specificity only for specific features of the stimulus through a high threshold. Nociceptors are triggered only when the chemical, temperature, or mechanical environment reaches a high threshold. Most nociceptors are classified according to the environmental modality to which they respond. Some nociceptors respond to more than one of these modalities and are therefore designated as polysynaptic. Other nociceptors do not respond to any of these modalities (although they may respond to stimuli under inflammatory conditions) and are called sleeping or silent.

[0338] There are two different types of axons in nociceptors. The first is the Aδ fiber axon. The Aδ fiber axon is myelinated and can carry action potentials to the CNS at a rate of about 20 meters per second. The other type is the slower conducting C fiber axon. C fiber axons can only conduct at a rate of about 2 meters per second. This is because C fiber axons are lightly myelinated or unmyelinated. Therefore, pain is divided into two stages. The first stage is mediated by the fast-conducting Aδ fibers, and the second stage is mediated by (polymodal) C fibers. Pain associated with Aδ fibers may be associated with an initial extreme sharp pain. The second stage is a longer, slightly less intense sensation of pain due to acute injury. If a large amount or a long period of input is given to the C fibers, it will gradually accumulate in the dorsal horn of the spinal cord; this phenomenon is similar to tetanus in muscles, but is called wind-up. If wind-up occurs, there is a possibility of increased sensitivity to pain.

[0339] Different classes of nerve fibers can be distinguished by biomarkers such as neurofilament 200 (NF200, also known as neurofilament heavy polypeptide), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4). Aα and Aβ fibers are usually positive for NF200, but negative for IB4. Aδ fibers are usually positive for both NF200 and IB4. C fibers are usually positive for IB4, but negative for NF200. Therefore, NF200+ neurons mainly include highly myelinated Aα or moderately myelinated Aβ fibers. In some cases, these neurons mediate signals for light touch and proprioception. But some NF200+ neurons may also mediate nociceptive signals. IB4+ neurons mainly include unmyelinated C fibers or Aδ fibers for nociception. And CGRP+ neurons mainly include unmyelinated C fibers or lightly myelinated Aδ fibers for nociception.

[0340] Characteristics of the expression cassette

[0341] In some embodiments, compared to a control expression cassette (e.g., a control with tissue-specific expression), the expression cassette of the present disclosure mediates a higher level of expression of the encoded transgene in one or more neurons. In some embodiments, the control expression cassette comprises all the regulatory elements of the expression cassette No. 18 in Table 2, and the expression cassette No. 18 comprises the hSyn-V2 promoter for neuron-specific expression. In some embodiments, such a control expression cassette comprises a polynucleotide sequence of SEQ ID NO: 88 excluding the sequence of the transgene (SEQ ID NO: 36). In some embodiments, compared to a control expression cassette with ubiquitous but not tissue-specific transgenic expression, the expression cassette of the present disclosure mediates an equivalent or higher level of expression of the encoded transgene in one or more neurons. In some embodiments, the control expression cassette comprises all the regulatory elements of the expression cassette No. 17 in Table 2, and the expression cassette No. 17 comprises a CAG (CMV enhancer, cβ actin promoter, and rβ actin-rGlob intron) regulatory element 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). The skilled artisan will readily recognize the appropriate control to use in each case, and generally the control expression cassette will have the same transgene polynucleotide sequence as the expression cassette being compared.

[0342] The expression level 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.

[0343] 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 a 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 a control expression cassette. In some embodiments, the control expression cassette comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 88 (expression cassette No. 18 in Table 2) excluding the sequence of the transgene.

[0344] In some embodiments, the expression cassette mediates expression of the encoded transgene at a comparable or higher level in one or more target neurons as compared to a control expression cassette having ubiquitous, but not 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 the transgene expression level of the control expression cassette, including all ranges and subranges therebetween. In some embodiments, the control expression cassette comprises a polynucleotide sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 87 (expression cassette No. 17 in Table 2) excluding the sequence of the transgene.

[0345] In some embodiments, transgene expression levels refer to mRNA expression levels. In some embodiments, transgene expression levels refer to protein expression levels.

[0346] In some embodiments, the target neuron is a hippocampal neuron. In some embodiments, the target neuron is an excitatory neuron.

[0347] In some embodiments, the target neuron is a DRG or TGG neuron.

[0348] In some embodiments, the target neurons include A fibers. In some embodiments, the target neurons include Aα or Aβ fibers. In some embodiments, the target neurons include Aδ fibers. In some embodiments, the target neurons include C fibers.

[0349] In some embodiments, the target neurons include nerve fibers that are NF200 positive and IB4 negative. In some embodiments, the target neurons include nerve fibers that are NF200 positive and IB4 positive. In some embodiments, the target neurons include nerve fibers that are NF200 negative and IB4 positive. In some embodiments, the target neurons include nerve fibers that are CGRP positive.

[0350] genetically modified

[0351] In some embodiments, the expression cassette of the present disclosure comprises a transgene. In some embodiments, the transgene encodes a protein, 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, thereby allowing co-expression of two or more biomolecules.

[0352] 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 form thereof. In some embodiments, the engineered LGIC can be activated by a small molecule ligand. In some embodiments, the administration of an expression cassette encoding an eLGIC delivers it to a subpopulation of neuronal cells of a subject and causes expression of the eLGIC in neurons. In some embodiments, the expression of the eLGIC allows the activity of these neurons to be regulated by administering a corresponding small molecule ligand.

[0353] In some embodiments, the transgene is operably linked to one or more control / regulatory elements that direct the transcription or expression of the transgene. Control elements (i.e., regulatory elements) may include appropriate 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 secretion of the encoded product when desired. The present disclosure describes exemplary regulatory elements.

[0354] Such control elements may include control sequences that are typically associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences may be used. Useful heterologous control sequences typically include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (AdMLP); herpes simplex virus (HSV) promoter, endogenous cellular promoter heterologous to the target gene, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMV-IE), Rous sarcoma virus (RSV) promoter, synthetic promoter, hybrid promoter, etc. In addition, sequences derived from non-viral genes (e.g., mouse metallothionein gene) may also be used.

[0355] In some embodiments, a cell type-specific or tissue-specific promoter can be operably linked to a transgene, allowing for the selective or preferential production of the corresponding gene product encoded by the transgene in a specific cell type or tissue. In some embodiments, an inducible promoter can be operably linked to a transgene.

[0356] In some embodiments, non-viral methods can be used to deliver an expression cassette encoding a transgene to neurons (eg, using non-viral vectors, synthetic nanoparticles, etc.).

[0357] Engineered ligand-gated ion channels (eLGIC)

[0358] The present disclosure describes receptors, mutants thereof, and methods for their 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.

[0359] 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 changed so that it is physically and / or functionally different from the corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. The term "wild-type receptor" as used herein refers to a receptor having a polypeptide sequence identical to the polypeptide sequence of a protein found in nature. Wild-type receptors include receptors naturally present in humans and orthologs naturally present in other eukaryotic organisms (e.g., protists, fungi, plants, or animals, such as yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates). In some embodiments, the parent receptor is a non-natural receptor; that is, it is a receptor that does not exist in nature, such as a receptor engineered from a wild-type receptor. For example, a parent receptor can be an engineered receptor that includes one or more subunits from a wild-type receptor and one or more subunits from a second wild-type receptor. Therefore, the resulting protein consists of subunits from two or more wild-type receptors. Therefore, in some embodiments, the parent receptor is a chimeric receptor. Engineered receptors of the present disclosure include, for example, parent receptor mutants and switched receptors.

[0360] In some embodiments, the engineered receptor of the present disclosure includes at least one amino acid mutation relative to the corresponding parent receptor, such as one or more mutations in one or more domains of a wild-type receptor. In some embodiments, mutation is an amino acid substitution. In some embodiments, the engineered receptor has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or lower sequence identity of the corresponding parent receptor, including all values ​​and subranges located therebetween. In some embodiments, the parent receptor mutant has 85% or higher sequence identity with the corresponding parent receptor, such as 90% or higher or 95% or higher, such as about 96%, about 97%, about 98% or about 99% identity with the corresponding parent receptor, including all values ​​and subranges located therebetween. In some embodiments, the engineered receptor (e.g., parent receptor mutant) is generated by error-prone PCR.

[0361] In some embodiments, the ligand binding domain (LBD) of the engineered receptor of the present disclosure includes at least one amino acid mutation relative to the corresponding ligand binding domain of the parent receptor, for example, one or more mutations in the ligand binding domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ligand binding domain of the engineered receptor has 85% or more sequence identity with the corresponding ligand binding domain of the parent receptor, for example, 90% or more or 95% or more with the corresponding ligand binding domain of the parent receptor, for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity, including all values ​​and sub-ranges therebetween. In some embodiments, the ligand binding domain of the engineered receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the corresponding ligand binding domain of the parent receptor, including all values ​​and sub-ranges therebetween.

[0362] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure includes at least one amino acid mutation relative to the corresponding ion pore domain of the parent receptor, for example, 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, for example, 90% or more or 95% or more with the corresponding ion pore domain of the parent receptor, for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity or 100% identity, including all values ​​and subranges therebetween. In some embodiments, the ion pore domain of the engineered receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the corresponding ion pore domain of the parent receptor, including all values ​​and subranges therebetween.

[0363] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation relative to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, significantly reduce, or eliminate the function of the engineered receptor relative to the parent receptor, such as by reducing the binding of endogenous ligands to the engineered receptor relative to the binding of endogenous ligands to the parent receptor, or by reducing the activity of a signaling pathway downstream of the engineered receptor that is normally activated in response to the binding of a ligand to the corresponding parent receptor. In some embodiments, the mutation is an amino acid substitution.

[0364] In some embodiments, amino acid mutations are functional gain amino acid mutations relative to corresponding parent receptors. "Function gain" amino acid mutations refer to one or more mutations: it changes the function of the engineered receptor relative to the parent receptor, such as by changing or enhancing the affinity of the engineered receptor to the ligand relative to the binding of the endogenous ligand to the parent receptor, or by changing or enhancing the activity of the signal transduction pathway activated in response to the binding of the ligand to the engineered receptor relative to the binding of the endogenous ligand to the corresponding parent receptor. In some embodiments, the functional gain mutation causes the affinity of the engineered receptor to the ligand to increase. In specific embodiments, the functional gain mutation causes the affinity of the engineered receptor to the agonist ligand to increase. In some embodiments, the functional gain mutation causes the antagonist ligand to act as an agonist ligand after binding to the engineered receptor (for example, causing the activation of the agonist signal transduction pathway instead of the antagonist signal transduction pathway). In some embodiments, the functional gain mutation causes the modulator ligand to act as an agonist ligand after binding to the engineered receptor. In some embodiments, mutations are amino acid substitutions.

[0365] In some embodiments, the subject engineered receptors or their ligand binding domains and / or ion pore domains of the present disclosure include one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations relative to the corresponding parent receptor. In some embodiments, the mutations are amino acid substitutions.

[0366] In some embodiments, loss-of-function mutations and gain-of-function mutations are at the same residue, i.e., they are the same mutations. In other embodiments, loss-of-function mutations and gain-of-function mutations are mutations at different amino acid residues. In some embodiments, mutations are amino acid substitutions. In some embodiments, the subject engineered receptor (or its ligand binding domain and / or its ion pore domain) including loss-of-function mutations and / or gain-of-function mutations has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% (including all ranges and subranges therebetween) or lower sequence identity with the corresponding parent receptor (e.g., wild-type receptor or non-natural receptor) (or its ligand binding domain and / or ion pore domain). In some embodiments, the subject engineered receptor (or its ligand binding domain and / or its ion pore domain) has about 85% or more sequence identity with the corresponding parent receptor (or its ligand binding domain and / or ion pore domain), for example, 85%, 90% or 95% or more sequence identity, in some cases 96%, 97%, 98% or more sequence identity, for example 99% or 99.5% or more sequence identity, including all values ​​and subranges therebetween.

[0367] In some aspects, the engineered receptors of the present disclosure include receptors produced by a combination of one or more amino acid sequences (e.g., subunits) derived from a wild-type receptor and one or more amino acid sequences (e.g., subunits) derived from a second wild-type receptor. In other words, the engineered receptors include amino acid sequences that are heterologous to each other, where "heterologous" means that they do not occur together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, the chimeric receptor acts as a parent receptor from which the engineered receptors of the present disclosure are produced. In some embodiments, the chimeric receptor includes a ligand binding domain from a first LGIC and an ion pore domain from a second LGIC.

[0368] In some embodiments, the parent receptor mutant exhibits increased affinity for the agonist ligand. In some embodiments, a ligand that functions as an antagonist or modulator when bound to a wild-type receptor functions as an agonist when bound to a parent receptor mutant.

[0369] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGIC refers to a large class of transmembrane proteins that allow ions to pass through after being activated by a specific ligand. LGICs are composed of at least two domains: a ligand binding domain and a transmembrane ion pore domain. Binding of a ligand to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding causes a dramatic change in the permeability of the channel to one or more specific ions; when the channel is inactive or closed, virtually no ions can pass through the channel, but up to 107 ions can pass through per second after ligand binding. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and promote the influx of ions into the cytosol. In some embodiments, LGICs respond to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and promote the outflow of ions from the cytosol to the extracellular environment. Importantly, activation of LGICs results in the transport of ions (e.g., Ca2+, Na+, K+, Cl-, etc.) across the cell membrane, and does not result in the transport of the ligand itself.

[0370] LGIC receptors are composed of multiple subunits and can be homomeric or heteromeric receptors. Homomeric receptors are composed of subunits that are all of the same type. Heteromeric receptors are composed of subunits in which at least one subunit is different from at least one other subunit included in the receptor. For example, glycine receptors are composed of 5 subunits, of which there are two types: α subunits, of which there are four isoforms (α1–α4) and β subunits, of which there is one known isoform. An exemplary homomeric GlyR is a GlyR composed of 5 α1-GlyR subunits. Similarly, homomeric GABAA receptors can be composed of β3-GABAA subunits, and nAchR receptors can be composed of α7-nAchR subunits. Exemplary heteromeric GlyRs can be composed of one or more α-subunits and one or more β-subunits (e.g., α1β-GlyR). The subunits of exemplary LGIC receptors are shown in Table 4.

[0371] Table 4: LGIC receptors and subunits

[0372]

[0373]

[0374]

[0375] Illustrative examples of LGIC families suitable for use in particular embodiments include, but are not limited to, Cys-loop receptors, such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), λ-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchRs); as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaCs), ionotropic glutamate receptors, IP3 receptors, P2X receptors, ranolidine receptors, and zinc-activated channels (ZACs).

[0376] 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; GABRR; RIA4; 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; CHRD; CHRNE; P2RX1; P2RX2; P2RX3; P2RX4; P2RX5; P2RX6; P2RX7; RYR1; RYR2; RYR3 and ZACN.

[0377] Non-limiting examples of sequences of wild-type LGIC receptors that can be used to generate engineered receptors of the present disclosure include the following. In the sequence, the signal peptide is italicized, the ligand binding domain is bolded, and the ion pore domain is underlined:

[0378] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor α7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 25), encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5):

[0379]

[0380]

[0381] In some embodiments, the wild-type LGIC receptor is the human α1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 26), encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1):

[0382]

[0383]

[0384] In some embodiments, the wild-type LGIC receptor is the human α2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO:27), encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1):

[0385]

[0386] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO: 28), encoded by the GLRA3 gene (GenBank Accession No. NM_006529.3):

[0387]

[0388] In some embodiments, the wild-type LGIC receptor is human α3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO:29), encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.3):

[0389]

[0390]

[0391] In some aspects, the engineered receptor is a chimeric LGIC receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence derived from at least a first LGIC and an ion pore conduction domain sequence (or more simply, an "ion pore domain sequence") derived from at least a second LGIC. In some embodiments, the derived amino acid sequence is identical to the corresponding region of the LGIC from which it is derived. In some embodiments, the derived amino acid sequence may contain a change in at least one amino acid position compared to the corresponding region of the LGIC from which it is derived. In some embodiments, the amino acid sequence derived from the LGIC sequence differs from the corresponding region of the original amino acid sequence by a maximum of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the derived amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% (including all ranges and subranges therebetween) sequence identity with the corresponding region of the LGIC amino acid sequence.

[0392] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequences and ion pore domain sequences of Cys loop receptors are well known in the art and can be easily identified from the literature using publicly available software such as PubMed, Genbank, Uniprot, etc. In some embodiments, the ligand binding domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with the ligand binding domain of the first LGIC. In some embodiments, the ion pore domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is represented by bold and the ion pore domain is represented by underline.

[0393] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO: 26). In some embodiments, the ligand binding domain includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with about 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, 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.

[0394] In some embodiments, the ligand binding domain of the chimeric receptor includes a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain includes about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 25) (e.g., 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 human α7-nAChR.

[0395] 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 includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with about amino acids 23-220 of human α7-nAChR (SEQ ID NO: 25) (e.g., 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 human α7-nAChR.

[0396] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of a human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with about amino acids 245-457 of GlyRα1 (SEQ ID NO: 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.

[0397] In some embodiments, the ion pore domain of the chimeric receptor comprises 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 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 that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα2 (SEQ ID NO: 27). In some embodiments, the ion pore domain sequence of human GlyRα2 includes, 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 includes, 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 includes, 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 includes, consists essentially of, or consists of amino acids 260-452 of SEQ ID NO: 27.

[0398] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 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 that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO: 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.

[0399] In some embodiments, the ion pore domain of the chimeric receptor comprises the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 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 that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence that is identical to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO: 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.

[0400] In some embodiments, the ion pore domain is derived from the ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ion pore domain includes an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with about 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.

[0401] In some embodiments, the ion pore domain of the subject chimeric ligand-gated ion channel includes an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ion pore domain. "M2-M3 linker domain" or "M2-M3 linker" refers to a sequence within the ion pore domain of the LGIC that is at its amino (N) terminus to the C-terminus of the transmembrane domain 2 (M2) of the acceptor and at its carboxyl (C) terminus to the N-terminus of the transmembrane domain 3 (M3) of the acceptor. The M2-M3 linker of the LGIC can be readily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). In some embodiments, when the ion pore domain of the chimeric receptor includes a heterologous M2-M3 linker, the M2-M3 linker is derived from the same receptor as the ligand binding domain of the chimeric receptor. For example, when the subject ligand-gated ion channel includes a ligand binding domain from an AChR and an ion pore domain from a GlyR, its ion pore domain sequence may include an M2-M3 linker sequence derived from an AChR. In some embodiments, the ion pore domain is derived from GlyRα1 and the M2-M3 linker is derived from α7-nAChR. In some embodiments, the native M2-M3 linker sequence removed from the ion pore domain corresponds to about amino acids 293-313 of GlyRα1 (SEQ ID NO: 26), such as 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 about amino acids 281-295 of 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 at least 100% identical to amino acids 281-295 or 283-295 of α7-nAChR (SEQ ID NO:25).

[0402] In some embodiments, the ligand binding domain of the subject chimeric ligand-gated ion channel includes a Cys loop domain sequence that is heterologous to the Cys loop sequence of the ligand binding domain. A "Cys loop domain sequence" or "Cys loop sequence" refers to a domain within the ligand binding domain of the Cys loop LGIC that forms a loop structure flanked by cysteines at the N-terminus and C-terminus. Without wishing to be bound by theory, it is believed that upon ligand binding to the ligand binding domain, the Cys loop structurally moves into close proximity with the M2-M3 loop, and this movement mediates the biophysical conversion of ligand binding in the extracellular domain to signal transduction in the ion pore domain (as reviewed in Miller and Smart, Trends in Pharmacological Sci 2009: 31(4)). Replacing the endogenous Cys loop sequence with a heterologous Cys loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases, at least 5 times or 6 times, and at certain doses, at least 7 times, 8 times, 9 times or 10 times. The Cys loop domain of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the chimeric receptor includes a heterologous Cys-loop sequence, the Cys-loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the subject chimeric ligand-gated ion channel includes a ligand binding domain from an AChR and an ion pore domain from a GlyR, the subject ligand-gated ion channel can include the ligand binding domain sequence from the AChR, but with the exception of the sequence of the Cys-loop domain, which is alternatively derived from the GlyR. In some embodiments, the ligand binding domain is derived from a7-nAChR and the Cys-loop sequence is derived from GLyR. In some embodiments, the Cys-loop sequence removed from the ligand binding domain corresponds to about amino acids 150-164 of α7-nAChR (SEQ ID NO: 25), such as amino acids 150-157 of α7-nAChR. In some embodiments, the inserted Cys-loop sequence is derived from about amino acids 166-180 of GlyRα1 (SEQ ID NO: 26), such as amino acids 166-172 of GlyRα1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 166-180 of GlyRα1. In some embodiments, the inserted Cys loop sequence is derived from about 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 about amino acids 171-185 of GlyRα3 (SEQ ID NO: 28 or 29), such as amino acids 171-177 of GlyRα3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 171-185 of GlyRα3.

[0403] In some embodiments, the ligand binding domain of the subject chimeric ligand-gated ion channel includes a β1-2 loop domain sequence that is heterologous to the β1-2 loop domain sequence of the ligand binding domain. "β1-2 loop domain sequence" or "β1-2 loop or β1-β2 loop" refers to a domain within the ligand binding domain of the Cys-loop LGIC that is flanked at its N-terminus by the C-terminus of the β1 fold and at its C-terminus by the N-terminus of the β2 fold. 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 ion influx in the case of GlyR). It is believed that after ligand binding, the β1-2 loop, together with the Cys loop, is very close to the M2-M3 loop to mediate the biophysical conversion of ligand binding in the extracellular domain to signal transduction in the ion pore domain where the M2-M3 loop is located (as reviewed in Miller and Smart, supra). Replacing the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of the LGIC by 1.5 times or more, such as at least 2 times, 3 times or 4 times, in some cases, at least 5 times or 6 times, and at certain doses, at least 7 times, 8 times, 9 times or 10 times. The β1-2 loop domain of the Cys loop receptor can be easily determined from the art and / or by using any publicly available protein analysis tool (e.g., Expasy, uniProt, etc.). Typically, when the ligand binding domain of the chimeric receptor includes 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 includes a ligand binding domain derived from an AChR and an ion pore domain derived from a GlyR, the sequence of the β1-2 loop domain of the ligand binding domain can be derived from GlyR. In some embodiments, the ligand binding domain is derived from α7-nAChR. In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to about amino acids 64-72 or 67-70 of α7-nAChR (SEQ ID NO: 25), such as amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the inserted β1-2 loop sequence is about amino acids 79-85 of GlyRα1 (SEQ ID NO: 26), such as amino acids 80-85, 81-84, 79-85, or 81-84 of GlyRα1, with up to 3, up to 2, up to 1 amino acid mutations, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα2 and the inserted β1-2 loop corresponds to about amino acids 86-91 of GlyRα2 (SEQ ID NO: 27), with up to 3, up to 2, up to 1 amino acid mutations, or no amino acid mutations.In some embodiments, the ion pore domain is derived from GlyRα3 and the inserted β1-2 loop corresponds to about amino acids 85-90 of GlyRα3 (SEQ ID NO: 28 or 29), with up to 3, up to 2, up to 1, or no amino acid mutations. In some embodiments, the mutations are amino acid substitutions.

[0404] In some embodiments, the present disclosure provides a chimeric LGIC receptor, the chimeric LGIC receptor comprising a ligand binding domain derived from a human α7-nAChR and an ion pore domain derived from a human glycine receptor, wherein the ligand binding domain comprises one or more amino acid substitutions of the present disclosure. In some embodiments, the human glycine receptor is a human glycine receptor α1, a human glycine receptor α2, or a human glycine receptor α3. In some embodiments, the ligand binding domain comprises a Cys loop domain derived from a human glycine receptor. In some embodiments, the ligand binding domain comprises a β1-2 loop domain derived from a human glycine receptor.

[0405] Non-limiting examples of chimeric LGIC receptor sequences disclosed herein include the sequences disclosed herein as SEQ ID NOs: 30-31 and 33. In some embodiments, the chimeric LGIC receptor has 85% or greater sequence identity to the sequences provided herein as SEQ ID NOs: 30-31 and 33, and has 90% or greater, 93% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity to the sequences provided herein as SEQ ID NOs: 30-31 and 33. In the sequences, the signal peptide is indicated in italics, the ligand binding domain is indicated in bold, and the ion pore domain is indicated in underline.

[0406] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, which includes a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold), wherein the ligand binding domain includes a GlyRα1Cys loop sequence (lowercase letters); which is fused to a human GlyRα1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor includes 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:

[0407]

[0408] (SEQ ID NO:33, encoded by SEQ ID NO:32).

[0409] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera, which includes a human α7-nAChR signal peptide (italics) and a ligand binding domain (bold), wherein the ligand binding domain includes a GlyRα1β1-2 loop sequence (lowercase letters) and a Cys loop sequence (lowercase letters); fused to a human GlyRα1 ion pore domain (underlined):

[0410]

[0411] (SEQ ID NO:30).

[0412] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 junction) comprising the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined):

[0413]

[0414] Amino acid mutations in ligand-gated ion channels

[0415] As discussed above, in some aspects, the subject engineered receptor comprises at least one amino acid mutation that changes the potency of the ligand to the engineered receptor relative to the potency of the ligand to the unmutated parent receptor. In other words, one or more amino acid mutations, such as loss-of-function mutations or gain-of-function mutations, shift the potency of the engineered receptor to the ligand relative to the potency of the unmutated parent receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, one or more mutations are located in the ligand binding domain of the engineered receptor. In some embodiments, when the ligand binding domain of the engineered receptor is a Cys loop receptor protein, one or more amino acid mutations are substitutions at residues corresponding to residues selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213, and Y217 corresponding to α7-nAChR (SEQ ID NO: 25). 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 selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188 of α7-nAChR (SEQ ID NO: 25). In certain embodiments, one or more substitutions are within the α7-nAChR sequence.

[0416] In some embodiments, one or more substitutions reduce the potency of the engineered receptor for acetylcholine and a non-natural ligand, e.g., by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In certain embodiments, one or more substitutions are substitutions corresponding to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of α7-nAChR. In other cases, one or more substitutions selectively reduce the efficacy of acetylcholine on the engineered receptor. In other words, the one or more substitutions reduce the effectiveness of the engineered receptor to acetylcholine while substantially maintaining the effectiveness of the non-natural ligand, or otherwise, the one or more substitutions reduce the effectiveness of the engineered receptor to acetylcholine by 2 times or more than the effectiveness of the engineered receptor to the non-natural ligand, for example 3 times, 4 times, 5 times or more, in some cases 10 times, 20 times, 50 times or 100 times or more. In some embodiments, the substitution corresponds to L131E, L131S, L131T, L131D or S172D of α7-nAChR. In other embodiments, the one or more substitutions selectively reduce the effectiveness of the non-natural ligand on the engineered receptor. In other words, the one or more substitutions reduce the effectiveness of the engineered receptor to the non-natural ligand while substantially maintaining the effectiveness of acetylcholine, or otherwise, the one or more substitutions reduce the effectiveness of the engineered receptor to the non-natural ligand by 2 times or more than the effectiveness of the engineered receptor to acetylcholine, for example 3 times, 5 times or more, in some cases 10 times, 20 times or 50 times or more. In some embodiments, the substitution corresponds to W77M, Y115W, S172T or S172C of α7-nAChR. In certain embodiments, one or more substitutions are located within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126 and Facinicline / RG3487.

[0417] In some embodiments, one or more substitutions increase the effectiveness of engineered receptors to acetylcholine and / or non-natural ligands, for example, by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In 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 effectiveness of both acetylcholine and non-natural ligands. In some embodiments, the substitution corresponds to L131N, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q or S188T of α7-nACh. In other cases, one or more substitutions selectively increase the effectiveness of acetylcholine on engineered receptors. In other words, the one or more substitutions increase the effectiveness of engineered receptors to acetylcholine, more than 2 times or more, for example 3 times, 4 times or 5 times or more, in some cases 10 times, 20 times, 50 times or 100 times the effectiveness of engineered receptors to non-natural ligands. In some embodiments, the substitution corresponds to L141W, S172T, S172C, S188P or S188W of α7-nAChR. In certain embodiments, one or more substitutions are located within 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 embodiments, the one or more substitutions selectively increase the potency of the non-natural ligand on the engineered receptor. In other words, the one or more substitutions increase the potency of the engineered receptor to the non-natural ligand, more than 2 times or more, for example 3 times, 5 times or more, in some cases 10 times, 20 times, 50 times or more than it increases the potency of the engineered receptor to acetylcholine.

[0418] In some embodiments, the amino acid residue mutated in the subject engineered receptor is not the amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of the wild-type a7nAChR (SEQ ID NO: 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 the wild-type a7nAChR (SEQ ID NO: 25). In some embodiments, the substitution does not correspond to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution is a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, when such a substitution is present in an engineered receptor, it is present in combination with one or more of the amino acid mutations described herein.

[0419] In some embodiments, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 25) mediate the binding of the natural ligand acetylcholine. In some embodiments, mutations at these residues can reduce the binding of acetylcholine and are therefore considered to be loss-of-function mutations. In some embodiments, residues W77, Y115, N129, V130, L131, Q139, L141, S170, Y210, C212, C213, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand AZD0328 to this receptor, and mutations of these residues can increase the affinity of AZD0328 and / or other ligands for this receptor and are therefore considered to be gain-of-function mutations. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues of the ligand binding domain of a chimeric receptor comprising the ligand binding domain of α7-nAChR (SEQ ID NO: 25), 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 of the α7-nAChR (SEQ ID NO: 25) or a chimeric receptor comprising the ligand binding domain of the α7-nAChR is a substitution at one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217.

[0420] In some embodiments, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 25) can mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues can reduce the binding of acetylcholine and / or nicotine. In some embodiments, R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand ABT126, and mutations at one or more of these residues can increase the affinity of ABT126 and / or other ligands for α7-nAChR. In some embodiments, the mutation is an amino acid substitution. In some embodiments, R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand TC6987, and mutation of one or more of these residues can increase the affinity of TC6987 and / or other ligands for α7-nAChR. In some embodiments, R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of α7-nAChR can mediate the binding of the non-natural ligand Facinicline / RG3487, and mutation of one or more of these residues can increase the affinity of Facinicline / RG3487 and / or other ligands for α7-nAChR. In some embodiments, the subject engineered receptor comprises a mutation in one or more amino acid residues of the ligand binding domain of a chimeric receptor comprising the ligand binding domain 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, the one or more amino acid residues alter the binding of acetylcholine and / or nicotine to an α7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212, and Y217 of an α7-nAChR. In certain such embodiments, the amino acid is selected from the group consisting of C212 and S170. In some embodiments, mutations in one or more amino acid residues alter the binding of ABT126 to α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR.In certain such embodiments, the amino acid is selected from R101, S188 and Y210. In some embodiments, the mutation in the one or more amino acid residues alters the binding of TC6987 to a7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210 and C213. In some embodiments, mutations in one or more amino acid residues alter the binding of Facinicline / RG3487 to α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of α7-nAChR. In certain such embodiments, the amino acids are selected from Y210, R101, and N129.

[0421] 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 includes 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 includes an ion pore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is a human glycine receptor α1, a human glycine receptor α2, or a human glycine receptor α3. In some embodiments, the ligand binding domain of the engineered receptor includes a Cys ring domain derived from a human glycine receptor. In some embodiments, the parent receptor includes an amino acid sequence of SEQ ID NO: 33. In some embodiments, compared to a parent receptor including an amino acid sequence of SEQ ID NO: 33, the engineered receptor includes two amino acid substitutions. In some embodiments, the ligand binding domain of the engineered receptor includes a β1-2 ring domain from a human glycine receptor α1 subunit.

[0422] 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).

[0423] In some embodiments, the two amino acid substitutions are located 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 includes 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 includes an amino acid substitution at residue L131 and an amino acid substitution at S172D. In some embodiments, the ligand binding domain includes an amino acid substitution at residue L131 and an amino acid substitution at Y115D. In some embodiments, the ligand binding domain includes 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 the amino acid substitution of L131E.

[0424] In some embodiments, the ligand binding domain includes one or more amino acid substitutions at an amino acid residue selected from the group consisting of Y140, R101, L131, Y115 and Y210, wherein the amino acid residue corresponds to an amino acid residue of α7-nAChR. In some embodiments, the ligand binding domain includes R101W and / or Y210V amino acid substitutions. In some embodiments, the ligand binding domain includes two or more amino acid substitutions at an amino acid residue selected from the group consisting of R101, L131, Y115, Y210 and Y140. In some embodiments, the ligand binding domain includes two amino acid substitutions at an amino acid residue selected from the group consisting of R101, L131, Y115, Y210 and Y140. In some embodiments, the ligand binding domain includes two amino acid substitutions at a pair of amino acid residues selected from the group consisting of: R101 and L131, Y115 and Y210, R101 and Y210. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of R101F and L131G, R101F and L131D, Y115E and Y210W, R101W and Y210V, R101F and Y210V, R101F and Y210F, R101M and L131A, and R101M and L131F. In some embodiments, the ligand binding domain comprises three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand binding domain comprises amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0425] In some embodiments, the ligand binding domain includes an amino acid substitution at residue L131 and an amino acid substitution of R101F or R101M. In some embodiments, the amino acid substitution at residue L131 is L131G, L131D, L131A, L131F, or L131N.

[0426] In some embodiments, the ligand binding domain includes 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.

[0427] Those skilled in the art will readily identify suitable control receptors for comparison with the engineered receptors of the present disclosure. In some embodiments, the control receptor is identical to the engineered receptor in sequence except for one or more distinguished amino acid mutations (e.g., substitutions). In all cases, reference to the control receptor is intended to indicate that the variation in the enumerated characteristics (e.g., effectiveness to the ligand) is the result of one or more amino acid mutations of the engineered receptor of the present disclosure.

[0428] The present disclosure provides engineered receptors, wherein the engineered receptor is a chimeric ligand-gated ion channel (LGIC) receptor and includes: (a) a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and including a Cys ring domain from a human glycine receptor α1 subunit; and (b) an ion pore domain derived from a human glycine receptor α1 subunit. In some embodiments, the engineered receptor is derived from a parent engineered receptor comprising or consisting of an amino acid sequence of SEQ ID NO: 33, and further includes one or more amino acid substitutions based on the parent engineered receptor.

[0429] In some embodiments, the potency of engineered receptors to acetylcholine is lower than that of human α7 nicotinic acetylcholine receptors (α7-nAChR) to acetylcholine. In some embodiments, the potency of engineered receptors to acetylcholine is at least about 1.5 times lower than that of human α7 nicotinic acetylcholine receptors (α7-nAChR) to acetylcholine (e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, based on the cell reporter assay using YFP fluorescence quenching, the potency of engineered receptors to acetylcholine is evaluated by the EC50 of engineered receptors to acetylcholine. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 100uM, at least 200uM, at least 300uM, at least 500uM, at least 700uM, at least 1mM, at least 2mM, at least 3mM, at least 4mM, at least 5mM, at least 6mM, at least 7mM, at least 8mM, at least 9mM or at least 10mM. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 1mM. In some embodiments, the EC50 of the engineered receptor to acetylcholine is at least 3mM. In some embodiments, having a higher EC50 for acetylcholine allows the engineered receptor to have a higher expression level in the cell or on the cell surface, and there will not be a large amount of current flowing into the cell in the presence of physiological concentrations of acetylcholine.

[0430] In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is roughly the same as that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands. In some embodiments, the effectiveness of the engineered receptor to non-natural ligands is at least about 1.5 times higher than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) to non-natural ligands (e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, determining the potency comprises 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.

[0431] In some embodiments, the efficacy of engineered receptors in the presence of non-natural ligands is higher than the efficacy of human α7 nicotinic acetylcholine receptors (α7-nAChR) in the presence of non-natural ligands. In some embodiments, the efficacy of engineered receptors in the presence of non-natural ligands is at least about 1.5 times higher than the efficacy of human α7 nicotinic acetylcholine receptors (α7-nAChR) in the presence of non-natural ligands (e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times or about 100 times, including all sub-ranges and values ​​therebetween). In some embodiments, determining efficacy includes determining the amount of current passing through engineered receptors in vitro in the presence of non-natural ligands.

[0432] In some aspects, the subject ligand-gated ion channel includes one or more non-desensitizing mutations. In some embodiments, the mutation is an amino acid substitution. When used in the context of a ligand-gated ion channel, "desensitization" refers to the gradual reduction of ion fluxes in the long-term presence of an agonist. This causes the neuron to gradually lose its effectiveness to the ligand. Non-desensitizing mutations refer to amino acid mutations that prevent LGIC from becoming desensitized to the ligand, thereby preventing neurons from becoming reduced or unresponsive to the ligand response. Non-desensitizing mutations can be easily identified by introducing LGICs carrying mutations into neurons and analyzing the current fluxes that vary over time during long-term exposure to the ligand. If the LGIC does not include a non-desensitizing mutation, the current will recover from the peak to a steady state during long-term exposure, and if the LGIC includes a non-desensitizing mutation, the current will remain at the peak flux during exposure to the ligand. Exemplary amino acid mutations that cause desensitization include the V322L mutation in human GlyRα1 (V294L post-processing of the original protein to remove the signal peptide). 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.

[0433] In some aspects, the subject ligand-gated ion channel includes one or more conversion mutations. In some embodiments, the mutation is an amino acid substitution. A conversion mutation refers to a mutation that changes the permeability of the ion pore domain of the LGIC, allowing the conductance of non-natural ions. A conversion mutation refers to a mutation that changes the permeability of the ion pore domain of the LGIC so that it allows the conduction of non-natural ions (i.e., ions that are not naturally allowed to pass through). In some cases, the mutation converts the permeability from a cation to an anion, such as replacing amino acid residues 260-281 (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 34) in human α7-nAChR (CHRNA7) with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 35) or the corresponding amino acids in another cation-permeable LGIC. In some cases, the mutation converts the permeability from anion to cation, for example, substitution of amino acid residue 279 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC with glutamate (E) (as with the A293E substitution in GLRA1, which converts the LGIC from anion-permeable to calcium-permeable), 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 glutamate (E), and substitution of amino acid residue 293 of GLRA1 or the corresponding amino acid in another anion-permeable LGIC with valine (V) (as with P278A, A279E, T293V in GLRA1, which convert the LGIC from anion-permeable to cation-permeable).

[0434] By in vitro screening and verification method, other engineered receptors except those described herein can be identified.In some embodiments, the library of parent receptor mutants is generated from a limited number of parent receptors.Parent receptors can be mutated using methods known in the art, including error-prone PCR for mutation.In some embodiments, then the library of parent receptor mutants is transfected into yeast or mammalian cells, and with high-throughput screening to identify functional receptors (for example, to identify the parent receptor mutants that can be carried out in response to part signal transduction).In some embodiments, then the functional parent receptor mutants identified in this preliminary screening will be expressed in mammalian cells, and for the effectiveness of part is screened, for example, by microplate reader described herein and / or electrophysiological determination to carry out.Then it is possible to select to show the binding affinity of increase to agonist ligands or to enable the use of antagonists or regulator ligands as agonist parent receptor mutants in secondary screening, and complete further in vitro and / or in vivo verification and characterization determination. Such screening assays are known in the art, e.g., Armbruster, B Net 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, J Met al. (2009) PNAS 106, 19197–19202; Ellefson J Wet 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) ACS Synth Biol. 20; 6(1): 108-119; Talwar S et al. (2014) ACS Synth Biol. 21; 6(1): 109-115; Talwar S et al. (2013) ACS Synth Biol. 21; 6(1): 111-116; Talwar S et al. (2013) ACS Synth Biol. 21; 6(1): 112-117; Talwar S et al. (2013) ACS Synth Biol. 21; 6(1): 113-119. 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.

[0435] 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.

[0436] Table 5: Exemplary Engineered Receptors

[0437]

[0438]

[0439] Ligand

[0440] In some embodiments, the ligand of the present disclosure refers to an exogenous drug or compound (for example, an agonist, antagonist or modulator known to act as a receptor) with a known mechanism of action to mammalian cells. Such ligands may also be referred to as "binding agents". The ligand of the present disclosure may include proteins, lipids, nucleic acids and / or small molecules. In some embodiments, the ligand includes a drug or compound approved by the U.S. Food and Drug Administration (FDA) for clinical use to treat a specific disease (for example, a nervous system disease). In some embodiments, the ligand includes a drug or compound that has not been approved by the FDA for clinical use, but the drug or compound has been tested in one or more clinical trials, is currently being tested in one or more clinical trials, and / or is expected to be tested in one or more clinical trials. In some embodiments, the ligand includes a drug or compound that has not been approved by the FDA for clinical use, but is routinely used in laboratory studies. In some embodiments, the ligand is an analog of one of the above-mentioned 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.

[0441] In certain embodiments, the ligand is an analog of cilansetron, for example as depicted by one of the following compound formulas 2-7, in its R or S enantiomer:

[0442]

[0443] In some embodiments, the ligand acts as an agonist. As used herein, the term "agonist" refers to a ligand that induces a signaling response. In some embodiments, the ligand acts as an antagonist. As used herein, the term antagonist refers to a ligand that inhibits a signaling response.

[0444] In some embodiments, the ligand is AZD-0328 according to the formula:

[0445]

[0446] In some embodiments, the ligand is TC-6987 according to the formula:

[0447]

[0448] In some embodiments, the ligand is ABT-126 according to the formula:

[0449]

[0450] In some embodiments, the ligand is TC-5619 / Bradanicline according to the formula:

[0451]

[0452] In some embodiments, the ligand is TC-6683 according to the formula:

[0453]

[0454] In some embodiments, the ligand is varenicline according to the formula:

[0455]

[0456] In some embodiments, the ligand is Facinicline / RG3487 according to the formula:

[0457]

[0458] 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 includes a ligand binding domain derived from human α7-nAChR. In some embodiments, the engineered receptor includes an ion pore domain derived from a human glycine receptor. In some embodiments, the human glycine receptor is a human glycine receptor α1. In some embodiments, the engineered receptor includes a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQID NO:33 except for the mutations shown in Table 6.

[0459] Table 6: Non-limiting examples of combinations of engineered receptors and non-natural ligands

[0460]

[0461]

[0462]

[0463] Composition

[0464] The present disclosure also provides compositions (e.g., pharmaceutical compositions). In some embodiments, compositions or pharmaceutical compositions include recombinant nucleic acids, and the recombinant nucleic acids include expression cassettes of the present disclosure. In some embodiments, the expression cassettes include transgenes encoding engineered receptors (e.g., ligand-gated ion channels) of the present disclosure. In some embodiments, transgenes encode SEQ ID NO:33 or mutants thereof. In some embodiments, compositions or pharmaceutical compositions include pharmaceutically acceptable carriers, diluents, excipients and / or buffers. In some embodiments, pharmaceutically acceptable carriers, diluents, excipients and / or buffers are suitable for use in humans.

[0465] Such excipients, carriers, diluents and / or buffers (together as "pharmaceutical vehicles") include any medicaments that can be administered without excessive toxicity. It can be approved by the regulatory agency of the U.S. federal government or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals such as humans. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to mammals, the compounds and compositions of the present disclosure and pharmaceutically acceptable vehicles, excipients or diluents can be sterile. In some cases, when the compounds of the present disclosure are administered intravenously, aqueous media are used as vehicles, such as water, saline solutions, and aqueous dextrose solutions and aqueous glycerol solutions.

[0466] The pharmaceutical composition can be in the form of a capsule, tablet, pill, granule, lozenge, powder, granule, syrup, elixir, solution, suspension, emulsion, suppository or its sustained release formulation, or any other form suitable for application to a mammal. In some cases, the pharmaceutical composition is formulated for use as a pharmaceutical composition suitable for oral or intravenous administration to a human according to conventional procedures. Examples of suitable pharmaceutical vehicles and methods of preparing them are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th edition, 1995, Chapters 86, 87, 88, 91 and 92, which are incorporated herein by reference.

[0467] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may be present in such vehicles. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. 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) AK Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[0468] In some embodiments, the pharmaceutical composition comprises a liquid comprising a recombinant nucleic acid in a solution, a 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.

[0469] 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 promoters or delay agents that are compatible with drug administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically 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) may also be incorporated into the composition.

[0470] In some embodiments, the pharmaceutical composition is formulated to be compatible with a specific route of administration or delivery. Thus, the pharmaceutical composition includes carriers, diluents and / or excipients suitable for administration by various routes.

[0471] The choice of excipient will depend in part on the particular carrier, as well as the particular method used to administer the composition.Thus, there are a variety of suitable formulations of the pharmaceutical compositions of the present disclosure.

[0472] For example, the carrier can be formulated into an injectable preparation by dissolving, suspending or emulsifying the carrier in an aqueous or non-aqueous solvent (such as vegetable oil or other similar oils, synthetic fatty acid glycerides, high-carbon fatty acid esters or propylene glycol); and if desired, with conventional additives (such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives).

[0473] Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound. The preparations are generally sterile and may be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal oils, vegetable oils or synthetic oils.

[0474] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents include hydroxyl or other polar groups, such as alcohols, such as isopropanol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants, such as soy lecithin and oleic acid; sorbitan esters, such as sorbitan trioleate; and polyvinyl pyrrolidone.

[0475] In some embodiments, the pharmaceutical composition of the present disclosure is suitable for parenteral administration, such as aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The preparation may be presented in unit doses or multi-dose sealed containers (such as ampoules and vials), and may be stored under freeze drying (lyophilization), requiring only the addition of sterile liquid excipients (e.g., water) for injection immediately before use. Extemporaneous injection solutions and suspensions may be prepared by sterile powders, granules, and tablets of the aforementioned types.

[0476] As another example, the compound can be formulated into a formulation suitable for oral administration, including (a) a liquid solution, such as an effective amount of the compound dissolved in a diluent (such as water or saline); (b) a capsule, sachet or tablet, each of which contains a predetermined amount of the active ingredient as a solid or granules; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. The tablet form can include one or more of the following: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, cross-linked carboxymethyl cellulose sodium, talc, magnesium stearate, stearic acid and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents and pharmacologically compatible excipients. Lozenge forms may include the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, as well as lozenges, emulsions, gels and the like comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, containing, in addition to the active ingredient, such excipients as described herein.

[0477] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the present disclosure in combination with a pharmaceutically acceptable diluent, carrier or vehicle, the predetermined amount being calculated to be sufficient to produce the desired effect. The specifications of the novel unit dosage forms of the present disclosure depend on the specific compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0478] method

[0479] In some aspects of the present disclosure, the compositions and methods disclosed herein can be used to treat a neurological disease or disorder in a subject in need thereof. 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 to measure. 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.

[0480] In some aspects of the present disclosure, a method for treating a neurological disease or condition in a subject is provided, the method 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 hippocampal neurons. In some embodiments, the recombinant nucleic acid is delivered to dorsal root ganglion neurons or trigeminal ganglion neurons. 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 administering a small molecule ligand / drug / agonist that activates the engineered LGIC, which in turn regulates the activity of the target neurons, and thereby treats the subject's neurological disease or condition (e.g., pain or epilepsy).

[0481] In some aspects of the present disclosure, a method for treating focal epilepsy in a subject is provided, the method comprising administering to the subject an effective amount of a recombinant nucleic acid comprising an expression cassette, wherein the expression cassette comprises a transgene encoding an engineered LGIC receptor of the present disclosure. In some embodiments, the recombinant nucleic acid is delivered to hippocampal neurons. In some embodiments, the method further comprises administering a small molecule ligand that activates the engineered LGIC, which in turn modulates the activity of target neurons, thereby treating focal epilepsy in the subject.

[0482] In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to prepare drugs for treating neurological diseases or disorders. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to prepare drugs for treating neurological diseases or disorders associated with hippocampal neuron disorders, such as focal epilepsy, schizophrenia, autism spectrum disorders, Alzheimer's disease, Rett syndrome, and fragile X syndrome. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat focal epilepsy. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat schizophrenia. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat autism spectrum disorders. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat Alzheimer's disease. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat Rett syndrome. In some aspects, the vectors, recombinant nucleic acids, or compositions disclosed herein are used to treat fragile X syndrome.

[0483] This disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating epilepsy in a subject. In some embodiments, the epilepsy is focal epilepsy. In some embodiments, the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0484] In some embodiments, the compositions and methods herein can be used to reduce the level of epileptic seizures in a subject. In some embodiments, the compositions and methods herein can be used to prevent or control the level of epileptic seizures in a subject. Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonic, absence, or atonic seizures.

[0485] In some embodiments, the compositions and methods herein may reduce the number or frequency of seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%, including all ranges and subranges therebetween. In some embodiments, the compositions and methods herein may reduce the number or frequency of seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, including all ranges and subranges therebetween.

[0486] In some embodiments, the compositions and methods herein may reduce the level and / or duration of seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%, including all ranges and subranges therebetween. In some embodiments, the compositions and methods herein may reduce the level and / or duration of seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, including all ranges and subranges therebetween.

[0487] 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 the use of an expression cassette comprising a transgene encoding an engineered LGIC disclosed herein to modulate neuronal activity to alleviate epilepsy in a subject.

[0488] In various embodiments, a vector and / or 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 that control epilepsy. In the presence of a ligand, the neuronal cells expressing the engineered receptor are activated and reduce sensitivity to epilepsy.

[0489] In some embodiments, the epilepsy is focal epilepsy (focal epileptic seizures). Focal epilepsy is a neurological condition in which the primary symptom is recurrent seizures affecting one hemisphere (half) of the brain.

[0490] Focal seizures are divided into four categories: (a) focal awareness-preserving seizures, in which the subject is aware of what is happening during the seizure; (b) focal impaired awareness seizures, in which the subject is confused or unaware of what is happening during the seizure or does not remember the seizure; (c) focal motor seizures, in which the subject moves to some degree - from twitching, spasming, to rubbing hands together to walking around; and (d) focal non-motor seizures, in which the subject does not twitch or make other movements during the seizure. Instead, it causes a change in the way the subject feels or thinks (e.g., feeling strong emotions, strange sensations, or symptoms such as a racing heart, goose bumps, or feeling hot or cold).

[0491] Focal epilepsy is characterized by seizures arising from a specific part (lobe) of the brain. Focal epilepsy includes idiopathic location-related epilepsy (ILRE), frontal lobe epilepsy, temporal lobe epilepsy, parietal lobe epilepsy, and occipital lobe epilepsy.

[0492] Idiopathic position-related epilepsy (ILRE) is caused by unknown factors.

[0493] Frontal lobe epilepsy is a term for recurring seizures that start in the frontal lobe (the area of ​​the brain behind the forehead).

[0494] Temporal lobe epilepsy is a term for recurrent seizures that begin in the temporal lobes (parts of the brain on both sides of the head behind the temples and cheekbones). The temporal lobe is the area in the brain that most often causes seizures. The mesial part (middle) of the two temporal lobes is very important in epilepsy - it is often the source of seizures and is easily damaged or scarred. Medial temporal lobe epilepsy (mTLE) is the most common form of human epilepsy. Many times, its pathophysiological basis is hippocampal sclerosis. Therefore, one strategy for treating focal epilepsy (such as mTLE) is to target hippocampal neurons.

[0495] Parietal lobe epilepsy. The parietal lobe is the part of the brain that is on the top and sides of the head. The parietal lobe is called the "association cortex" and is responsible for associating meaning with brain functions. It is here that the brain creates visual images, sounds are recognized as language, and touch sensations are associated with specific objects. In some ways, the parietal lobe is where perception coincides with physical reality.

[0496] Occipital lobe epilepsy. Occipital lobe epilepsy is a term for recurring seizures that begin in the occipital lobe (the part of the brain at the back of the head that is primarily responsible for vision).

[0497] In some aspects of the present disclosure, a method for treating neuropathic pain (e.g., peripheral neuropathy and trigeminal neuralgia) in a subject is provided, the method comprising administering to the subject an effective amount of a recombinant nucleic acid, wherein the recombinant nucleic acid comprises an expression cassette comprising a transgene encoding an engineered LGIC receptor of the present disclosure. In some embodiments, the recombinant nucleic acid is delivered to DRG or TGG neurons. In some embodiments, the method further comprises administering a small molecule ligand that activates the engineered LGIC, which in turn regulates the activity of the target neurons, and thereby treats the subject's neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia.

[0498] In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to prepare medicines for treating nervous system diseases or conditions. In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to prepare medicines for treating neurological diseases or conditions (such as neuropathic pain) associated with DRG / TGG neuron disorders. In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to prepare medicines for treating spinal cord related diseases such as spasms, spinal cord injuries and avulsions. In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to treat spasms. In certain embodiments, treating spasms includes transducing neurons in the spine. In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to treat spinal cord injuries. In some aspects, the vectors, recombinant nucleic acids or compositions disclosed herein are used to treat avulsions. In some embodiments, treating spinal cord injuries or avulsions includes transducing neurons in the dorsal horn.

[0499] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing or treating pain in a subject. In some embodiments, pain is neuropathic pain. "Pain" refers to an uncomfortable feeling and / or unpleasant sensation in a subject. The range of pain sensations can be from mild and occasional to severe and continuous. Pain can be divided into acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain or psychogenic pain. In some cases, pain is caused by or associated with a disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include: neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, carpal tunnel syndrome, central pain after stroke, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, pain associated with Parkinson's disease, epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and nociceptive pain, including pain associated with central nervous system trauma, strains / sprains, and burns; myocardial infarction, acute pancreatitis, postoperative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0500] The compositions and methods herein can be used to alleviate the pain level of a subject. In some cases, the pain level of a subject is alleviated 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 99% or about 100%, including all ranges and sub-ranges therebetween. The pain level of a subject can be assessed by a variety of methods. In some cases, the pain level is assessed by self-report (i.e., the pain level that a human subject verbally reports he / she is experiencing). In some cases, the pain level is assessed by the behavioral indicators of pain, such as facial expressions, limb movements, vocalization, agitation and defense. For example, when a subject cannot self-report (e.g., infants, unconscious subjects, non-human subjects), these types of assessments may be useful. Pain levels can be assessed following treatment with a composition of the present disclosure as compared to the level of pain the subject was experiencing prior to treatment with the composition.

[0501] 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 modulating neuronal activity using vectors and / or recombinant nucleic acids encoding the engineered LGIC disclosed herein to relieve pain in a subject.

[0502] 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) that reduce pain sensation. In the presence of a ligand, the neuronal cells expressing the engineered receptor are activated and reduce sensitivity to pain, thereby enhancing the analgesic effect of stimulating these neuronal cells.

[0503] 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 increase pain sensation or sensitivity to pain (e.g., nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc.). In the presence of a ligand, the neuronal cell expressing the engineered receptor is inactivated and sensitivity to pain is reduced, and analgesia is enhanced.

[0504] In some embodiments, the compositions and methods of the present disclosure are effective in alleviating pain. Illustrative examples of pain suitable for treatment with 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, burns, back pain, eye pain, visceral pain, cancer pain (e.g., bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, postherpetic neuralgia, postoperative pain, poststroke pain, and menstrual pain.

[0505] Pain can be classified as acute or chronic. "Acute pain" refers to pain that starts suddenly and is usually sharp in degree. Acute pain may be mild and last only a few moments, or it may be severe and last for weeks or months. In most cases, acute pain does not last more than three months, and it disappears when the underlying cause of the pain has been treated or has healed. However, unrelieved acute pain may lead to chronic pain. "Chronic pain" refers to persistent or recurring pain that lasts longer than the common course of an acute illness or injury or lasts more than three to six months, and which adversely affects the individual's health status. In some embodiments, the term "chronic pain" refers to pain that persists when it should not. Chronic pain can be nociceptive pain or neuropathic pain.

[0506] In some embodiments, the development of pain is expected or anticipated to be associated with or caused by injury, infection, or medical intervention. In some embodiments, infection causes nerve damage. In some embodiments, medical intervention is surgery, such as surgery on the central core of the body. In some embodiments, medical intervention is surgery to remove part or all of one or more tissues, tumors, or organs in the body. In some embodiments, medical intervention is amputation. In some embodiments, the compositions and methods of the present disclosure are effective in alleviating acute pain. In some embodiments, the compositions and methods of the present disclosure are effective in alleviating chronic pain.

[0507] Clinical pain exists when discomfort and abnormal sensitivity are features of the patient's symptoms. Individuals may present with various pain symptoms. Such symptoms include: 1) spontaneous pain, which may be dull, burning or tingling; 2) excessive pain response to noxious stimuli (hyperalgesia); and 3) pain produced by usually harmless stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Although patients with various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different, and different treatment strategies may therefore be required. Therefore, pain can also be divided into many different subtypes according to different pathophysiologies, including nociceptive pain, inflammatory pain and neuropathic pain.

[0508] 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.

[0509] Nociceptive pain is induced by tissue damage or by strong stimulation that may cause damage. Moderate to severe acute nociceptive pain is the prominent feature of pain from central nervous system trauma, strain / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical operation), post-traumatic pain, renal colic, cancer pain and back pain. Cancer pain can be chronic pain, such as tumor-related pain (such as bone pain, headache, facial pain or visceral pain) or pain related to cancer therapy (such as post-chemotherapy syndrome, chronic postoperative pain syndrome or radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormone therapy or radiotherapy. Back pain may be due to disc herniation or rupture, or lumbar intervertebral facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament abnormalities. Back pain can subside naturally, but in some patients, when back pain continues for more than 12 weeks, back pain becomes a chronic condition that may make people particularly weak.

[0510] Neuropathic pain can be defined as pain that is initiated or caused by a primary lesion or functional disorder in the nervous system. Causes of neuropathic pain include, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0511] Neuropathic pain may be associated with a pain disorder, the term pain disorder refers to a disease, disorder or condition associated with or caused by pain. Illustrative examples of pain disorders include arthritis, allodynia, classic trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia of pain, causalgia, sciatica pain disorder, degenerative joint disease, fibromyalgia, visceral disease, chronic pain disorder, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis, or pain associated with cancer.

[0512] In some embodiments, the neuropathic pain is peripheral neuropathy. Peripheral neuropathy refers to the condition caused when the nerves that carry information back and forth between the brain and spinal cord and other parts of the body are damaged or sick. The range of various peripheral neuropathies is from carpal tunnel syndrome (traumatic injury common after long-term repeated use of hands and wrists such as using computers) to nerve damage related to diabetes. Generally, peripheral neuropathy can be divided into mononeuropathy and polyneuropathy. Mononeuropathy includes carpal tunnel syndrome, ulnar nerve palsy, radial nerve palsy and peroneal nerve palsy. When multiple peripheral nerves of the whole body fail at the same time, polyneuropathy will occur. Polyneuropathy may have a variety of causes, including exposure to certain toxins such as alcohol abuse, malnutrition (especially vitamin B deficiency) and complications caused by diseases such as cancer or renal failure. One of the most common forms of chronic polyneuropathy is diabetic neuropathy (a condition that occurs in diabetic patients). It is more serious for people with poor blood sugar level control. Although less common, diabetes can also cause mononeuropathy. One of the most serious polyneuropathies is Guillain-Barré syndrome, a rare disease that develops when the body's immune system attacks nerves in the body as they leave the spinal cord. Symptoms tend to come on quickly and get worse quickly, sometimes leading to paralysis. Early symptoms include weakness and tingling, which may eventually spread up the arms. More severe cases may cause blood pressure problems, heart rhythm problems, and breathing difficulties. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic form of Guillain-Barré, with symptoms that last for months or even years. Early diagnosis and treatment are crucial for people with CIDP, 30% of whom are at risk of eventually being confined to a wheelchair.

[0513] In some embodiments, the neuropathic pain is trigeminal neuralgia. Trigeminal neuralgia (TN), also known as cramps, is a chronic pain condition that affects one of the most widespread nerves in the head, the trigeminal nerve or the 5th cranial nerve. The trigeminal nerve is a group of cranial nerves in the head. It is the nerve responsible for providing sensation to the face. One trigeminal nerve extends to the right side of the head, and the other extends to the left side. Each of these nerves has three different branches. After the trigeminal nerve leaves the brain and enters the skull, the trigeminal nerve divides into three smaller branches that control sensation throughout the face: Ophthalmic nerve (V1): The first branch controls sensation in a person's eyes, upper eyelids, and forehead. Maxillary nerve (V2): The second branch controls sensation in the lower eyelids, cheeks, nostrils, upper lip, and upper gums. Mandibular nerve (V3): The third branch controls sensation in the lower jaw, lower lip, lower gums, and some muscles used for chewing.

[0514] TN is a form of neuropathic pain. The typical or "classic" form of the condition (called "type 1" or TN1) causes extreme, diffuse, sudden burning or shock-like facial pain, with each episode lasting from a few seconds to up to two minutes. These episodes can occur in rapid succession, lasting up to two hours in a row. The "atypical" form of the condition (called "type 2" or TN2) is characterized by constant pain, burning, stabbing pain, and is slightly less intense than type 1. Both forms of pain can occur in the same person, sometimes at the same time. The intensity of the pain can be physically and mentally incapacitating. TN is associated with a variety of conditions. TN can be caused by blood vessels pressing on the trigeminal nerve as it exits the brainstem. This compression causes the protective coating (myelin) around the nerve to wear away or become damaged. People with multiple sclerosis may also experience symptoms of TN, which causes the myelin sheath of the trigeminal nerve to degenerate. Rarely, symptoms of TN can be caused by compression of the nerve by a tumor or by a tangle of arteries and veins called an arteriovenous malformation. Injury to the trigeminal nerve (which may be the result of sinus surgery, oral surgery, stroke, or facial trauma) can also produce neuropathic facial pain.

[0515] The inflammatory process is a complex series of biochemical and cellular events that are activated in response to tissue damage or the presence of foreign substances, which results in swelling and pain. Arthritis pain is a common inflammatory pain.

[0516] Other types of pain suitable for treatment with the compositions and methods of the present disclosure include, but are not limited to, pain caused by musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatoid) arthropathy, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis, and pyomyositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma, and skeletal muscle ischemia; headaches, such as migraine (including migraine with aura and without aura), cluster headaches, tension-type headaches, mixed headaches, and headaches associated with vascular disorders; and maxillofacial pain, including dental pain, ear pain, burning mouth syndrome, and temporomandibular myofascial pain.

[0517] A variety of pain scales can be used to determine the effective amount of the compositions and methods of the present disclosure to reduce the amount of pain experienced by a human subject. Patient self-reports can be used to assess whether pain is reduced; see, e.g., Katz and Melzack (1999) Surg. Clin. North Am. 79: 231. Alternatively, an observational pain scale can be used. The LANSS pain scale can be used to assess whether pain is reduced; see, e.g., Schmader (2002) Clin. A visual analog pain scale can be used; see, e.g., Schmader (2002) Clin. J. Pain 18: 350. A Likert pain scale can be used; e.g., where 0 represents no pain, 5 represents moderate pain, and 10 represents the worst possible pain. Self-report pain scales for children include, e.g., the Facial Pain Scale; the Wong-Baker Facial Expression Pain Rating Scale; and the Color Analog Scale. Self-report pain scales for adults include, e.g., the Visual Analog Scale; the Verbal Numerical Rating Scale; the Verbal Description Scale; and the Brief Pain Questionnaire.Pain measurement scales include, for example, the Alder Hey Triage Pain Score (Stewart et al. (2004) Arch. Dis. Child. 89:625); Behavioral Pain Rating Scale (Payen et al. (2001) Critical Care Medicine 29:2258); Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129); Nonverbal Pain Index Checklist (Feldt (2000) Pain Manager. Nurs. 1:13); Critical Care Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); Comfort Scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); Dallas Pain Questionnaire (Ozguler et al. (2002) Spine Pain Questionnaire 27:1783); Algometer pain index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Revised Face Expression Pain Scale (Hicks et al. (2001) Pain 93:173); Expression, Leg, Activity, Crying, Soothing Scale; McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptive Discrimination Scale (Gracely and Kwilosz (1988) Pain 35:279); Numerical 1 1 Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numerical Rating Scale (Hartrick et al. (1989) Clin. J. Pain 5:153); Numerical Rating Scale (Hartrick et al. (2001) Pain 93:173); Expression, Leg, Activity, Crying, Soothing Scale (Hicks et al. (2001) Pain 93:173); McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptive Discrimination Scale (Gracely and Kwilosz (1988) Pain 35:279); Numerical 1 1 Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numerical Rating Scale (Hartrick et al. (2001) Pain 93:17 ... al. (2003) Pain Pract. 3:310); Wong-Baker Facial Expression Pain Rating Scale; and Visual Analog Scale (Huskisson (1982) J. Rheumatol. 9:768).

[0518] In some embodiments, a method for relieving pain in a subject is provided, the method 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 to control the activity of the cell by providing an effective amount of a ligand that activates the engineered receptor, thereby alleviating the pain in the subject. The method provides significant analgesia for pain without off-target effects, such as general central nervous system inhibition. 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) of neuropathic pain reduction in a subject compared to an untreated subject. In some embodiments, the method includes a step of measuring the pain of the subject before and after administering the ligand, wherein the pain of 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 a case, the measurement can be performed 4 hours or more after administering the ligand, for example, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, or 4 days or more after administering the ligand.

[0519] In some cases, the compositions and methods are used to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumor), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that can be treated by the compositions and methods herein include: avolition, agraphia, alcoholism, alexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), happy puppet syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, attention deficit hyperactivity disorder, auditory processing disorder, autism spectrum, bipolar disorder, Bell's palsy, brachial plexus injury, brain damage, brain injury, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, causalgia, central nervous system disease, cerebral infarction ... Neuralgia syndrome, central pontine myelinolysis, centronuclear myopathy, head disease, cerebral aneurysm, cerebral arteriosclerosis, brain atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cerebral spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, Coffin-Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital bilateral facial palsy, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt- Jacob's disease, strain disorder, Cushing's syndrome, cyclothymic disorder, cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, De Morse's syndrome, Dejerine-Kromke's palsy, Dejerine-Sotas disease, delayed sleep phase syndrome, dementia, dermatomyositis, developmental coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, articulation disorders, autonomic disorders, dyscalculia, dysgraphia, movement disorders, alexia, dystonia, empty sella syndrome, encephalitis, encephalocele, cerebral trigeminal nerve hemorrhage Angiomatosis, encopresis, enuresis, epilepsy, intellectual disorder of female epilepsy, Erb's palsy, erythromelalgia, head explosion syndrome, Fabry disease, Fall syndrome, syncope, familial spastic paralysis, febrile seizures, Fisher syndrome, Friedreich's ataxia, fibromyalgia, Fauville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy with febrile seizures plus, Gerstmann syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, gray matter heterotopia, Guillain-Barré syndrome, generalized anxiety disorder,HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary ataxia polyneuritis-like disease, herpes zoster oticus, herpes zoster, Hirayama syndrome, Hirschsprung disease, Holmes-Adie syndrome, holoprosencephaly, Huntington disease, hydrocephalus, hydrocephalus, hypercortisolism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmenti, infantile Refsum disease, infantile spasms, inflammatory myopathy, intracranial cysts, intracranial hypertension, iso-dicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sell syndrome, Kinsbourne syndrome, Kleine-Levin syndrome, Kleine-Levin syndrome Lippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kloefner syndrome, dorsolateral medullary (Wallenberg) syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, ablative leukoencephalopathy, dementia with Lewy bodies, lissencephaly, locked-in syndrome, lumbar disc disease, lumbar spinal stenosis, Lyme disease - neurologic sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, macrocephaly, Landing syndrome, megalencephaly with subcortical cysts, megalencephaly, May-Row syndrome, Meniere's disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, macrocephaly disease, migraine, Miller-Fe syndrome, mini-stroke (transient ischemic attack), misophonia, mitochondrial myopathy, Moebius syndrome, monolimb muscular atrophy, motor skill disorders, moyamoya disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, diffuse myelin-destructive sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neuro-Behcet's disease, neurofibromatosis, neuroleptic malignant syndrome, neurologic manifestations of AIDS, neurologic sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neurosis, Niemann- Pick's disease, non-24-hour sleep-wake disorder, non-verbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, Ohtahara syndrome, olivopontocerebellar atrophy, strabismic opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, persistent afterimages, paresthesias, Parkinson's disease, paramyotonia congenita, paraneoplastic disease, paroxysmal seizures, Parry-Romberg syndrome, PANDAS, Pelizeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, generalized developmental disorder, light sneeze reflex, phytanic acid storage disease, Pick's disease, nerve compression, pituitary tumor, PMG, polyneuropathy, poliomyelitis, polymicrogyria,Polymyositis, cerebral puncta, postpoliomyelitis syndrome, postherpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrantanopia, quadriplegia, rabies, radiculopathy, Ramsay-Hunt syndrome type I, Ramsay-Hunt syndrome type II, Ramsay-Hunt syndrome type III, Rasmussen Encephalitis, reflex neurovascular dystrophy, Refsum disease, rapid eye movement sleep behavior disorder, repetitive stress injury, restless leg syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg syndrome, St. Vitus' chorea, Sandhoff disease, Schilder disease, schizencephaly, sensory processing disorder, septo-optic dysplasia, shaken baby syndrome, herpes zoster, Shy-German syndrome, Sjögren's syndrome, sleep apnea, sleep sickness, sneezing fullness and, megalencephaly syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinobulbar muscular atrophy, spinocerebellar ataxia, split-brain, Still-Richardson-Olszewski syndrome, stiff-person syndrome, stroke, Stitch-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, chorea, syncope, synaesthesia, syringomyelia, tarsal tunnel syndrome, tardive movement disorder, tardive psychosis encephalopathy, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, head injury, tremor, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, myoclonic epilepsy (Unverricht-Lundborg disease), Hippel-Lindau disease (VHL), Viliuisk encephalomyelitis (VE), Wallenberg syndrome, West syndrome, whiplash, Williams syndrome, Wilson's disease, or Zellweger syndrome.

[0520] The subject treated by the methods and compositions disclosed herein can be a person, or a non-human animal. In some embodiments, the subject is a person. In some embodiments, the person is an adult (≥18 years old). Non-limiting examples of non-human animals include non-human primates, livestock animals, domestic pets, and experimental animals. Non-human animals can be apes (e.g., chimpanzees, baboons, gorillas, or orangutans), old world monkeys (e.g., rhesus monkeys), new world monkeys, dogs, cats, bison, camels, cattle, deer, pigs, donkeys, horses, mules, llamas, sheep, goats, buffaloes, reindeers, yaks, mice, rats, rabbits, or any other non-human animals. Compositions and methods described herein are suitable for treating veterinary animals. Veterinary animals can include, but are not limited to, dogs, cats, horses, cattle, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some aspects, contacting tissues or cell groups with compositions includes applying compositions to cell groups or subjects. In some embodiments, application is performed in vitro, such as by adding compositions to a cell culture system. In some aspects, administration is carried out in vivo, for example, by specific route administration. When more than one composition is administered, the composition can be administered at the same time (e.g., on the same day) via the same route, or at different times via the same route. Alternatively, the composition can be administered at the same time (e.g., on the same day) via different routes or at different times via different routes.

[0521] Application

[0522] The present disclosure provides dosing regimens for administering a recombinant nucleic acid comprising an expression cassette of the present disclosure or a pharmaceutical composition thereof. In some embodiments, the recombinant nucleic acid is incorporated into a vector (eg, an AAV vector) for administration.

[0523] In some embodiments, the recombinant nucleic acid is administered to or introduced into one or more neuronal cells. The neuronal cells can be of the same type, or a mixed population of different types of neuronal cells.

[0524] 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 to or introduced into one or more hippocampal neuronal cells.

[0525] In some embodiments, the neuronal cell is a nociceptor or a peripheral sensory neuron. Illustrative 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 a neuronal pain circuit. In some embodiments, the recombinant nucleic acid of the present disclosure is administered to or introduced into one or more DRG neuronal cells. In some embodiments, the recombinant nucleic acid of the present disclosure is administered to or introduced into one or more TGG neuronal cells.

[0526] The non-limiting examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglion administration, intraspinal administration, cerebellomedullary cisterna administration and intraneural administration. In some embodiments, administration may involve injection of a liquid preparation of a recombinant nucleic acid or a carrier comprising the recombinant nucleic acid. In some embodiments, administration may involve oral delivery of a solid preparation of a carrier or recombinant nucleic acid. In some embodiments, oral formulations may be administered with food. In some embodiments, the recombinant nucleic acid of the present disclosure is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneurally, intraganglionically, intraspinally or intraventricularly to a subject, to introduce the recombinant nucleic acid into one or more neuronal cells.

[0527] In some embodiments, a recombinant nucleic acid of the present disclosure is administered to neurons intracranially, intrathecally (IT), intracerebrally, intraventricularly, or by direct injection into an epileptic focus in the hippocampus.

[0528] In some embodiments, a recombinant nucleic acid of the present disclosure is administered intrathecally (IT) or intraganglionically (IG) to sensory neurons or nociceptors, such as DRG neurons, TGG neurons, and the like.

[0529] Intrathecal (IT) administration includes delivery through the spine or through the cisterna magna. The IT route delivers the recombinant nucleic acid to the cerebrospinal fluid (CSF). This route of administration can be suitable for treatment, such as chronic pain or other peripheral nervous system (PNS) or central nervous system (CNS) indications. In animals, IT administration is achieved by inserting an IT catheter into the cisterna magna and advancing its tail to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a conventional bedside procedure with excellent safety.

[0530] In some embodiments, the recombinant nucleic acid of the present disclosure is administered to a subject by intraganglionic administration. Intraganglionic administration may involve direct injection into one or more ganglia. The IG approach may deliver the recombinant nucleic acid directly to the DRG or TGG substance. In some embodiments, IG is administered to the DRG by open neurosurgery. In some embodiments, open neurosurgery is invasive and undesirable in humans. In some embodiments, minimally invasive, CT imaging-guided techniques may be used to safely target the DRG, for example, for human subjects. A custom needle assembly for convection-enhanced delivery (CED) may be used to deliver the recombinant nucleic acid to the DRG substance. In a non-limiting example, the recombinant nucleic acid of the present disclosure may be delivered to one or more dorsal root ganglia and / or trigeminal ganglia to treat chronic pain. In another non-limiting example, the recombinant nucleic acid of the present disclosure may be delivered to the nodose ganglion (vagus nerve) to treat epilepsy.

[0531] In some embodiments, the recombinant nucleic acid of the present invention is administered to a subject by intracranial administration (i.e., directly into the brain). In the non-limiting example of intracranial administration, the recombinant nucleic acid of the present invention can be delivered to the cerebral cortex to treat, for example, epileptic seizure lesions, delivered to the paraventricular hypothalamus to treat, for example, satiety disorders, or delivered to the central nucleus of the amygdala to treat, for example, satiety disorders. In some embodiments, the recombinant nucleic acid can be administered to a subject by intraneural injection (i.e., directly into the nerve). Nerves can be selected based on the indication to be treated, for example, injected into the sciatic nerve to treat chronic pain or injected into the vagus nerve to treat epilepsy or satiety disorders. In another specific case, the recombinant nucleic acid can be administered to a subject by injection, for example, injected into sensory nerve endings to treat chronic pain. In some embodiments, the recombinant nucleic acid of the present invention is administered to a subject by direct injection into the epileptic lesions in the hippocampus.

[0532] The dosage may vary and depends on whether the treatment is preventive or therapeutic, the type, initiation, progression, severity, frequency, duration or probability of the disease treatment involved, the desired clinical endpoint, previous or concurrent treatment, the general health, age, sex, race or immune competence of the subject, and other factors that the skilled artisan will understand. The dosage, amount, frequency or duration may be proportionally increased or decreased as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the state of the subject. The skilled artisan will understand the factors that may affect the dosage and time required to provide an amount sufficient to provide a therapeutic or preventive benefit.

[0533] 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 the subject. The size of a single vector genome generally depends on the type of viral vector used. The vector genome of the present disclosure may be about 1.0 kilobases, 1.5 kilobases, 2.0 kilobases, 2.5 kilobases, 3.0 kilobases, 3.5 kilobases, 4.0 kilobases, 4.5 kilobases to 5.0 kilobases, or more than 5.0 kilobases, including all ranges and subranges therebetween.

[0534] In some embodiments, the dose of the vector administered has a value of about 1 x 10 6 , about 2x10 6 , about 3x10 6 , about 4x10 6 , about 5x10 6 , about 6x10 6 , about 7x10 6 , about 8x10 6 , about 9x10 6 , about 1x10 7 , about 2x10 7 , about 3x10 7 , about 4x10 7 , about 5x10 7 , about 6x10 7 , about 7x10 7 , about 8x10 7 , about 9x10 7 , about 1x10 8 , about 2x10 8 , about 3x10 8 , about 4x10 8 , about 5x10 8 , about 6x10 8 , about 7x10 8 , about 8x10 8 , about 9x10 8 , about 1x10 9 , about 2x10 9 , about 3x10 9 , about 4x10 9 , about 5x10 9 , about 6x10 9 , about 7x10 9 , about 8x10 9 , about 9x10 9 , about 1x10 10 , about 2x10 10 , about 3x10 10 , about 4x10 10 , about 5x1010 , about 6x10 10 , about 7x10 10 , about 8x10 10 , about 9x10 10 , about 1x10 11 , about 2x10 11 , about 3x10 11 , about 4x10 11 , about 5x10 11 , about 6x10 11 , about 7x10 11 , about 8x10 11 , about 9x10 11 , about 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , about 7x10 12 , about 8x10 12 , about 9x10 12 , about 1x10 13 , about 2x10 13 , about 3x10 13 , about 4x10 13 , about 5x10 13 , about 6x10 13 , about 7x10 13 , about 8x10 13 , about 9x10 13 , about 1x10 14 , about 2x10 14 , about 3x10 14 , about 4x10 14 , about 5x10 14 , about 6x10 14 , about 7x10 14 , about 8x10 14 , about 9x10 14 , about 1x10 15 , about 2x10 15 , about 3x10 15 , about 4x10 15 , about 5x10 15 , about 6x10 15 , about 7x10 15 , about 8x10 15 , about 9x10 15 , about 1x10 16 , about 2x10 16 , about 3x10 16, about 4x10 16 , about 5x10 16 , about 6x10 16 , about 7x10 16 , about 8x10 16 , about 9x10 16 , about 1x10 17 or more vector genome units, including all ranges and subranges therebetween.

[0535] In some embodiments, the dose of vector administered has a value of at least 1×10 6 , at least 2x10 6 , at least 3x10 6 , at least 4x10 6 , at least 5x10 6 , at least 6x10 6 , at least 7x10 6 , at least 8x10 6 , at least 9x10 6 , at least 1x10 7 , at least 2x10 7 , at least 3x10 7 , at least 4x10 7 , at least 5x10 7 , at least 6x10 7 , at least 7x10 7 , at least 8x10 7 , at least 9x10 7 , at least 1x10 8 , at least 2x10 8 , at least 3x10 8 , at least 4x10 8 , at least 5x10 8 , at least 6x10 8 , at least 7x10 8 , at least 8x10 8 , at least 9x10 8 , at least 1x10 9 , at least 2x10 9 , at least 3x10 9 , at least 4x10 9 , at least 5x10 9 , at least 6x10 9 , at least 7x10 9 , at least 8x10 9 , at least 9x10 9 , at least 1x10 10 , at least 2x10 10 , at least 3x10 10 , at least 4x10 10, at least 5x10 10 , at least 6x10 10 , at least 7x10 10 , at least 8x10 10 , at least 9x10 10 , at least 1x10 11 , at least 2x10 11 , at least 3x10 11 , at least 4x10 11 , at least 5x10 11 , at least 6x10 11 , at least 7x10 11 , at least 8x10 11 , at least 9x10 11 , at least 1x10 12 , at least 2x10 12 , at least 3x10 12 , at least 4x10 12 , at least 5x10 12 , at least 6x10 12 , at least 7x10 12 , at least 8x10 12 , at least 9x10 12 , at least 1x10 13 , at least 2x10 13 , at least 3x10 13 , at least 4x10 13 , at least 5x10 13 , at least 6x10 13 , at least 7x10 13 , at least 8x10 13 , at least 9x10 13 , at least 1x10 14 , at least 2x10 14 , at least 3x10 14 , at least 4x10 14 , at least 5x10 14 , at least 6x10 14 , at least 7x10 14 , at least 8x10 14 , at least 9x10 14 , at least 1x10 15 , at least 2x10 15 , at least 3x10 15 , at least 4x10 15 , at least 5x10 15 , at least 6x10 15 , at least 7x10 15 , at least 8x10 15 , at least 9x10 15, at least 1x10 16 , at least 2x10 16 , at least 3x10 16 , at least 4x10 16 , at least 5x10 16 , at least 6x10 16 , at least 7x10 16 , at least 8x10 16 , at least 9x10 16 or at least 1x10 17 vector genome units, including all ranges and subranges therebetween.

[0536] In some embodiments, the dose of vector administered has no more than 1 x 10 6 , not more than 2x10 6 , not more than 3x10 6 , not more than 4x10 6 , no more than 5x10 6 , not more than 6x10 6 , no more than 7x10 6 , no more than 8x10 6 , not more than 9x10 6 , not more than 1x10 7 , not more than 2x10 7 , not more than 3x10 7 , not more than 4x10 7 , no more than 5x10 7 , not more than 6x10 7 , no more than 7x10 7 , no more than 8x10 7 , not more than 9x10 7 , not more than 1x10 8 , not more than 2x10 8 , not more than 3x10 8 , not more than 4x10 8 , no more than 5x10 8 , not more than 6x10 8 , no more than 7x10 8 , no more than 8x10 8 , not more than 9x10 8 , not more than 1x10 9 , not more than 2x10 9 , not more than 3x10 9 , not more than 4x10 9 , no more than 5x10 9 , not more than 6x10 9 , no more than 7x10 9 , no more than 8x10 9, not more than 9x10 9 , not more than 1x10 10 , not more than 2x10 10 , not more than 3x10 10 , not more than 4x10 10 , no more than 5x10 10 , not more than 6x10 10 , no more than 7x10 10 , no more than 8x10 10 , not more than 9x10 10 , not more than 1x10 11 , not more than 2x10 11 , not more than 3x10 11 , not more than 4x10 11 , no more than 5x10 11 , not more than 6x10 11 , no more than 7x10 11 , no more than 8x10 11 , not more than 9x10 11 , not more than 1x10 12 , not more than 2x10 12 , not more than 3x10 12 , not more than 4x10 12 , no more than 5x10 12 , not more than 6x10 12 , no more than 7x10 12 , no more than 8x10 12 , not more than 9x10 12 , not more than 1x10 13 , not more than 2x10 13 , not more than 3x10 13 , not more than 4x10 13 , no more than 5x10 13 , not more than 6x10 13 , no more than 7x10 13 , no more than 8x10 13 , not more than 9x10 13 , not more than 1x10 14 , not more than 2x10 14 , not more than 3x10 14 , not more than 4x10 14 , no more than 5x10 14 , not more than 6x10 14 , no more than 7x10 14 , no more than 8x10 14 , not more than 9x10 14 , not more than 1x10 15 , not more than 2x1015 , not more than 3x10 15 , not more than 4x10 15 , no more than 5x10 15 , not more than 6x10 15 , no more than 7x10 15 , no more than 8x10 15 , not more than 9x10 15 , not more than 1x10 16 , not more than 2x10 16 , not more than 3x10 16 , not more than 4x10 16 , no more than 5x10 16 , not more than 6x10 16 , no more than 7x10 16 , no more than 8x10 16 , not more than 9x10 16 or no more than 1x10 17 vector genome units, including all ranges and subranges therebetween.

[0537] In some embodiments, the vector dosage is expressed as vector genome units per kilogram of subject body weight (vg / kg). In some embodiments, the above vector dosage amounts are based on a subject weighing 50 kg, and the dosage can be converted to vg / kg accordingly and applied to another subject based on the subject's body weight. For example, about 5x10 for a subject weighing 50 kg 14 The dose of vector genome units can be converted to approximately 1x10 per kg 13 Vector genome units (vg / kg).

[0538] In some embodiments, the vector dose is expressed in terms of the concentration or titer of the vector administered to a subject. In some embodiments, the vector dose can be expressed as the number of units per volume (eg, genomic units / volume) multiplied by the volume.

[0539] 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 L, 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 more, including all ranges and sub-ranges therebetween. In some embodiments, the vector is administered in a volume of 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 0.10 mL, at least 0.11 mL, at least 0.12 mL, at least 0.13 mL, at least 0.14 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 0.16 mL, at least 0.17 mL, at least 0.18 mL, at least 0.19 mL, at least 0.20 mL, at least 0.21 mL, at least 0.23 mL, at least 0.24 mL, at least 0.25 mL At least 1.0 mL, 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 administered in a volume of no more than 0.01 mL, no more than 0.02 mL, no more than 0.03 mL, no more than 0.04 mL, no more than 0.05 mL, no more than 0.06 mL, no more than 0.07 mL, no more than 0.08 mL, no more than 0.09 mL, no more than 0.1 mL, no more than 0.15 mL, no more than 0.2 mL, no more than 0.25 mL, no more than 0.3 mL, no more than 0.4 mL, no more than 0.5 mL, no more than 0.6 mL, no more than 0.7 mL, no more than 0.8 mL, no more than 0.9 mL, no more than No more than 1.0 mL, no more than 2.0 mL, no more than 3.0 mL, no more than 4.0 mL, no more than 5.0 mL, no more than 6.0 mL, no more than 7.0 mL, no more than 8.0 mL, no more than 9.0 mL, no more than 10.0 mL, no more than 11.0 mL, no more than 12.0 mL, no more than 13.0 mL, no more than 14.0 mL, no more than 15.0 mL, no more than 16.0 mL, no more than 17.0 mL, no more than 18.0 mL, no more than 19.0 mL, no more than 20.0 mL or no more than 25.0 mL, including all ranges and subranges therebetween.

[0540] In some embodiments, the vectors contemplated herein are administered to a subject at a titer of at least 1 x 10 9 genome units / mL, at least 1x10 10 genome units / mL, at least 5x10 10 genome units / mL, at least 1x10 11 genome units / mL, at least 5x10 11 genome units / mL, at least 1x10 12 genome units / mL, at least 5x10 12 genome units / mL, at least 6x10 12 genome units / mL, at least 7x10 12 genome units / mL, at least 8x10 12 genome units / mL, at least 9x10 12 genome units / mL, at least 10x10 12 genome units / mL, at least 15x10 12 genome units / mL, at least 20x10 12 genome units / mL, at least 25x10 12 genome units / mL, at least 50x10 12 genome units / mL or at least 100x10 12In some embodiments, the vectors herein are administered to a subject at a titer of about 1 x 10 9 genome units / mL, about 1x10 10 genome units / mL, about 5x10 10 genome units / mL, about 1x10 11 genome units / mL, about 5x10 11 genome units / mL, about 1x10 12 genome units / mL, about 5x10 12 genome units / mL, about 6x10 12 genome units / mL, about 7x10 12 genome units / mL, about 8x10 12 genome units / mL, about 9x10 12 genome units / mL, about 10x10 12 genome units / mL, about 15x10 12 genome units / mL, about 20x10 12 genome units / mL, about 25x10 12 genome units / mL, about 50x10 12 genome units / mL or approximately 100x10 12 In some embodiments, the disclosed vector is administered to a subject at a titer of no more than 1 x 10 9 genome units / mL, no more than 1x10 10 genome units / mL, no more than 5x10 10 genome units / mL, no more than 1x10 11 genome units / mL, no more than 5x10 11 genome units / mL, no more than 1x10 12 genome units / mL, no more than 5x10 12 genome units / mL, no more than 6x10 12 genome units / mL, no more than 7x10 12 genome units / mL, no more than 8x10 12 genome units / mL, no more than 9x10 12 genome units / mL, no more than 10x10 12 genome units / mL, no more than 15x10 12 genome units / mL, no more than 20x10 12genome units / mL, no more than 25x10 12 genome units / mL, no more than 50x10 12 genome units / mL or no more than 100x10 12 genomic units / mL, including all ranges and subranges therebetween.

[0541] In some embodiments, the vectors contemplated herein are administered to a subject at a titer of about 5 x 10 9 Infectious units / mL, about 6x10 9 Infectious units / mL, about 7x10 9 Infectious units / mL, about 8x10 9 Infectious units / mL, about 9x10 9 Infectious units / mL, about 1x10 10 Infectious units / mL, about 1.5x10 10 Infectious units / mL, about 2x10 10 Infectious units / mL, about 2.5x10 10 Infectious units / mL, about 5x10 10 Infectious units / mL, about 1x10 11 Infectious units / mL, about 2.5x10 11 Infectious units / mL, about 5x10 11 Infectious units / mL, about 1x10 12 Infectious units / mL, about 2.5x10 12 Infectious units / mL, about 5x10 12 Infectious units / mL, about 1x10 13 Infectious units / mL, about 5x10 13 infectious units / mL or about 1x10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of at least 5 x 10 9 infectious units / mL, at least 6x10 9 infectious units / mL, at least 7x10 9 Infectious units / mL, at least 8x10 9 infectious units / mL, at least 9x10 9 infectious units / mL, at least 1x10 10 infectious units / mL, at least 1.5x10 10 infectious units / mL, at least 2x10 10 infectious units / mL, at least 2.5x10 10 infectious units / mL, at least 5x1010 infectious units / mL, at least 1x10 11 infectious units / mL, at least 2.5x10 11 infectious units / mL, at least 5x10 11 infectious units / mL, at least 1x10 12 infectious units / mL, at least 2.5x10 12 infectious units / mL, at least 5x10 12 infectious units / mL, at least 1x10 13 infectious units / mL, at least 5x10 13 infectious units / mL or at least 1x10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of no more than 5 x 10 infectious units / mL, including all ranges and subranges therebetween. 9 Infectious units / mL, no more than 6x10 9 Infectious units / mL, no more than 7x10 9 Infectious units / mL, no more than 8x10 9 Infectious units / mL, no more than 9x10 9 Infectious units / mL, no more than 1x10 10 infectious units / mL, no more than 1.5x10 10 Infectious units / mL, no more than 2x10 10 infectious units / mL, no more than 2.5x10 10 Infectious units / mL, no more than 5x10 10 Infectious units / mL, no more than 1x10 11 infectious units / mL, no more than 2.5x10 11 Infectious units / mL, no more than 5x10 11 Infectious units / mL, no more than 1x10 12 infectious units / mL, no more than 2.5x10 12 Infectious units / mL, no more than 5x10 12 Infectious units / mL, no more than 1x10 13 Infectious units / mL, no more than 5x10 13 infectious units / mL or no more than 1x10 14 infectious units / mL, including all ranges and su...

Claims

1. 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.

2. The recombinant nucleic acid of claim 1, wherein 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.

3. The recombinant nucleic acid of claim 1, wherein the expression cassette does not comprise a 5' enhancer.

4. The recombinant nucleic acid of any one of claims 1-3, wherein the expression cassette comprises a promoter comprising a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 41-51.

5. The recombinant nucleic acid according to any one of claims 1 to 4, wherein the promoter is a neuron-specific promoter.

6. The recombinant nucleic acid of any one of claims 1-5, wherein the expression cassette comprises an intron located between the promoter and the transgene.

7. The recombinant nucleic acid of any one of claims 1-5, wherein the expression cassette comprises an intron located between the 5'UTR and the transgene.

8. The recombinant nucleic acid according to any one of claims 1 to 5, wherein the expression cassette comprises an intron located between the promoter and the 5'UTR.

9. The recombinant nucleic acid of any one of claims 6-8, wherein the intron comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 57-61.

10. The recombinant nucleic acid of any one of claims 1-5, wherein the expression cassette does not comprise an intron.

11. The recombinant nucleic acid of any one of claims 1-10, wherein 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.

12. The recombinant nucleic acid of any one of claims 1-11, wherein the expression cassette comprises a 3' enhancer comprising a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 62-65.

13. The recombinant nucleic acid of any one of claims 1-12, wherein the expression cassette comprises polyA comprising a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 67-70.

14. The recombinant nucleic acid of any one of claims 1-13, wherein the expression cassette comprises a non-neuronal silencing element embedded in the promoter, and wherein the non-neuronal silencing element comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:

40.

15. The recombinant nucleic acid of any one of claims 1-13, wherein the expression cassette comprises a non-neuronal silencing element located between the 5' enhancer and the promoter, and wherein the non-neuronal silencing element comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:

40.

16. The recombinant nucleic acid of any one of claims 1-15, wherein the expression cassette comprises a 3'UTR located between the 3' enhancer and the polyA, and wherein the 3'UTR comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

66.

17. The recombinant nucleic acid of any one of claims 1-16, wherein the transgene encodes a ligand-gated ion channel (LGIC).

18. The recombinant nucleic acid of claim 17, wherein the ligand-gated ion channel comprises a ligand binding domain derived from human α7 nicotinic acetylcholine receptor (α7-nAChR).

19. The recombinant nucleic acid of claim 18, wherein 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.

20. The recombinant nucleic acid of claim 18 or 19, wherein the ligand binding domain comprises one or more amino acid mutations listed in Table 5.

21. The recombinant nucleic acid of any one of claims 18-20, wherein the ligand-gated ion channel comprises an ion pore domain derived from a human glycine receptor.

22. The recombinant nucleic acid of claim 21, wherein the ion pore domain comprises an amino acid sequence that is at least 85% identical to amino acids 255-457 of SEQ ID NO:26, amino acids 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.

23. The recombinant nucleic acid of claim 21 or 22, wherein the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from a human glycine receptor.

24. The recombinant nucleic acid of any one of claims 21-23, wherein 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.

25. The recombinant nucleic acid of any one of claims 21-23, wherein the human glycine receptor is human glycine receptor α1, and wherein the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

33.

26. The recombinant nucleic acid according to any one of claims 1 to 25, wherein the transgene comprises or consists of the following polynucleotide sequence: The polynucleotide sequence is 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:

32.

27. The recombinant nucleic acid of any one of claims 1-26, wherein the transgene is codon-optimized for expression in human cells.

28. The recombinant nucleic acid of claim 27, wherein the human cell is a neuron.

29. The recombinant nucleic acid of any one of claims 1 to 28, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43; (iii) an hCaMKII 5'UTR comprising a polynucleotide sequence that is 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:53; (iv) an hCMV+rGlob intron comprising a polynucleotide sequence that is 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:58; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64; (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and (viii) hGH polyA comprising a polynucleotide sequence that is 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:

68.

30. The recombinant nucleic acid of claim 29, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 173-1877 of SEQ ID NO: 108, the transgene, and a sequence that is 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 nucleotides 3237-4315 of SEQ ID NO:

108.

31. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48; (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54; (iv) an hSyn intron comprising a polynucleotide sequence that is 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:59; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64; (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and (viii) hGH PolyA comprising a polynucleotide sequence that is 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:

68.

32. The recombinant nucleic acid of claim 31 , wherein the expression cassette comprises, in 5′ to 3′ order: a sequence that is 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 nucleotides 173-1159 of SEQ ID NO: 106, the transgene, and a sequence that is 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 nucleotides 2519-3597 of SEQ ID NO:

106.

33. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence that is 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:38; (ii) an hSyn-V2 promoter comprising a polynucleotide sequence that is 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:49; (iii) the transgene; (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and (v) hGH-V2 PolyA comprising a polynucleotide sequence that is 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:

69.

34. The recombinant nucleic acid of claim 33, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 149-946 of SEQ ID NO: 125, the transgene, and a sequence that is 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 nucleotides 2285-3416 of SEQ ID NO:

125.

35. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV-V2 5' enhancer comprising a polynucleotide sequence that is 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:38; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43; (iii) the transgene; (iv) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and (v) hGH-V2 PolyA comprising a polynucleotide sequence that is 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:

69.

36. The recombinant nucleic acid of claim 35, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 149-1527 of SEQ ID NO: 126, the transgene, and a sequence that is 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 nucleotides 2848-3979 of SEQ ID NO:

126.

37. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) an hSyn-V2 5' enhancer comprising a polynucleotide sequence that is 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:49; (ii) the transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and (iv) hGH-V2 PolyA comprising a polynucleotide sequence that is 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:

69.

38. The recombinant nucleic acid of claim 37, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 153-661 of SEQ ID NO: 127, the transgene, and a sequence that is 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 nucleotides 1982-3113 of SEQ ID NO:

127.

39. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43; (ii) the transgene; (iii) a WPREx-V2 3' enhancer comprising a polynucleotide sequence that is 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:65; and (iv) hGH-V2 PolyA comprising a polynucleotide sequence that is 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:

69.

40. The recombinant nucleic acid of claim 39, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 153-1224 of SEQ ID NO: 128, the transgene, and a sequence that is 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 nucleotides 2545-3676 of SEQ ID NO:

128.

41. The recombinant nucleic acid of any one of claims 1-28, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48; (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54; (iv) an hTPI intron comprising a polynucleotide sequence that is 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:60; (v) the transgene; (vi) a FullEES 3' enhancer comprising a polynucleotide sequence that is 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:63; and (vii) alpha globulin PolyA, comprising a polynucleotide sequence that is 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:

70.

42. The recombinant nucleic acid of claim 41, wherein the expression cassette comprises, in 5' to 3' order: a sequence that is 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 nucleotides 173-1188 of SEQ ID NO:98, the transgene, and a sequence that is 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 nucleotides 2548-3824 of SEQ ID NO:

98.

43. The recombinant nucleic acid of any one of claims 1-42, wherein the expression cassette comprises (i) a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs:71-93 excluding the sequence of the transgene (SEQ ID NO:36), or (ii) a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs:121-124 excluding the sequence of the transgene (SEQ ID NO:32).

44. The recombinant nucleic acid of any one of claims 1-42, wherein the expression cassette comprises a polynucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 71-93 and 121-124.

45. The recombinant nucleic acid of any one of claims 1-44, comprising adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking each end of the expression cassette.

46. ​​The recombinant nucleic acid of claim 45, comprising a 5'ITR sequence at least 90% identical to SEQ ID NO:94 or 119 and a 3'ITR sequence at least 90% identical to SEQ ID NO:95 or 120.

47. The recombinant nucleic acid of any one of claims 1-45, comprising (i) a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 96-118 excluding the sequence of the transgene (SEQ ID NO: 36), or (ii) a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 125-128 excluding the sequence of the transgene (SEQ ID NO: 32).

48. The recombinant nucleic acid of any one of claims 1-45, comprising a polynucleotide sequence at least 90% identical to any one of SEQ ID NOs: 96-118 and 125-128.

49. A vector comprising the recombinant nucleic acid of any one of claims 1-48.

50. The vector of claim 49, wherein the vector is a non-viral vector.

51. The vector of claim 49, wherein the vector is a viral vector.

52. The vector of claim 51, wherein the vector comprises or consists of an AAV vector genome.

53. AAV comprising the vector of claim 51 or 52.

54. The AAV of claim 53, wherein the AAV is of the AAV9 serotype.

55. The AAV of claim 53 or 54, wherein the AAV is a self-complementary AAV or a single-stranded AAV.

56. The AAV of any one of claims 53-55, wherein the AAV is a wild-type AAV or a modified AAV.

57. The AAV of any one of claims 53-56, wherein the AAV comprises a capsid protein that is at least 95% identical to an AAV9 capsid protein (SEQ ID NO: 8) or an AAV9-TV capsid protein (SEQ ID NO: 9).

58. A host cell comprising the nucleic acid of any one of claims 1-48, or the vector of any one of claims 49-52.

59. A method of producing the AAV of any one of claims 53-57.

60. A kit comprising the recombinant nucleic acid of any one of claims 1-48, the vector of any one of claims 49-52, or the AAV of any one of claims 53-57.

61. A method of expressing a transgene in a cell, comprising delivering to the cell the recombinant nucleic acid of any one of claims 1-48 or the vector of any one of claims 49-52.

62. A method of transducing a cell, comprising contacting the cell with the AAV of any one of claims 53-57.

63. The method of claim 61 or 62, wherein the cell is a neuron.

64. The method of claim 63, wherein the neurons are hippocampal neurons.

65. The method of claim 64, wherein the neuron is an excitatory neuron.

66. The method of claim 65, wherein the neurons are CAMK2-positive neurons.

67. The method of claim 64, wherein the neuron is an inhibitory neuron.

68. The method of claim 67, wherein the neuron is a GABAergic neuron.

69. The method of claim 63, wherein the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron.

70. The method of claim 69, wherein the neurons comprise isolectin B4 (IB4)-positive nerve fibers.

71. The method of claim 69 or 70, wherein the neurons comprise NF200-positive nerve fibers.

72. The method of any one of claims 69-71, wherein the neurons comprise CGRP-positive nerve fibers.

73. The method of any one of claims 69-72, wherein the neurons comprise C fibers.

74. The method of any one of claims 69-73, wherein the neurons comprise Aδ fibers.

75. The method of any one of claims 61-74, wherein the cell is an ex vivo cell.

76. The method of any one of claims 61-74, wherein the cell is an in vivo cell of a subject, optionally wherein the subject is a human.

77. The method of any one of claims 61-76, wherein the cell comprising the expression cassette has a higher expression level of the transgene compared to a corresponding cell comprising a control expression cassette, optionally wherein the higher expression 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 two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-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.

78. The method of any one of claims 61-76, wherein the cell comprising the expression cassette has a comparable or higher level of expression of the transgene compared to a corresponding cell 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 two-fold, at least three-fold, at least four-fold, at least five-fold, or at least ten-fold the transgene expression level of a control expression cassette comprising the polynucleotide sequence of SEQ ID NO: 87 excluding the transgene sequence.

79. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant nucleic acid of any one of claims 1-48, the vector of any one of claims 49-52, or the AAV of any one of claims 53-57.

80. The method of claim 79, wherein the disease or disorder is epilepsy, schizophrenia, an autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome.

81. The method of claim 79, wherein the disease or disorder is focal epilepsy.

82. The method of claim 79, wherein the disease or disorder is mesial temporal lobe epilepsy (mTLE).

83. The method of any one of claims 61-82, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hCaMKIIa promoter comprising a polynucleotide sequence that is 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:43; (iii) an hCaMKII 5'UTR comprising a polynucleotide sequence that is 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:53; (iv) an hCMV+rGlob intron comprising a polynucleotide sequence that is 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:58; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64; (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and (viii) hGH PolyA comprising a polynucleotide sequence that is 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: 68, Optionally wherein 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.

84. The method of any one of claims 61-82, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48; (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54; (iv) an hSyn intron comprising a polynucleotide sequence that is 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:59; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64; (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and (viii) hGH PolyA comprising a polynucleotide sequence that is 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: 68, Optionally 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.

85. The method of any one of claims 79-84, wherein the recombinant nucleic acid, the vector or the AAV is administered intracranialy, intrathecally (spinal), intrathecally (cisternar) intrathecally, intracerebrally, intraventricularly, or directly injected into an epileptic focus in the hippocampus.

86. The method of any one of claims 79-84, wherein the recombinant nucleic acid, the vector or the AAV is administered by direct injection into an epileptic focus in the hippocampus.

87. The method according to any one of claims 79 to 86, wherein 1×10 9 -1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

88. The method of any one of claims 79-87, wherein the AAV comprises the capsid protein of AAV9 (SEQ ID NO: 8).

89. The method of any one of claims 79-88, wherein the method reduces the duration, intensity and / or frequency of seizures by at least 10%.

90. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a recombinant nucleic acid of any one of claims 1-48, a vector of any one of claims 49-52, or an AAV of any one of claims 53-57, wherein the disease or condition is neuropathic pain, spasticity, spinal cord injury, or avulsion.

91. The method of claim 90, wherein the disease or condition is neuropathic pain.

92. The method of claim 91, wherein the neuropathic pain is peripheral neuropathy.

93. The method of claim 91, wherein the neuropathic pain is trigeminal neuralgia.

94. The method of any one of claims 61-78 and 90-93, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48; (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54; (iv) an hTPI intron comprising a polynucleotide sequence that is 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:60; (v) the transgene; (vi) a FullEES 3' enhancer comprising a polynucleotide sequence that is 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:63; and (vii) rβ globulin 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, Optionally 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.

95. The method of any one of claims 61-78 and 90-93, wherein the expression cassette comprises, in 5' to 3' order: (i) a CMV 5' enhancer comprising a polynucleotide sequence that is 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:37; (ii) an hSyn promoter comprising a polynucleotide sequence that is 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:48; (iii) hSyn 5'UTR comprising a polynucleotide sequence that is 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:54; (iv) an hSyn intron comprising a polynucleotide sequence that is 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:59; (v) the transgene; (vi) a WPREx 3' enhancer comprising a polynucleotide sequence that is 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:64; (vii) an alpha globulin 3'UTR comprising a polynucleotide sequence that is 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:66; and (viii) hGH PolyA comprising a polynucleotide sequence that is 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: 68, Optionally 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.

96. The method of any one of claims 90-95, wherein the recombinant nucleic acid, the vector, or the AAV is administered by intrathecal (IT) or intraganglionic (IG) administration.

97. The method of any one of claims 90-95, wherein the recombinant nucleic acid, the vector, or the AAV is administered by intraganglionic (IG) administration directly into a dorsal root ganglion or a trigeminal ganglion.

98. The method according to any one of claims 90-97, wherein 1×10 9 -1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

99. The method of any one of claims 90-98, wherein the AAV comprises the capsid protein of AAV9-TV (SEQ ID NO:9).

100. The method of any one of claims 91-99, wherein the method reduces pain levels by at least 10%.

101. The method of any one of claims 79-100, wherein the recombinant nucleic acid, the vector or the AAV is administered by systemic, parenteral, intravenous, brain, cerebrospinal, intrathecal, intracisternal, intraputical, intrahippocampal, intrastriatal or intracerebroventricular injection.

102. The method according to any one of claims 79 to 101, wherein 1×10 9 -1×10 14 A copy of the recombinant nucleic acid or AAV vector genome is administered to the subject.

103. The method of any one of claims 79-102, wherein the method comprises administering a ligand of a ligand-gated ion channel encoded by the transgene.

104. The method of claim 103, wherein the ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and Facinicline / RG3487.

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