Adeno-associated virus capsids and engineered ligand-gated ion channels for treating focal epilepsy and neuropathic pain

Abstract

The present disclosure provides compositions and methods for treating neuropathic pain or focal epilepsy.Furthermore, AAV capsid polypeptides and / or nucleic acids suitable for transducing neurons associated with neuropathic pain or focal epilepsy are provided.The present disclosure relates to AAV vectors that comprise specific capsid polypeptides that provide desirable transduction efficiency and / or tropism for neurons responsible for the management of neuropathic pain or focal epilepsy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 141,124, filed January 25, 2021, U.S. Provisional Application No. 63 / 141,121, filed January 25, 2021, and U.S. Provisional Application No. 63 / 285,929, filed December 3, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer-readable copy of the sequence listing (file name: SWCH_037_01WO_SeqList_ST25.txt, creation date: January 23, 2022, file size: approximately 143 kilobytes). [Background technology]

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

[0004] The most commonly used treatment for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to dependency and can cause side effects such as drug dependence, tolerance, respiratory depression, sedation, cognitive impairment, hallucinations, and other systemic side effects.Despite the widespread use of pharmaceutical drugs, their effectiveness in pain relief has a surprisingly low success rate.More invasive options for pain treatment include nerve blocks and electrical stimulation.The most invasive and least preferred method for managing pain is the complete surgical removal of the nerve or part thereof that is causing pain.

[0005] Focal epilepsy is a chronic, debilitating neurological disorder that affects over 2 million people in the United States and is characterized by unpredictable seizures that initiate from specific locations in the brain. Recurrent seizures result in cognitive and emotional deficits, and current interventions have limited efficacy and multiple side effects. Although focal seizures result from abnormal firing in a subset of neurons, many antiepileptic drugs rely on systemic compound administration to alter whole-brain activity. Thus, patients with epilepsy suffer substantial side effects due to unintended modulation of neurons involved in normal cognition and undesirable off-target changes in other biological systems.

[0006] An ideal pain and focal epilepsy treatment would only alter the activity of specific neurons that are responsible for the occurrence of pain and / or seizures. Thus, a new strategy for treating pain or focal epilepsy is to deliver heterologous proteins encoded by recombinant nucleic acids to subpopulations of neurons to control their abnormal activity. As an example, target neurons can be transduced with adeno-associated virus (AAV) vectors that encode engineered ligand-gated ion channels (chemogenetic receptors) that are highly responsive to specific small molecule ligands but are otherwise inactive, resulting in tunable efficacy.

[0007] Such strategies can be used to treat neuropathic pain, particularly peripheral neuropathy and trigeminal neuralgia. The former is a common neurological disorder resulting from damage to, or dysfunction of, the peripheral nervous system. It is characterized by numbness, tingling and pain, and often begins in the hands and feet, but other areas of the body can also be affected. Caused by damage to the myelin sheath that protects the trigeminal cranial nerve, trigeminal neuralgia, also known as suicide disease, results in extreme, diffuse, shock-like facial pain and can last from seconds to minutes. Current pharmaceutical and surgical approaches provide little relief and potential for dependency, but have significant side effects. Because the dorsal root ganglion (DRG) and trigeminal ganglion (TGG) are active structures in the process of pain transmission, engineered chemogenetic receptors can be expressed in neurons of the DRG and TGG by AAV-mediated gene transfer.

[0008] One consideration of this strategy is the transduction efficiency and tropism of AAV vectors to target populations of neurons (e.g., in DRG and TGG, or hippocampus).There is a need in the art for a safe and efficient strategy to effectively transduce specific subpopulations of neurons using AAV vectors.

[0009] Another consideration of this strategy is the selection of heterologous proteins to control neuronal activity. Recently, designer receptors activated only by designer drugs to modify seizure activity (DREADDs) have shown promising results in animal epilepsy models. However, inhibitory DREADDs rely on G protein-coupled receptors that indirectly activate ion channels through second messengers, and can inconsistently change neuronal potential. There is a need in the art for alternative proteins (engineered ligand-gated ion channels) to control neuronal activity (e.g., in DRG and TGG) for the treatment of neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia, or to control neuronal activity in the treatment of focal epilepsy. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides such AAV vectors, engineered ligand-gated ion channels, and others.

[0011] Abstract In one aspect, the present disclosure provides a method for treating neuropathic pain in a subject that requires treatment of neuropathic pain, comprising administering an effective amount of adeno-associated virus (AAV) vector to the subject, wherein the AAV vector transduces the neuron in the subject, and the neuron is dorsal root ganglion neuron or trigeminal ganglion neuron.In some embodiments, the neuropathic pain is peripheral neuropathy.In some embodiments, the neuropathic pain is trigeminal neuralgia.

[0012] In one aspect, the present disclosure provides a method for treating a neurological disease or disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces the neurons in the subject. In some embodiments, the neurological disease or disorder is neuropathic pain, spasticity, spinal cord injury, or avulsion injury.

[0013] In some embodiments, the AAV vector is an AAV9-TV vector comprising a capsid polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9.

[0014] In one aspect, the present disclosure provides a method of transducing a neuron, comprising contacting the neuron with an adeno-associated virus (AAV) vector, wherein the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron. In some embodiments, the method transduces the neuron in vitro. In some embodiments, the method transduces the neuron in vivo.

[0015] In some embodiments, the AAV vector comprises a heterologous nucleic acid.

[0016] In one aspect, the disclosure provides a method of delivering a heterologous nucleic acid to a neuron, the method comprising contacting the neuron with an adeno-associated virus (AAV) vector comprising a capsid polypeptide, wherein the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron.

[0017] In some embodiments, the AAV vector is an AAV vector capable of transducing iPSC-derived neurons in vitro. In some embodiments, the method comprises selecting the AAV vector as an AAV vector capable of transducing iPSC-derived neurons in vitro.

[0018] In some embodiments, the AAV vector is AAV2, AAV2.5, AAV2.5-TV, AAV2.5-2YF, AAV2.5-TV2YF, AAV5, AAV6, AAV9, AAV9-TV, AAV9-2YF, AAV9-TV2YF, or AAV-PHP.S vector. In some embodiments, the AAV vector is AAV9-TV vector. In some embodiments, the AAV vector is AAV6 vector. In some embodiments, the AAV vector is AAV5 vector. In some embodiments, the AAV vector is AAV2.5-TV2YF vector.

[0019] In some embodiments, the AAV vector comprises a capsid polypeptide that comprises, or consists of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the capsid polypeptide comprises, or consists of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3 and 5-9. In some embodiments, the capsid polypeptide comprises, or consists of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 5-7 and 9.

[0020] In some embodiments, the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and the capsid polypeptide comprises a non-threonine mutation at a position corresponding to T492 of SEQ ID NO: 8. In some embodiments, the non-threonine mutation is a valine, isoleucine, or leucine substitution. In some embodiments, the non-threonine mutation is a valine substitution.

[0021] In some embodiments, the neuron is a dorsal root ganglion neuron. In some embodiments, the neuron is a trigeminal ganglion neuron. In some embodiments, the neuron comprises isolectin B4 (IB4) positive nerve fibers. In some embodiments, the neuron comprises NF200 positive nerve fibers. In some embodiments, the neuron comprises CGRP positive nerve fibers. In some embodiments, the neuron comprises C fibers. In some embodiments, the neuron comprises Aδ fibers.

[0022] In some embodiments, the AAV vector is administered by intrathecal (IT) or intraganglionic (IG) administration. In some embodiments, the AAV vector is administered by intraganglionic (IG) administration directly into the dorsal root ganglion or trigeminal ganglion.

[0023] In one aspect, the disclosure provides a method of treating focal epilepsy in a subject in need thereof, the method comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces neurons in the subject, and the neurons are hippocampal neurons.

[0024] In one aspect, the present disclosure provides a method of treating a neurological disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces hippocampal neurons in the subject. In some embodiments, the neurological disease or disorder is focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome.

[0025] In one aspect, the present disclosure provides a method of transducing a neuron, comprising contacting the neuron with an adeno-associated virus (AAV) vector, wherein the neuron is a hippocampal neuron. In some embodiments, the method transduces the neuron in vitro. In some embodiments, the method transduces the neuron in vivo.

[0026] In some embodiments, the AAV vector comprises a heterologous nucleic acid.

[0027] In one aspect, the disclosure provides a method of delivering a heterologous nucleic acid to a neuron, the method comprising contacting the neuron with an adeno-associated virus (AAV) vector comprising a capsid polypeptide, wherein the neuron is a hippocampal neuron.

[0028] In some embodiments, the AAV vector is an AAV vector capable of transducing embryonic hippocampal neurons in vitro. In some embodiments, the method comprises selecting the AAV vector as an AAV vector capable of transducing embryonic hippocampal neurons in vitro.

[0029] In some embodiments, the AAV vector is AAV2, AAV2.5, AAV2.5-TV, AAV2.5-2YF, AAV2.5-TV2YF, AAV5, AAV6, AAV9, AAV9-TV, AAV9-2YF, AAV9-TV2YF, or AAV-PHP.S vector. In some embodiments, the AAV vector is AAV9 vector. In some embodiments, the AAV vector is AAV9-TV vector. In some embodiments, the AAV vector is AAV6 vector. In some embodiments, the AAV vector is AAV5 vector.

[0030] In some embodiments, the AAV vector comprises a capsid polypeptide that comprises, or consists of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:1-12.

[0031] The method of any one of claims 30-40, wherein the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3 and 5-9. In some embodiments, the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6-9. In some embodiments, the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8.

[0032] In some embodiments, the neuron is an excitatory neuron. In some embodiments, the neuron is a CAMK2 positive neuron. In some embodiments, the neuron is an inhibitory neuron. In some embodiments, the neuron is a GABAergic neuron.

[0033] In some embodiments, the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0034] In some embodiments, the AAV vector is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cisternal) administration, intracerebral administration, intraventricular administration, or direct injection into the epileptogenic focus of the hippocampus.In some embodiments, the AAV vector is administered by direct injection into the epileptogenic focus of the hippocampus.

[0035] In some embodiments, the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel. In some embodiments, the ligand-gated ion channel comprises a ligand-binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor. In some embodiments, the ligand-binding domain comprises an amino acid sequence having at least 85% identity to amino acid residues 23-220 of SEQ ID NO:25. In some embodiments, the ligand-binding domain comprises an amino acid mutation at a residue selected from those corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172 and Y210 of SEQ ID NO:25. In some embodiments, the ligand-binding domain comprises one or more amino acid mutations listed in Table 3. In some embodiments, the ligand-binding domain comprises mutations corresponding to R101W, Y115E and Y210W in SEQ ID NO:33. In some embodiments, the ligand-binding domain comprises mutations corresponding to R101W and Y210V in SEQ ID NO:33. In some embodiments, the ligand binding domain comprises mutations corresponding to R101M and L131F in SEQ ID NO:33.

[0036] In some embodiments, the human glycine receptor is human glycine receptor alpha 1, human glycine receptor alpha 2, or human glycine receptor alpha 3. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity to amino acids 255-457 of SEQ ID NO:26, 260-452 of SEQ ID NO:27, amino acids 259-464 of SEQ ID NO:28, or amino acids 259-449 of SEQ ID NO:29. In some embodiments, the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from a human glycine receptor. In some embodiments, the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:26. In some embodiments, the Cys loop domain comprises amino acids 166-180 of SEQ ID NO:26. In some embodiments, the ligand binding domain of the engineered receptor comprises a β1-2 loop domain derived from a human glycine receptor alpha 1 subunit. In some embodiments, the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:26.

[0037] In some embodiments, the human glycine receptor is the human glycine receptor alpha 1 and the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

[0038] In some embodiments, the method comprises administering a ligand of a ligand-gated ion channel. 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. In some embodiments, the ligand is ABT-126.

[0039] In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult.

[0040] In one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 12; (b) Instructions for administering an AAV vector to transduce dorsal root ganglion neurons or trigeminal ganglion neurons. A kit comprising:

[0041] In some embodiments, the kit further comprises a device adapted for administration of the AAV vector by intrathecal (IT) or intraganglionic (IG) administration, hi some embodiments, the device is adapted for administration directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

[0042] In one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 12; (b) Instructions for administering AAV vectors to transduce hippocampal neurons. A kit is provided, comprising:

[0043] In some embodiments, the kit further comprises a device adapted for administration of the AAV vector by intracranial administration, intrathecal (spinal), intrathecal (cisternal), intracerebral, intraventricular administration, or direct injection into the epileptogenic focus of the hippocampus. In some embodiments, the device is adapted for administration by direct injection into the epileptogenic focus of the hippocampus.

[0044] In some embodiments, the AAV vector comprises a heterologous nucleic acid encoding a ligand-gated ion channel. In some embodiments, the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the kit comprises a ligand of the ligand-gated ion channel. In some embodiments, the ligand is TC-5619. In some embodiments, the ligand is ABT-126.

[0045] In one aspect, the present disclosure provides a method for treating neuropathic pain in a subject in need of such treatment, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to a dorsal root ganglion neuron or a trigeminal ganglion neuron of the subject, and the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel. In some embodiments, the neuropathic pain is peripheral neuropathy. In some embodiments, the neuropathic pain is trigeminal neuralgia.

[0046] In one aspect, the disclosure provides a method of treating a neurological disease or disorder in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, the heterologous nucleic acid comprising a polynucleotide sequence encoding a ligand-gated ion channel. In some embodiments, the neurological disease or disorder is neuropathic pain, spasticity, spinal cord injury, or avulsion injury.

[0047] In some embodiments, the heterologous nucleic acid is administered by intrathecal (IT) or intraganglionic (IG) administration. In some embodiments, the heterologous nucleic acid is administered directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

[0048] In some embodiments, the heterologous nucleic acid is contained within a vector, hi some embodiments, the vector is a viral vector, and the viral vector transduces dorsal root ganglion neurons or trigeminal ganglion neurons.

[0049] In some embodiments, the neuron comprises isolectin B4 (IB4) positive nerve fibers. In some embodiments, the neuron comprises NF200 positive nerve fibers. In some embodiments, the neuron comprises CGRP positive nerve fibers. In some embodiments, the neuron comprises C fibers. In some embodiments, the neuron comprises Aδ fibers.

[0050] In one aspect, the disclosure provides a method of treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to hippocampal neurons of the subject, and wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0051] In one aspect, the disclosure provides a method of treating a neurological disease or disorder in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to a hippocampal neuron of the subject, and the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel. In some embodiments, the neurological disease or disorder is focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome.

[0052] In some embodiments, the heterologous nucleic acid is administered by intracranial administration, intrathecal (spinal) administration, intrathecal (cisternal) administration, intracerebral administration, intraventricular administration, or direct injection into the epileptogenic focus of the hippocampus.In some embodiments, the heterologous nucleic acid is administered by direct injection into the epileptogenic focus of the hippocampus.

[0053] In some embodiments, the heterologous nucleic acid is contained within a vector, hi some embodiments, the vector is a viral vector, and the viral vector transduces hippocampal neurons.

[0054] In some embodiments, the neuron is an excitatory neuron. In some embodiments, the neuron is a CAMK2 positive neuron. In some embodiments, the neuron is an inhibitory neuron. In some embodiments, the neuron is a GABAergic neuron.

[0055] In some embodiments, the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0056] In some embodiments, the ligand-gated ion channel comprises a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85% identity to amino acid residues 23-220 of SEQ ID NO:25. In some embodiments, the ligand binding domain comprises an amino acid mutation at a residue selected from those corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172 and Y210 of SEQ ID NO:25. In some embodiments, the ligand binding domain comprises one or more amino acid mutations listed in Table 3. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85% identity to amino acids 255-457 of SEQ ID NO:26, 260-452 of SEQ ID NO:27, amino acids 259-464 of SEQ ID NO:28, or amino acids 259-449 of SEQ ID NO:29.

[0057] In some embodiments, the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from a human glycine receptor. In some embodiments, the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:26. In some embodiments, the Cys loop domain comprises amino acids 166-180 of SEQ ID NO:26. In some embodiments, the ligand binding domain of the engineered receptor comprises a β1-2 loop domain derived from a human glycine receptor α1 subunit. In some embodiments, the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:26. In some embodiments, the human glycine receptor is human glycine receptor α1 and the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

[0058] In some embodiments, the method includes administering a ligand of a ligand-gated ion channel. 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. In some embodiments, the ligand is ABT-126.

[0059] In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult.

[0060] The patent or application file contains at least one drawing executed in color. [Brief description of the drawings]

[0061] [Figure 1] FIG. 1 shows cytofluorescence images of GFP expression in iPSC-derived sensory neuron progenitor cells transduced with AAV vectors containing the indicated capsids 5 days after transduction.

[0062] [Diagram 2]FIG. 2 shows ddPCR assessment of GFP mRNA levels in iPSC-derived sensory neuron progenitor cells transduced with AAV vectors containing the indicated capsids 5 days after transduction.

[0063] [Diagram 3] FIG. 3 shows cytofluorescence images of GFP expression in iPSC-derived sensory neuron progenitor cells transduced with AAV vectors containing the indicated capsids 5 days after transduction.

[0064] [Figure 4] FIG. 4 shows the results of ELISA of GFP protein expression levels in iPSC-derived sensory neuron progenitor cells transduced with AAV vectors containing the indicated capsids, 5 days after transduction.

[0065] [Diagram 5] FIG. 5 shows ddPCR assessment of GFP mRNA levels in iPSC-derived sensory neuron progenitor cells transduced with AAV vectors containing the indicated capsids, 5 days post-transduction.

[0066] [Figure 6] FIG. 6 shows ddPCR assessment of GFP mRNA levels in rat DRG tissue transduced with AAV vectors containing the indicated capsids by direct injection in vivo.

[0067] [Figure 7] FIG. 7 shows the results of ELISA of GFP protein expression levels in rat DRG tissue transduced with AAV vectors containing the indicated capsids by direct injection in vivo.

[0068] [Figure 8] FIG. 8 shows representative immunofluorescence images analyzing the tropism of AAV capsids following direct injection of AAV vectors into rat DRG.

[0069] [Figure 9] FIG. 9 shows a quantitative analysis of the results of immunofluorescence imaging of rat DRG tissue transduced with AAV vectors containing the indicated capsids.

[0070] [Figure 10] FIG. 10 shows a pie chart depicting the tropism of AAV vectors containing the indicated capsids by the percentage of different sensory neurons in rat DRG showing positive GFP staining.

[0071] [Figure 11] FIG. 11 shows ddPCR assessment of GFP mRNA levels in non-human primate (NHP) DRG tissue transduced with AAV vectors containing the indicated capsids by direct injection in vivo in study V01220.

[0072] [Figure 12A] FIG. 12A shows the results of ELISA of GFP protein expression levels in NHP DRG tissues transduced with AAV vectors containing the indicated capsids by direct injection in vivo in Study V01220. [Figure 12B] FIG. 12B shows the results of ELISA of GFP protein expression levels in NHP DRG tissues transduced with AAV vectors containing the indicated capsids by direct injection in vivo in Study V01331.

[0073] [Figure 13A] FIG. 13A shows representative immunofluorescence images analyzing the tropism of the indicated AAV capsids following direct injection in NHP DRG in study V01220. [Figure 13B-1] FIG. 13B shows zoomed-in immunofluorescence images analyzing the tropism of the indicated AAV capsids following direct injection in NHP DRG in study V01220. [Figure 13B-2]FIG. 13B shows zoomed-in immunofluorescence images analyzing the tropism of the indicated AAV capsids following direct injection in NHP DRG in study V01220. [Figure 13C] FIG. 13C shows representative immunofluorescence images analyzing the tropism of the indicated AAV capsids following direct injection in NHP DRG in study V01331.

[0074] [Figure 14] FIG. 14 shows cytofluorescence images of GFP expression in E18 (embryonic day 18) rat mixed hippocampal cultures transduced with AAV vectors containing the indicated capsids, 5 days after transduction.

[0075] [Figure 15] FIG. 15 shows ddPCR assessment of GFP mRNA levels in E18 rat mixed hippocampal cultures transduced with AAV vectors containing the indicated capsids, 5 days post-transduction.

[0076] [Figure 16] FIG. 16 shows cytofluorescence images of GFP expression in E18 rat mixed hippocampal cultures transduced with AAV vectors containing the indicated capsids, 5 days after transduction.

[0077] [Figure 17] FIG. 17 shows ddPCR assessment of GFP mRNA levels in E18 rat mixed hippocampal cultures transduced with AAV vectors containing the indicated capsids, 5 days post-transduction.

[0078] [Figure 18] Figure 18 shows ddPCR assessment of GFP mRNA levels in rat hippocampal tissue transduced with AAV vectors containing the indicated capsids. All data points correspond to either the left or right hemisphere of separate animals. Each animal within a group is represented by a unique color.

[0079] [Figure 19]Figure 19 shows the results of ELISA of GFP levels in rat hippocampal tissue transduced with AAV vectors containing the indicated capsids. All data points correspond to either the left or right hemisphere of separate animals. Each animal within a group is represented by a unique color.

[0080] [Figure 20] FIG. 20 shows representative immunofluorescence images of rat hippocampi transduced with AAV vectors containing the indicated capsids.

[0081] [Figure 21A] Figures 21A-21B show semi-quantitative immunofluorescence analysis of rat hippocampi transduced with AAV vectors containing the indicated capsids by sections through the left and right hemispheres. Figure 21A shows the GFP coverage per hippocampal section for each hemisphere. Figure 21B shows the GFP intensity per positive pixel in each hippocampus per hemisphere. [Figure 21B] Figures 21A-21B show semi-quantitative immunofluorescence analysis of rat hippocampi transduced with AAV vectors containing the indicated capsids by sections through the left and right hemispheres. Figure 21A shows the GFP coverage per hippocampal section for each hemisphere. Figure 21B shows the GFP intensity per positive pixel in each hippocampus per hemisphere.

[0082] [Figure 22A] FIG. 22A shows ddPCR assessment of GFP mRNA levels in hippocampal tissue of non-human primates (NHPs) transduced with AAV vectors containing the indicated capsids by direct injection in vivo in study V01220. [Figure 22B] FIG. 22B shows ddPCR assessment of GFP mRNA levels in hippocampal tissue of non-human primates (NHPs) transduced with AAV vectors containing the indicated capsids by direct injection in vivo in study V01331.

[0083] [Figure 23A]FIG. 23A shows the results of ELISA of GFP protein expression levels in NHP hippocampal tissue transduced with AAV vectors containing the indicated capsids by direct injection in vivo in Study V01220. [Figure 23B] FIG. 23B shows the results of ELISA of GFP protein expression levels in NHP hippocampal tissue transduced with AAV vectors containing the indicated capsids by direct injection in vivo in Study V01331.

[0084] [Figure 24A] FIG. 24A shows representative immunofluorescence images of NHP hippocampi from mice directly injected with AAV9 in study V01220. [Figure 24B] FIG. 24B shows representative immunofluorescence images of NHP hippocampi from mice directly injected with AAV5 or AAV9 in study V01331.

[0085] [Diagram 25] Figure 25 contains images showing hippocampal expression of AAV9-hSyn-CODA71-GFP (green) 12 weeks after the first injection. Neuronal cell bodies are labeled with NeuN (blue).

[0086] [Figure 26] Figure 26 shows action potentials of CA1 neurons transduced with either CODA71 or a control vector and bathed with the agonist TC-5619. The black trace (bottom) depicts the protocol of the ramp test.

[0087] [Figure 27A]Figure 27A shows the RMP (mV) readout (top) and input resistance (MΩ) readout (bottom) of neurons after application of the agonist TC-5619. Figure 27B shows the rheobase (pA) readout (top) and AP count (baseline %) readout (bottom) of neurons after application of the agonist TC-5619. In both Figures 27A and 27B, the red lines represent neurons transduced with the CODA71 vector and the blue lines represent neurons transduced with the control (scrambled) vector. [Figure 27B] Figure 27A shows the RMP (mV) readout (top) and input resistance (MΩ) readout (bottom) of neurons after application of the agonist TC-5619. Figure 27B shows the rheobase (pA) readout (top) and AP count (baseline %) readout (bottom) of neurons after application of the agonist TC-5619. In both Figures 27A and 27B, the red lines represent neurons transduced with the CODA71 vector and the blue lines represent neurons transduced with the control (scrambled) vector.

[0088] [Figure 28] Figure 28 shows RMP (mV) readout (top left), input resistance (MΩ) readout (top right), rheobase (pA) readout (bottom left) and AP count (% baseline) readout (bottom right) of neurons after application of the agonist TC-5619 in the presence of bungarotoxin (BTX). The red line represents neurons transduced with the CODA71 vector and the blue line represents neurons transduced with the control (scrambled) vector.

[0089] [Figure 29] FIG. 29 shows a schematic diagram of the surgical and electrical stimulation procedures in mice.

[0090] [Diagram 30] FIG. 30 shows examples of electrical elicitation of CA1 responses with 60 Hz stimulation of perforant pathway inputs at various amplitudes in CODA71-transduced mice 45 min after TC-5619 injection.

[0091] [Diagram 31] FIG. 31 shows a seizure threshold graph for mice injected with CODA71 vector (blue line) or scrambled control vector (red line).

[0092] [Diagram 32] FIG. 32 shows a schematic of the surgical and recording procedures in mice.

[0093] [Diagram 33] FIG. 33 shows spontaneous seizure recordings in mice following unilateral kainic acid injection into CA1.

[0094] [Figure 34A-1] FIG. 34A shows seizure density graphs of mice injected with either the CODA71 vector (red line) or the scrambled control vector (blue line) upon administration of either TC-5619 or vehicle. [Figure 34A-2] FIG. 34A shows seizure density graphs of mice injected with either the CODA71 vector (red line) or the scrambled control vector (blue line) upon administration of either TC-5619 or vehicle. [Figure 34B] FIG. 34B shows seizure density graphs for mice injected with either the CODA71 vector or the scrambled control vector upon administration of either TC-5619 or vehicle control.

[0095] [Diagram 35] FIG. 35 shows seizure frequency graphs for the indicated mice treated with either TC-5619 or vehicle control.

[0096] [Diagram 36] FIG. 36 shows seizure duration graphs for the indicated mice treated with either TC-5619 or vehicle control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] Detailed Description Details of the present disclosure are described in the attached description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described here. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In this specification and the appended claims, the singular form includes the plural form unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications cited herein are incorporated herein by reference in their entirety.

[0098] The embodiments according to the present disclosure will be described more completely hereafter. However, aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Unless otherwise expressly indicated, all specific embodiments, features, and terms are intended to include both the described embodiments, features, or terms and their biological equivalents. The terms used herein are intended to describe only specific embodiments and are not intended to be limiting.

[0099] Before describing the method and composition of the present invention, it should be understood that the present disclosure is not limited to the specific method or composition described, and therefore, of course, may vary.It should also be understood that the terms used herein are for describing only specific embodiments, and are not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.

[0100] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0101] definition Throughout this disclosure, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, e.g., polypeptides, and the like.

[0102] Throughout this specification, the term "and / or" is used in the present disclosure to refer to either "and" or "or," unless otherwise indicated.

[0103] Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of a stated element or integer or group of elements or integers but not to the exclusion of any other element or integer or group of elements or integers. Moreover, recitations of numerical ranges throughout this specification specifically include all integers and decimal points therebetween.

[0104] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting essentially of" refers to a limitation of the scope of a described composition, method, or kit to specified materials or steps that do not materially affect the basic and novel characteristics of the subject disclosure. For example, a polypeptide "consisting essentially of" a disclosed sequence has an amino acid sequence that is the disclosed sequence plus or minus about 5 amino acid residues of the boundary of the sequence, e.g., about 5, 4, 3, 2, or about 1 residue less than the stated boundary amino acid residue, or about 1, 2, 3, 4, or 5 residues more than the stated boundary amino acid residue.

[0105] Throughout this disclosure, unless the context requires otherwise, the phrase "consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a polypeptide "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0106] The terms "about" and "approximately" are used as equivalents. Any numerical values ​​used in this application, including or not including about / approximately, are meant to encompass any normal variation that would be understood by a person skilled in the relevant technical field. In certain embodiments, the term "about" or "about" refers to a range of values ​​that is within 10% (greater or less) in either direction of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number exceeds 100% of the possible value).

[0107] The term "isolated" refers to material that is substantially or essentially free from components that normally accompany it as found in its natural state. In some embodiments, the term "obtained" is used synonymously with isolated.

[0108] The terms "subject", "individual" and "patient" are used interchangeably and refer to a vertebrate such as a mammal. A mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., a cynomolgus monkey, a chimpanzee) or a human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject may be an adult, a teenager, a child (2-14 years), an infant (1-24 months), or a newborn (up to 1 month). In some embodiments, an adult is about 65 years of age or older, or an elderly person about 60 years of age or older.

[0109] The term "sample" refers to a volume and / or mass of biological material that is subjected to analysis. In some embodiments, a sample includes a tissue sample, a cell sample, a fluid sample, and the like. In some embodiments, a sample is taken from or provided by a subject (e.g., a human subject). In some embodiments, a sample includes a portion of tissue taken from any internal organ, cancerous, precancerous, or noncancerous tumor, brain, skin, hair (including hair roots), eye, muscle, bone marrow, cartilage, white adipose tissue, and / or brown adipose tissue. In some embodiments, a fluid sample includes a buccal swab, blood, umbilical cord blood, saliva, semen, urine, peritoneal fluid, pleural fluid, cerebrospinal fluid, lung lavage, tears, sweat, and the like. One of skill in the art will understand that in some embodiments, a "sample" is a "primary sample" in that it is obtained directly from a source (e.g., a subject). In some embodiments, a "sample" is the result of processing a primary sample, e.g., to remove certain potentially contaminating components, to isolate certain components, and / or to purify certain components of interest. In some embodiments, the sample is a cell or a population of cells (e.g., neural cells). A cell sample may be derived directly from a subject (e.g., a primary sample) or may be a cell line. A cell line may include non-mammalian cells (e.g., insect cells, yeast cells, and / or bacterial cells) or mammalian cells (e.g., an immortalized cell line).

[0110] The terms "treating", "treatment" and grammatical equivalents as used herein generally refer to the use of a composition or method to reduce, eliminate, or prevent symptoms of a disease, including achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit means slowing down, stopping, reversing, or eradicating or ameliorating the progression of the symptoms of the disorder or condition being treated. A prophylactic benefit of treatment includes reducing the risk of a condition, delaying the progression of a condition, or reducing the likelihood of occurrence of a condition. In some embodiments, treating refers to delivering a composition (e.g., an AAV vector) to a subject and / or a population of cells to affect a physiological outcome. In some embodiments, treatment results in an improvement (e.g., reduction, amelioration, or correction) of one or more disease symptoms. The improvement may be an observable or measurable improvement, or an improvement in the subject's general sense of well-being. Treatment of a disease may refer to a reduction in the severity of disease symptoms. In some embodiments, treatment may refer to a reduction in the severity of disease symptoms to a level equivalent to that before the onset of the disease. In some embodiments, treatment may refer to short-term (e.g., temporary or acute) and / or long-term (e.g., sustained or chronic) reduction of disease symptoms. In some embodiments, treatment may refer to the amelioration of disease symptoms. In some embodiments, treatment may refer to prophylactic treatment of a subject at risk of developing a particular disease to prevent the onset of the disease. Prevention of disease onset may refer to preventing disease symptoms altogether, delaying the onset of the disease, reducing the severity of symptoms in a disease that has subsequently developed, or reducing the likelihood of disease onset.

[0111] The terms "management" 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.

[0112] The term "effective amount" refers to an amount capable of achieving a stated result (e.g., transduction of target neurons). In some embodiments, an effective amount for administration in vivo refers to a dose capable of achieving a desired result, such as, for example, transducing a sufficient amount of target neurons for therapeutic purposes.

[0113] A "therapeutically effective amount" is the amount of a composition required to achieve a desired therapeutic result. The therapeutically effective amount may vary depending on factors such as, but not limited to, the disease state and the subject's age, sex, and weight. In general, a therapeutically effective amount is also one in which any toxic or harmful effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" includes the amount of a composition that is effective for treating a subject.

[0114] "Increase" refers to an increase in value (e.g., an increase in transduction efficiency) of at least 5% compared to a reference or control level. For example, an increase may include an increase of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. Increase also refers to an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) over a reference or control level.

[0115] "Decrease," "reduce," "diminish" or synonyms thereof refer to a decrease in value (e.g., a decrease in transduction efficiency) of at least 5% compared to a reference or control level. For example, a decrease may include a decrease of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000% or more. A decrease also refers to a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) lower than a reference or control level.

[0116] The terms "reference" or "control" level are used interchangeably throughout this disclosure and refer to the value of a particular physiological and / or therapeutic effect in a subject or sample that is not treated with a composition of the present disclosure, or in a subject or sample that is treated with a vehicle control. In some embodiments, a reference level refers to the value of a particular physiological and / or therapeutic effect that is a measure in a subject or sample prior to administration of a composition of the present disclosure (e.g., a baseline level).

[0117] The term "ligand" refers to a molecule that binds to another, larger molecule. In some embodiments, a ligand binds to a receptor. In some embodiments, the binding of a ligand to a receptor alters the function of the receptor to activate or inhibit its function. In some embodiments, the binding of a ligand to a receptor, such as a ligand-gated ion channel (LGIC), results in the opening or closing of an ion channel. The term "ligand" may refer to an endogenous or naturally occurring ligand. For example, in some embodiments, a ligand refers to a neurotransmitter (e.g., lambda-aminobutyric acid (GABA), acetylcholine, serotonin, etc.) and a signaling intermediate (e.g., phosphatidylinositol 4,5-bisphosphate (PIP2)), an amino acid (e.g., glycine), or a nucleotide (e.g., ATP). In some embodiments, a ligand may refer to a non-natural, i.e., synthetic, or non-naturally occurring ligand (e.g., a binding agent). For example, in some embodiments, a ligand refers to a small molecule. Ligand binding can be measured by various methods known in the art (e.g., detecting 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.

[0118] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a receptor and a ligand. Unless otherwise indicated, as used 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., receptor and ligand). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described in this disclosure.

[0119] The term "wild-type" or "native" is a term of the art understood by those of skill in the art and refers to the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a wild-type protein is the typical form of that protein as it occurs in nature.

[0120] The terms "non-naturally occurring," "variant," and "mutant" are used interchangeably throughout the specification and claims to refer to mutants of a naturally occurring or wild-type composition, e.g., variant polypeptides having less than 100% sequence identity with the naturally occurring or wild-type sequence.

[0121] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids. The terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, methylation, carboxylation, deamidation, acetylation, or conjugation with a labeling moiety.

[0122] The amino acid modification may be an amino acid substitution, an amino acid deletion, and / or an amino acid insertion. The amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative substitution (also called a conservative mutation, a conservative substitution, or a conservative variation) is an amino acid replacement in a protein that changes a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). As used throughout this disclosure, a "conservative variation" refers to the replacement of an amino acid residue with another, biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another; or the replacement of one polar residue with another, such as the replacement of arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine. Other examples of conservative substitutions include alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to praline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamic acid; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine, and the like.

[0123] The term "engineered" is used throughout this specification and claims to refer to a non-naturally occurring composition or protein that has properties that differ from the parent composition or protein from which it is derived.

[0124] Generally, "sequence identity" or "sequence homology" refers to the correspondence between nucleotides or amino acids of two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity". The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, an improved version of the BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. This program can be used to determine the percent identity over the entire length of the proteins being compared. For example, default parameters are provided for optimizing searches on short query sequences using the BLAST program.The program also allows the use of SEG filters to unmask segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Desirable ranges of sequence identity are approximately 80% to 100% and intervening integer values. Typically, the percent identity between the disclosed and claimed sequences is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0125] As used throughout this disclosure, "substantially identical" refers to having a sequence identity that is 85% or greater, e.g., 90% or greater, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, where the activity of the composition is not altered by modifications in the sequence that result in differences in sequence identity.

[0126] As used throughout this disclosure, an "amino acid mutation" refers to any difference in an amino acid sequence compared to the corresponding parent sequence, such as an amino acid substitution, deletion, and / or insertion.

[0127] As used throughout this disclosure, the term "promoter" refers to one or more nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the start site of transcription, such as a TATA element. A promoter may also include a cis-acting polynucleotide sequence that can be bound by a transcription factor. A "constitutive" promoter is a promoter that is active under many environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0128] As used throughout this disclosure, the term "viral vector", "viral vector" or "gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors of this disclosure include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, and retroviral vectors.

[0129] "AAV virion" or "AAV virus" or "AAV virus particle" or "AAV vector particle" or "AAV vector" refers to a viral particle that contains at least one AAV capsid polypeptide and a polynucleotide (AAV vector genome) enclosed in the capsid. The polynucleotide may contain heterologous nucleic acid (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell). AAV vectors are often named based on the name of the capsid polypeptide (serotype). For example, an AAV5 vector indicates that the AAV vector contains an AAV5 capsid polypeptide.

[0130] When used in reference to viral titer, the term "vector genome unit", "genomic unit", "genomic particle (gp)" or "genomic copy" (gc) refers to the number of vector genomes encapsidated in virions, regardless of infectivity or function. The number of genome particles in a particular vector preparation can be measured by methods well understood in the art, such as quantitative PCR of genomic DNA, or as described, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0131] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0132] An "infectious" virion, virus or virus particle is one that contains a polynucleotide component that can be delivered into a cell that is tropic for the virus species. This term does not necessarily mean the replicative ability of the virus. As used herein, an "infectious" virus or virus particle is one that can infect a target cell and express heterologous nucleic acid in the target cell when it approaches the target cell. Thus, "infectivity" refers to the ability of a virus particle to approach, enter, and express heterologous nucleic acid in the target cell. Infectivity may refer to in vitro infectivity or in vivo infectivity. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles.

[0133] The term "transducing units (tu)" when used with reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in a functional assay such as that described in, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0134] The ability of viral particles to express heterologous nucleic acid in cells can be referred to as "transduction".The ability of viral particles to express heterologous nucleic acid in cells can be assayed using several techniques, including marker gene evaluation, such as green fluorescent protein (GFP) assay (for example, when the virus contains the nucleotide sequence encoding GFP), in which green fluorescent protein (GFP) is produced and detected and / or measured in the cells infected with viral particles; or measurement of produced protein, for example, by enzyme-linked sorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).

[0135] The term "tropism" refers to the ability of an AAV vector to infect one or more specified cell types, but can also encompass the manner in which the vector functions to transduce cells in one or more specified cell types; that is, tropism refers to the preferential entry of AAV virions into a particular cell or tissue type and / or the entry into a particular cell or tissue type, and optionally followed by expression (e.g., transcription and optionally translation) of sequences carried by the AAV virion in the cell, e.g., for recombinant viruses, preferential interaction with the cell surface that facilitates expression of heterologous nucleotide sequences.

[0136] The term "tropism profile" refers to the pattern of transduction of one or more target cells, tissues and / or organs by the AAV vectors described herein.

[0137] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more transduction or tropism, respectively, of a control). A suitable control will depend on a variety of factors, including the desired tropism profile. Similarly, whether a capsid and / or virus "does not efficiently transduce" or "does not have efficient tropism" or similar terms for a target tissue can be determined by reference to a suitable control.

[0138] As used throughout this disclosure, "neuronal activity," "neuronal activity," "neuronal firing," and variations and synonyms thereof, refer to electrical activity resulting from stimulation or excitation of a neuron. In some embodiments, neuronal activity is measured using automated or manual patch clamp techniques. In some embodiments, determining neuronal activity includes determining an excitatory postsynaptic potential (EPSP), an inhibitory postsynaptic potential (IPSP), and / or an action potential of the neuron. In some embodiments, the level of neuronal activity depends on or is influenced by the excitatory postsynaptic potential (EPSP), the inhibitory postsynaptic potential (IPSP), and / or the action potential.

[0139] 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.

[0140] As used throughout this disclosure, a "sign" of a disease refers to a physical or mental characteristic that is considered to be indicative of a disease state. In some embodiments, the sign is an objective sign of the disease. In some embodiments, the sign is objectively assessed, examined, observed, or measured by a person other than the patient, such as a physician.

[0141] As used throughout this disclosure, a "symptom" of a disease refers to a physical or mental feature that is considered to be indicative of a disease state, particularly one that is evident to the patient. In some embodiments, a symptom is subjectively assessed by the patient. For example, in some embodiments, a symptom is pain; in some embodiments, a symptom is epilepsy.

[0142] As used throughout this disclosure with respect to amino acid positions, the term "corresponding to" or "corresponding to" refers to an amino acid in a first polypeptide sequence that aligns with a given amino acid in a reference polypeptide sequence when the first and reference polypeptide sequences are aligned. Alignment is performed by one of skill in the art using software designed for this purpose, such as Clustal Omega version 1.2.4, with the default parameters for that version.

[0143] Overview The present disclosure provides an adeno-associated virus (AAV) vector comprising a specific AAV capsid polypeptide for transducing dorsal root ganglion and / or trigeminal ganglion neurons. Such AAV vectors show enhanced transduction and / or tropism to dorsal root ganglion and / or trigeminal ganglion neurons compared to AAV vectors comprising control capsid polypeptides. In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure is delivered to DRG and / or TGG neurons to treat neuropathic pain. In some embodiments, the AAV vector further comprises a polynucleotide encoding an engineered ligand-gated ion channel (eLGIC) receptor or a chimeric version thereof.

[0144] In some embodiments, the AAV vectors comprising the capsid polypeptides of the present disclosure exhibit enhanced transduction in dorsal root ganglion and / or trigeminal ganglion neurons compared to AAV vectors comprising control capsid polypeptides. In some embodiments, the AAV vectors comprising the capsid polypeptides of the present disclosure exhibit enhanced tropism to dorsal root ganglion and / or trigeminal ganglion neurons compared to AAV vectors comprising control capsid polypeptides.

[0145] In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human dorsal root ganglion and / or trigeminal ganglion neurons in vivo compared to AAV vectors comprising a control capsid polypeptide. In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human dorsal root ganglion and / or trigeminal ganglion neurons in vitro compared to AAV vectors comprising a control capsid polypeptide. In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human dorsal root ganglion and / or trigeminal ganglion neurons ex vivo compared to AAV vectors comprising a control capsid polypeptide.

[0146] The present disclosure also provides an adeno-associated virus (AAV) vector comprising a specific AAV capsid polypeptide for transducing hippocampal neurons. Such an AAV vector exhibits enhanced transduction and / or tropism to hippocampal neurons compared to the AAV vector comprising a control capsid polypeptide. In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure is delivered to hippocampal neurons to treat focal epilepsy. In some embodiments, the AAV vector further comprises a polynucleotide encoding an engineered ligand-gated ion channel (eLGIC) receptor or a chimeric version thereof.

[0147] In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure exhibits enhanced transduction in hippocampal neurons compared to the AAV vector comprising a control capsid polypeptide.In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure exhibits enhanced tropism to hippocampal neurons compared to the AAV vector comprising a control capsid polypeptide.

[0148] In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human hippocampal neurons in vivo compared to AAV vectors comprising a control capsid polypeptide. In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human hippocampal neurons in vitro compared to AAV vectors comprising a control capsid polypeptide. In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction of human hippocampal neurons ex vivo compared to AAV vectors comprising a control capsid polypeptide.

[0149] In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure is part of a functional AAV capsid, said functional AAV capsid packaging a recombinant nucleic acid molecule comprising a polynucleotide selected from the group consisting of a non-coding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a gene targeting sequence, and a therapeutic expression cassette. In some embodiments, the functional AAV capsid packages a recombinant nucleic acid molecule comprising a polynucleotide encoding an engineered LGIC receptor or a chimeric version thereof.

[0150] In some embodiments, the polynucleotide is contained within an AAV vector. In some embodiments, the polynucleotide is a genome targeting cassette. In some embodiments, the expression cassette is a CRISPR / CAS expression system. In some embodiments, the therapeutic expression cassette encodes a therapeutic protein or antibody.

[0151] The present disclosure provides methods of using AAV vectors comprising the capsid polypeptides of the present disclosure in therapeutic treatment regimens, vaccines, or research tool development modalities.

[0152] The present disclosure also provides a method of using an AAV vector comprising a capsid polypeptide of the present disclosure to reduce the amount of total nucleic acid administered to a subject, the method comprising administering to the subject a lesser amount of total nucleic acid when the nucleic acid is transduced using an AAV vector comprising a control capsid polypeptide, compared to the amount of nucleic acid administered to the subject when the nucleic acid is transduced using such an AAV vector, to obtain a similar therapeutic effect.

[0153] The present disclosure provides an AAV vector comprising an AAV capsid polypeptide of the present disclosure, wherein the capsid polypeptide causes the AAV vector to exhibit increased transduction and / or tropism in human dorsal root ganglion and / or trigeminal ganglion neurons compared to a control capsid polypeptide.

[0154] The present disclosure also provides a method of treating neuropathic pain, such as peripheral neuropathy and trigeminal neuralgia, in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel or a chimeric version thereof.

[0155] The present disclosure also provides an AAV vector comprising an AAV capsid polypeptide of the present disclosure, wherein the capsid polypeptide causes the AAV vector to exhibit increased transduction and / or tropism in human hippocampal neurons compared to a control capsid polypeptide.

[0156] The present disclosure also provides a method of treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel or a chimeric version thereof.

[0157] Adeno-associated virus (AAV) vectors Adeno-associated virus (AAV), a member of the Parvoviridae family, is a small, non-enveloped, icosahedral virus with a single-stranded linear DNA genome of approximately 4.7 kilobases (kb). AAV was found as a contaminant in purified adenovirus stocks, and therefore the virus has been assigned to the Dependovirus genus (DM Knipe, PM Howley, Field's Virology, Lippincott Williams & Wilkins, Philadelphia, ed. Sixth, 2013). In its wild-type state, AAV is naturally replication-deficient, so AAV depends on a helper virus, typically an adenovirus, to provide the necessary protein factors for replication. The 4.7 kb genome of AAV is flanked by two inverted terminal repeats (ITRs) that fold into a hairpin shape that is important for replication. Being naturally replication-deficient and able to transduce nearly all cell types in the human body, AAV is an ideal vector for therapeutic use in gene therapy or vaccine delivery. In its wild type state, the life cycle of AAV includes a latent phase in which AAV genome is site-specifically integrated into host chromosome after infection, and an infectious phase in which the integrated genome is rescued, replicated, and packaged into infectious virus after infection with either adenovirus or herpes simplex virus.When vectorized, the viral Rep and Cap genes of AAV are removed and provided in trans during virus production, making ITR the only remaining viral DNA (A. Vasileva, R. Jessberger, Nature reviews. Microbiology, 3, 837-847 (2005)).Rep and Cap are then replaced with an array of possible transfer vector configurations to perform gene addition or gene targeting.These vectorized AAVs transduce both dividing and non-dividing cells and show robust and stable expression in quiescent tissues.Various published U.S. applications describe AAV vectors and virions, including U.S. Publication Nos. 2015 / 0176027, 2015 / 0023924, 2014 / 0348794, 2014 / 0242031, and 2012 / 0164106, all of which are incorporated by reference in their entireties herein.

[0158] However, despite the excellent ability of AAV vectors to transduce various tissues and cell types, there is still a need in the art for AAV vectors that show improved transduction of specific neuronal cells, both in terms of transduction efficiency and desired tropism.In addition, administration method may also affect the location, amount and type of cells that are transduced.There are physical limitations to how much AAV can be delivered in a single injection, and because desired results (e.g., therapeutic results) are related to stimulating or suppressing the activity of the correct group of neurons in a subject, high levels of transduction and correct tropism are required for delivery to neurons.

[0159] The present disclosure provides AAV capsid polypeptides that demonstrate significantly improved transduction of subpopulations of neurons in a subject in vivo, in vitro and / or ex vivo.

[0160] The genomic organization of all known AAV serotypes is similar. The genome of AAV is a linear single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) long. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences of the nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, 2 and 3) form the capsid and contribute to the tropism of the virus. The terminal 145 nt ITRs are self-complementary and organized such that energetically stable intramolecular duplexes that form T-shaped hairpins can form. These hairpin structures serve as origins for viral DNA replication and as primers for the cellular DNA polymerase complex. After wild-type (wt) AAV infection in mammalian cells, the Rep genes are expressed and function in the replication of the viral genome.

[0161] In some embodiments, the envelope protein "capsid" of the viral vector is naturally occurring, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the capsid is synthetically engineered (e.g., by directed evolution or rational design) to have certain unique characteristics that do not occur in nature, such as altered tropism, increased transduction efficiency, and / or immune evasion. One example of a rationally designed capsid is the mutation of one or more surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on the VP3 viral capsid polypeptide.

[0162] Non-limiting examples of AAV ITRs that can be used in an AAV vector include ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16.

[0163] AAV vectors that contain two ITRs have a payload capacity of about 4.4 kB. Self-complementary AAV (scAAV) vectors contain a third ITR and package two strands of the recombinant portion of the vector, leaving only about 2.1 kB for the polynucleotide contemplated herein. In one embodiment, the AAV vector is a scAAV vector.

[0164] Expanded packaging capacity, roughly double that of AAV (about 9 kB), has been achieved using dual AAV vector strategy. Dual vector strategies useful for producing AAV contemplated herein include, but are not limited to, splicing (trans-splicing), homologous recombination (overlap), or a combination of the two (hybrid). In dual AAV trans-splicing strategy, splice donor (SD) signal is placed at the 3' end of the 5' half of the vector, and splice acceptor (SA) signal is placed at the 5' end of the 3' half of the vector. Upon co-infection of the same cell with dual AAV vectors and two halves of inverted terminal repeat (ITR)-mediated head-to-tail concatenation, trans-splicing leads to the production of mature mRNA and full-size protein (Yan et al., 2000). Trans-splicing has been successfully used to express large genes in muscle and retina (Reich et al., 2003; Lai et al., 2005). Alternatively, the two halves of a large transgene expression cassette contained in a dual AAV vector may contain homologous overlapping sequences (the 3' end of the 5' half of the vector and the 5' end of the 3' half of the vector overlapping dual AAV), which will mediate the reconstitution of a single large genome by homologous recombination (Duan et al., 2001). This strategy relies on the recombinogenic properties of the transgene overlapping sequences (Ghosh et al., 2006). The third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene (i.e., alkaline phosphatase; Ghosh et al., 2008, Ghosh et al., 2011) to the trans-splicing vector. The added region is placed downstream of the SD signal in the 5' half of the vector and upstream of the SA signal in the 3' half of the vector to increase recombination between the dual AAVs.

[0165] "Hybrid AAV" refers to an AAV genome packaged in a capsid of a different AAV serotype (and preferably a different serotype derived from one or more AAV ITRs), and can be alternatively referred to as a pseudotype AAV. For example, a genome of AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 can be encapsidated within a capsid of AAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a variant thereof, provided that the AAV capsid and genome (and preferably one or more AAV ITRs) are of different serotypes. In certain embodiments, pseudotyped AAV particles can be referred to as being of the "x / y" type, where "x" indicates the source of the ITRs and "y" indicates the serotype of the capsid, e.g., a 2 / 5 AAV particle has ITRs derived from AAV2 and a capsid derived from AAV5.

[0166] "Host cells" include cells that are transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present disclosure. Host cells may include virus-producing cells and cells infected with viral vectors. In some embodiments, in vivo host cells are infected with viral vectors contemplated herein. In certain embodiments, the term "target cell" is used interchangeably with host cell and refers to an infected cell of a desired cell type.

[0167] High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. Nos. 5,658,776; 6,566,118; 6,989,264; and 6,995,006; US2006 / 0188484; WO98 / 22607; WO2005 / 072364; and WO / 1999 / 011764; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003; Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; lnoue et al., (1998) J. Virol. 72, 7024. Methods for producing pseudotyped AAV vectors (e.g., WO00 / 28004) have also been reported, as well as various modifications or formulations of AAV vectors to reduce their immunogenicity upon administration in vivo (see, e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562).

[0168] In some embodiments, an AAV vector comprising an AAV capsid polypeptide (or a mutant of the present disclosure) contributes to targeted expression of an engineered receptor to a subpopulation of cells or neurons of a subject. In some embodiments, the neurons are nociceptors.

[0169] Capsid Polypeptide In some embodiments, the present disclosure provides capsid polypeptides that allow corresponding encapsidated AAV vectors to achieve enhanced transduction and / or targeting to at least a subpopulation of neuronal cells. Exemplary capsid polypeptides of the present disclosure are listed in Table 1 below: [Table 1-1] [Table 1-2]

[0170] In some embodiments, the capsid polypeptide of the present disclosure is AAV2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:1. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:13.

[0171] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:14.

[0172] In some embodiments, the capsid polypeptide of the disclosure is AAV2.5-TV. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492V in SEQ ID NO:2. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:3. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:3, and the capsid polypeptide comprises a valine at a position corresponding to V492 in SEQ ID NO:3. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 3, and the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 3. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 15.

[0173] In some embodiments, the capsid polypeptide of the disclosure is AAV2.5-2YF. In some embodiments, the capsid polypeptide of the 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 disclosure comprises an amino acid substitution at a position corresponding to Y705F and / or Y731F of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:4. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:4, and the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO:4. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 4, and the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO: 4. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 16.

[0174] In some embodiments, the capsid polypeptide of the present disclosure is AAV2.5-TV2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492, Y705, and / or Y731 of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492V, Y705F, and / or Y731F of SEQ ID NO:2. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:5. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:5, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO:5, and the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO:5. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 5, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 5, and wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO: 5. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 17.

[0175] In some embodiments, the capsid polypeptide of the present disclosure is AAV5. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:18.

[0176] In some embodiments, the capsid polypeptide of the present disclosure is AAV6. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:7. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:7. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:7. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:19.

[0177] In some embodiments, the capsid polypeptide of the present disclosure is AAV9. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:20.

[0178] In some embodiments, the capsid polypeptide of the disclosure is AAV9-TV. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution corresponding to T492V in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:9. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:9, and the capsid polypeptide comprises a valine at a position corresponding to V492 in SEQ ID NO:9. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 9, and the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 9. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:21.

[0179] In some embodiments, the capsid polypeptide of the disclosure is AAV9-2YF. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to Y705 and / or Y731 of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises an amino acid substitution at a position corresponding to Y705F and / or Y731F of SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence set forth in SEQ ID NO:10. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:10, and the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO:10. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 10, and the capsid polypeptide comprises a valine at a position corresponding to F705 and / or F731 of SEQ ID NO: 10. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:22.

[0180] In some embodiments, the capsid polypeptide of the present disclosure is AAV9-TV2YF. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492, Y705, and / or Y731 of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure comprises an amino acid substitution at a position corresponding to T492V, Y705F, and / or Y731F of SEQ ID NO: 8. In some embodiments, the capsid polypeptide of the present disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:11, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO:11, and the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO:11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence that is mutated (substituted, deleted, and / or added) by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids compared to the sequence of SEQ ID NO: 11, wherein the capsid polypeptide comprises a valine at a position corresponding to V492 of SEQ ID NO: 11, and wherein the capsid polypeptide comprises a phenylalanine at a position corresponding to F705 and / or F731 of SEQ ID NO: 11. In some embodiments, a capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:23.

[0181] In some embodiments, the capsid polypeptide of the disclosure is AAV-PHP.S. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 12. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO: 12. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:24.

[0182] In some embodiments, the AAV vector comprises an AAV capsid polypeptide comprising an amino acid mutation at one or more positions corresponding to T492, Y705, and / or Y731 of the AAV9 capsid polypeptide (SEQ ID NO: 8), which is of serotype AAV2, AAV2.5, AVV5, AAV6, AAV9, AAV-PHP.S, or another AAV serotype. In some embodiments, the one or more positions are two or more positions, two positions, or three positions. In some embodiments, the viral vector comprises an AAV capsid polypeptide comprising one or more amino acid substitutions corresponding to T492V, Y705F, and / or Y731F of the AAV9 capsid polypeptide (SEQ ID NO: 8), which is of serotype AAV2, AAV2.5, AVV5, AAV6, AAV9, AAV-PHP.S, or another AAV serotype. In some embodiments, the one or more substitutions is two or more substitutions, two substitutions, or three substitutions.

[0183] 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.

[0184] In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 99.7% sequence identity to SEQ ID NO:8, and the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the capsid polypeptide of the disclosure comprises, consists essentially of, or consists of an amino acid sequence in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are mutated (substituted, deleted, and / or added) compared to the sequence of SEQ ID NO:8, and the capsid polypeptide comprises an amino acid substitution at a position corresponding to T492 in SEQ ID NO:8. In some embodiments, the substitution is T492V. In some embodiments, the substitution is T492I. In some embodiments, the substitution is T492L. In some embodiments, the T492 residue is substituted with a hydrophobic amino acid selected from valine (Val), leucine (Leu), and isoleucine (Ile). In some embodiments, the T492 residue is substituted with a hydrophobic amino acid selected from glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).

[0185] Neurons and Nerve Fibers In some embodiments, the present disclosure provides methods of transducing target neurons located in the hippocampus, i.e., hippocampal neurons.

[0186] In some embodiments, the hippocampal neuron is an excitatory neuron. In some embodiments, the hippocampal neuron is a Ca2+ / calmodulin-dependent protein kinase II (CaMKII) positive neuron. In some embodiments, the hippocampal neuron is an inhibitory neuron. In some embodiments, the hippocampal neuron is a GABAergic neuron.

[0187] In some embodiments, the present disclosure provides a method of transducing target neurons located in the dorsal root ganglion (or spinal ganglion; also known as dorsal root ganglion). Dorsal root ganglion (DRG) is a mass of neurons (ganglion) in the dorsal root of spinal nerve. The cell bodies of sensory neurons, known as primary neurons, are located in the dorsal root ganglion. The axons of dorsal root ganglion neurons are known as afferent nerves. In the peripheral nervous system, afferent nerves refer to axons that relay sensory information to the central nervous system (i.e., brain and spinal cord).

[0188] In some embodiments, the present disclosure provides a method of transducing target neurons located in the trigeminal ganglion (or Gasserian ganglion, or semilunar ganglion, or Gasserian ganglion). The trigeminal ganglion (TGG) is a sensory ganglion of the trigeminal nerve (CNV) that occupies a cavity in the dura (Meckel's space), overlying the trigeminal nerve impingement near the apex of the petrous part of the temporal bone.

[0189] Axons (also known as nerve fibers) are the long, thin projections of neuronal cells in vertebrates that typically conduct electrical impulses known as action potentials away from the nerve cell body. Each neuron has only one axon. Thus, neuronal cells can be classified by their axons (nerve fibers).

[0190] Three general classes of nerve fibers (groups A, B, and C) have been classified by Erlanger and Gasser: group A nerve fibers are heavily myelinated, group B nerve fibers are moderately myelinated, and group C nerve fibers are unmyelinated.

[0191] There are four subdivisions of group A nerve fibers (A fibers): alpha (α), beta (β), gamma (γ), and delta (δ). These subdivisions have different amounts of myelination and axon thickness and therefore transmit signals at different rates. Aα type fibers include the alternative classification system of type Ia and type Ib sensory fibers, which are fibers originating from muscle spindle endings and Golgi tendons, respectively. Aβ type fibers, and Aγ type fibers, are type II afferent fibers originating from stretch receptors. Many of the Aβ type fibers originating from the skin are dedicated to touch. Aδ type fibers are nociceptor afferent fibers. Aδ fibers carry information from peripheral mechanoreceptors and thermoreceptors to the dorsal horn of the spinal cord. Aδ fibers serve to receive and transmit information primarily associated with acute pain (sharp, immediate, and relatively short-lasting). This type of pain can result from several stimuli classifications: temperature-induced, mechanical, and chemical. This may be part of a withdrawal reflex initiated by Aδ fibers in the reflex arc that activate a withdrawal response. Aδ fibers carry cold pain, pressure pain, and acute pain signals; because they are thin (2-5 μm in diameter) and myelinated, they transmit impulses faster than unmyelinated C fibers, but more slowly than the other, thicker myelinated A group nerve fibers. Their conduction velocity is moderate.

[0192] Group B nerve fibers (B fibers) are axons that are moderately myelinated, meaning that they are less myelinated than group A nerve fibers and more myelinated than group C nerve fibers. They are usually general visceral afferent fibers and preganglionic nerve fibers of the autonomic nervous system.

[0193] Group C nerve fibers (C fibers) are unmyelinated and have small diameters and low conduction velocities. Group C fibers include postganglionic fibers in the autonomic nervous system (ANS) and dorsal root nerve fibers (IV fibers). These fibers carry sensory information. Damage or injury to these nerve fibers causes neuropathic pain.

[0194] The peripheral end of the mature nociceptor is where the stimulus is detected and converted into electrical energy. When the electrical energy reaches a threshold, an action potential is induced and driven toward the central nervous system (CNS). This results in a chain of events that allows for the subjective awareness of pain. The sensory specificity of the nociceptor is established only by a high threshold for a particular feature of the stimulus. The nociceptor is triggered only when a high threshold is reached by either the chemical, thermal, or mechanical environment. Most nociceptors are classified by the environmental modality to which they respond. Some nociceptors respond to more than one of these modalities and are consequently designated as multimodal. Other nociceptors do not respond to any of these modalities (but they can respond to stimuli under inflammatory conditions) and are referred to as inactive or silent.

[0195] Nociceptors have two different types of axons. The first are A-delta fiber axons. They are myelinated and can transmit action potentials toward the CNS at a speed of about 20 meters per second. The other type is the slower conducting C-fiber axon. These only conduct at a speed of about 2 meters per second. This is due to light myelination or non-myelination of the axon. As a result, pain comes in two phases. The first phase is mediated by the fast conducting A-delta fibers and the second part due to the (polymodal) C-fibers. Pain associated with A-delta fibers may be associated with an initial extremely sharp pain. The second phase is a more prolonged and slightly less intense sensation of pain as a result of acute injury. When there is a large or prolonged input to the C-fibers, there is a progressive potentiation in the spinal dorsal horn; this phenomenon is similar to tetanus in muscles, but is called winding. When winding occurs, there is the possibility of increased sensitivity to pain.

[0196] Different classes of nerve fibers can be distinguished by biomarkers such as neurofilament 200 (NF200, also known as neurofilament heavy chain polypeptide), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4). Aα and Aβ fibers are typically NF200 positive but IB4 negative. Aδ fibers are typically positive for both NF200 and IB4. C fibers are typically IB4 positive but NF200 negative. Thus, many of the NF200+ neurons contain heavily myelinated Aα or moderately myelinated Aβ fibers. In some cases, these neurons mediate signals for light touch and proprioception. However, some NF200+ neurons may also mediate signals for nociception. Many of the IB4+ neurons contain unmyelinated C fibers or Aδ fibers for nociception. And many of the CGRP+ neurons contain unmyelinated C fibers or lightly myelinated Aδ fibers for nociception.

[0197] Properties of the Capsid Polypeptide In one aspect of the present disclosure, an AAV vector comprising a capsid polypeptide of the present disclosure exhibits increased transduction and / or tropism in one or more neurons compared to an AAV vector comprising a control capsid polypeptide. In some embodiments, the control capsid polypeptide is an AAV2 capsid polypeptide (SEQ ID NO: 1). In some embodiments, the control capsid polypeptide is an AAV2.5 capsid polypeptide (SEQ ID NO: 2). In some embodiments, the control capsid polypeptide is an AAV9 capsid polypeptide (SEQ ID NO: 8). In some embodiments, the control capsid polypeptide is an AAV5 capsid polypeptide (SEQ ID NO: 6). Those skilled in the art will readily recognize suitable control capsid polypeptides for testing, for example, as exemplified in the Examples section of the present disclosure.

[0198] Transduction / tropism can be measured by techniques known in the art, such as, 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.

[0199] In some embodiments, AAV vectors comprising the capsid polypeptides of the present disclosure exhibit increased transduction in one or more target neurons compared to AAV vectors comprising a control capsid polypeptide. In some embodiments, transduction 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). In some embodiments, transduction 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).

[0200] In some embodiments, the AAV vector comprising the capsid polypeptide of the present disclosure shows increased tropism in one or more target neurons compared to the AAV vector comprising a control capsid polypeptide. In some embodiments, the tropism is measured by the percentage of transduced target neurons in the total number of transduced neurons at a particular location of a subject. In some embodiments, the tropism 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%, or more than 95% (including all ranges and subranges therebetween). In some embodiments, directionality 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%, or at least 95% (including all ranges and subranges therebetween).

[0201] In some embodiments, the target neuron is a hippocampal neuron, hi some embodiments, the target neuron is an excitatory neuron.

[0202] In some embodiments, the target neuron is a DRG or TGG neuron.

[0203] In some embodiments, the target neuron comprises A fibers. In some embodiments, the target neuron comprises Aα or Aβ fibers. In some embodiments, the target neuron comprises Aδ fibers. In some embodiments, the target neuron comprises C fibers.

[0204] In some embodiments, the target neuron comprises nerve fibers that are NF200 positive and IB4 negative. In some embodiments, the target neuron comprises nerve fibers that are NF200 positive and IB4 positive. In some embodiments, the target neuron comprises nerve fibers that are NF200 negative and IB4 positive. In some embodiments, the target neuron comprises nerve fibers that are CGRP positive.

[0205] heterologous nucleic acid In some embodiments, the AAV vector of the present disclosure further comprises a heterologous nucleic acid, the heterologous nucleic acid comprising a nucleotide sequence encoding a receptor. In some embodiments, the receptor is an engineered ligand-gated ion channel (LGIC) or a chimeric version thereof. In some embodiments, the engineered LGIC can be activated by a small molecule ligand. In some embodiments, administration of the AAV vector delivers the heterologous nucleic acid encoding eLGIC to a subpopulation of neuronal cells in the subject, causing expression of eLGIC in the neurons. In some embodiments, expression of eLGIC can modulate the activity of these neurons by administration of a corresponding small molecule ligand.

[0206] In some embodiments, heterologous nucleic acid is operably linked to one or more control elements that direct its transcription or expression.The control elements include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance the secretion of encoded products.A large number of expression control sequences are known in the art and can be used, including promoters selected from natural, constitutive, inducible and / or tissue-specific.

[0207] Such control elements may include control sequences normally associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be used. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, endogenous cellular promoters heterologous to the gene of interest, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMV-IE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Additionally, sequences derived from non-viral genes, such as the mouse metallothionein gene, can also be used.

[0208] In some embodiments, a cell type-specific or tissue-specific promoter is operably linked to the heterologous nucleic acid to selectively or preferentially produce the corresponding gene product encoded by the heterologous nucleic acid in a particular cell type or tissue, hi some embodiments, an inducible promoter is operably linked to the heterologous nucleic acid.

[0209] In some embodiments, heterologous nucleic acids of the present disclosure can be delivered to neurons using non-AAV methods (e.g., using non-AAV viral vectors, non-viral vectors, synthetic nanoparticles, etc.).

[0210] In some embodiments, the heterologous nucleic acid of the present disclosure is incorporated into a vector.In some embodiments, the vector is a viral vector.In some embodiments, the viral vector is derived from a replication-defective virus.In some embodiments, the viral vector is an adenovirus, a retrovirus (e.g., lentivirus), an adeno-associated virus (AAV), a poxvirus, an alphavirus, a vaccinia virus, or a herpes simplex virus (e.g., HSV-1).

[0211] Engineered Ligand-Gated Ion Channels (eLGICs) The present disclosure describes a receptor, its mutants, and methods for its 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.

[0212] In some embodiments, the receptor is an engineered receptor (e.g., eLGIC). The term "engineered receptor" as used herein refers to a receptor that has been experimentally altered to be physically and / or functionally different from its corresponding parent receptor. In some embodiments, the parent receptor is a wild-type receptor. The term "wild-type receptor" as used herein refers to a receptor that has a polypeptide sequence that is identical to the polypeptide sequence of a protein found in nature. Wild-type receptors include receptors that naturally occur in humans as well as orthologues that naturally occur in other eukaryotes, e.g., protists, fungi, plants or animals, e.g., yeast, insects, nematodes, sponges, mammals, non-mammalian vertebrates. In some embodiments, the parent receptor is a non-natural receptor; that is, it is a receptor that does not occur in nature, e.g., a receptor that has been engineered from a wild-type receptor. For example, the parent receptor may be an engineered receptor that includes one or more subunits from one wild-type receptor along with one or more subunits from a second wild-type receptor. The resulting protein thus includes subunits from two or more wild-type receptors. Thus, in some embodiments, the parent receptor is a chimeric receptor. Engineered receptors of the present disclosure include, for example, parent receptor mutants, and switch receptors.

[0213] In some aspects, the engineered receptors of the present disclosure comprise at least one amino acid mutation compared to the corresponding parent receptor, e.g., one or more mutations in one or more domains of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptor has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, or less sequence identity (including all values ​​and subranges therebetween) with the corresponding parent receptor. In some embodiments, the parent receptor mutant has 85% or more sequence identity with the corresponding parent receptor, e.g., 90% or more, or 95% or more, e.g., about 96%, about 97%, about 98%, or about 99% identity (including all values ​​and subranges therebetween) with the corresponding parent receptor. In some embodiments, the engineered receptor (e.g., parent receptor mutant) is generated by error-prone PCR.

[0214] In some embodiments, the ligand binding domain (LBD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ligand binding domain of the parent receptor, 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, 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 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% identity sequence identity (including all values ​​and subranges therebetween) with the corresponding ligand binding domain of the parent receptor.

[0215] In some embodiments, the ion pore domain (IPD) of the engineered receptor of the present disclosure comprises at least one amino acid mutation compared to the corresponding ion pore domain of the parent receptor, e.g., one or more mutations in the ion pore domain of the wild-type receptor. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the ion pore domain of the engineered receptor has 85% or more sequence identity with the corresponding ion pore domain of the parent receptor, e.g., 90% or more, or 95% or more, e.g., about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity, or 100% identity (including all values ​​and subranges therebetween) with the corresponding ion pore domain of the parent receptor. In some embodiments, the ion pore domain of the engineered receptor has a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity (including all values ​​and subranges therebetween) to the corresponding ion pore domain of the parent receptor.

[0216] In some embodiments, the amino acid mutation is a loss-of-function amino acid mutation compared to the corresponding parent receptor. A "loss-of-function" amino acid mutation refers to one or more mutations that reduce, substantially reduce, or abolish the function of the engineered receptor compared to the parent receptor, for example, by reducing the binding of endogenous ligand to the engineered receptor compared to the binding of endogenous ligand to the parent receptor, or by reducing the activity of the downstream signaling pathway of the engineered receptor that is typically activated in response to the binding of ligand to the corresponding parent receptor. In some embodiments, the mutation is an amino acid substitution.

[0217] In some embodiments, the amino acid mutation is a gain-of-function amino acid mutation compared to the corresponding parent receptor. A "gain-of-function" amino acid mutation refers to one or more mutations that modify the function of the engineered receptor compared to the parent receptor, for example, by altering or enhancing the affinity of the engineered receptor for a ligand compared to the binding of the endogenous ligand to the parent receptor, or by altering or enhancing the activity of a signaling pathway that is activated in response to the binding of a ligand to the engineered receptor compared to the binding of the endogenous ligand to the corresponding parent receptor. In some embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for a ligand. In certain embodiments, the gain-of-function mutation results in an increase in the affinity of the engineered receptor for an agonist ligand. In some embodiments, the gain-of-function mutation results in an antagonist ligand that acts as an agonist ligand upon binding to the engineered receptor (e.g., resulting in the activation of an agonist signaling pathway instead of an antagonist signaling pathway). In some embodiments, the gain-of-function mutation results in a modulator ligand that acts as an agonist ligand upon binding to the engineered receptor. In some embodiments, the mutation is an amino acid substitution.

[0218] In some embodiments, the engineered receptors subject to the present disclosure, or their ligand binding domains and / or ion pore domains, contain one or more loss-of-function amino acid mutations and one or more gain-of-function amino acid mutations compared to the corresponding parent receptor, in some embodiments, the mutations are amino acid substitutions.

[0219] In some embodiments, the loss-of-function mutation and the gain-of-function mutation are at the same residue, i.e., they are the same mutation. In other embodiments, the loss-of-function mutation and the gain-of-function mutation are mutations at different amino acid residues. In some embodiments, the mutation is an amino acid substitution. In some embodiments, the engineered receptor of interest (or its ligand-binding domain and / or ion pore domain) containing the loss-of-function mutation and / or the gain-of-function mutation has about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about 60%, about 50% or less sequence identity (including all ranges and subranges therebetween) with the corresponding parent receptor, e.g., wild-type receptor or non-natural receptor (or its ligand-binding domain and / or ion pore domain). In some embodiments, the engineered receptor of interest (or its ligand binding domain and / or ion pore domain) has 85% or more sequence identity, e.g., 85%, 90%, or 95% or more sequence identity, and in some examples, 96%, 97%, 98% or more sequence identity, e.g., 99% or 99.5% or more sequence identity (including all values ​​and subranges therebetween), to the corresponding parent receptor (or its ligand binding domain and / or ion pore domain).

[0220] In some aspects, the engineered receptor of the present disclosure comprises a receptor resulting from the combination of one or more amino acid sequences, e.g., subunits, from one wild-type receptor with one or more amino acid sequences, e.g., subunits, from a second wild-type receptor. In other words, the engineered receptor comprises amino acid sequences that are heterologous to each other, where "heterologous" means that they do not exist together in nature. Such receptors are referred to herein as "chimeric receptors". In some embodiments, the chimeric receptor serves as a parent receptor from which the engineered receptor of the present disclosure is generated. In some embodiments, the chimeric receptor comprises a ligand binding domain from a first LGIC and an ion pore domain from a second LGIC.

[0221] In some embodiments, the parent receptor mutant exhibits increased affinity for an agonist ligand, hi some embodiments, a ligand that functions as an antagonist or modulator when bound to the wild-type receptor functions as an agonist when bound to the parent receptor mutant.

[0222] In some embodiments, the engineered receptor is a "ligand-gated ion channel" or LGIC. LGICs refer to a large group of transmembrane proteins that allow the passage of ions upon activation by a specific ligand. LGICs are composed of at least two domains: a ligand-binding domain and a transmembrane ion pore domain. Ligand binding to a LGIC results in activation of the LGIC and opening of the ion pore. Ligand binding causes a dramatic change in the permeability of the channel to a specific ion or ions; when it is inactive or closed, ions cannot effectively pass through the channel, but upon ligand binding, up to 107 ions / sec can pass through. In some embodiments, LGICs respond to extracellular ligands (e.g., neurotransmitters) and facilitate the influx of ions into the cytosol. In some embodiments, LGICs respond to intracellular ligands (e.g., nucleotides such as ATP and signaling intermediates such as PIP2) and facilitate the efflux of ions from the cytosol to the extracellular environment. Importantly, activation of LGIC results in the transport of ions (e.g., Ca2+, Na+, K+, Cl-, etc.) across the cell membrane and not the transport of the ligand itself.

[0223] LGIC receptors contain multiple subunits and may be either homomeric or heteromeric receptors. Homomeric receptors contain subunits that are all of the same type. Heteromeric receptors contain subunits where at least one subunit is different from at least one other subunit contained within the receptor. For example, glycine receptors contain five subunits where there are two types: α-subunits where there are four isoforms (α1-α4) and β-subunits where there is a single known isoform. An exemplary homomeric GlyR is a GlyR that contains five α1-GlyR subunits. Similarly, homomeric GABAA receptors may contain β3-GABAA subunits and nAchR receptors may contain α7-nAchR subunits. An exemplary heteromeric GlyR may contain one or more α-subunits and one or more β-subunits (e.g., α1β-GlyR). Exemplary LGIC receptor subunits are shown in Table 2. [Table 2-1] [Table 2-2]

[0224] Illustrative examples of families of LGICs suitable for use in certain embodiments include, but are not limited to, Cys-loop receptors such as glycine receptors (GlyRs), serotonin receptors (e.g., 5-HT3 receptors), lambda-aminobutyric acid A (GABA-A) receptors, and nicotinic acetylcholine receptors (nAchRs); as well as acid-sensing (proton-gated) ion channels (ASICs), epithelial sodium channels (ENaCs), ionotropic glutamate receptors, IP3 receptors, P2X receptors, ryanodine receptors, and zinc-activated channels (ZACs).

[0225] 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; GRIA4;GRID1;GRID2;GRIK1;GRIK2;GRIK3;GRIK4;GRIK5;GRIN1;GRIN2A;GRIN2B;GRIN2C;GRIN2D;GRIN3A;GRIN3B;ITPR1;ITPR2;ITPR3;CHRNA1;CHRNA2;CHRNA3;CHRNA4;CHRNA5;CHRNA6;CHRNA7;CHRNA9;CHRNA10;CHRNB1;CHRNB2;CHRNB3;CHRNB4;CHRNG;CHRND;CHRNE;P2RX1;P2RX2;P2RX3;P2RX4;P2RX5;P2RX6;P2RX7;RYR1;RYR2;RYR3; and ZACN.

[0226] Non-limiting examples of wild-type LGIC receptor sequences that are useful in generating engineered receptors of the present disclosure include the following, in which the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is underlined:

[0227] In some embodiments, the wild-type LGIC receptor is the human nicotinic cholinergic receptor alpha 7 subunit (α7-nAchR) (GenBank Accession No. NP_000737.1, SEQ ID NO: 25), encoded by the CHRNA7 gene (GenBank Accession No. NM_000746.5): [ka]

[0228] In some embodiments, the wild-type LGIC receptor is the human alpha 1 glycine receptor (GlyRα1) (GenBank Accession No. NP_001139512.1, SEQ ID NO: 26), encoded by the GLRA1 gene (GenBank Accession No. NM_001146040.1): [ka]

[0229] In some embodiments, the wild-type LGIC receptor is the human alpha 2 glycine receptor (GlyRα2) (GenBank Accession No. NP_001112357.1, SEQ ID NO: 27), encoded by the GLRA2 gene (GenBank Accession No. NM_001118885.1): [ka]

[0230] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform L (GenBank Accession No. NP_006520.2, SEQ ID NO: 28), encoded by the GLRA3 gene (GenBank Accession No. NM_006529.3): [ka]

[0231] In some embodiments, the wild-type LGIC receptor is the human alpha 3 glycine receptor (GlyRα3) isoform K (GenBank Accession No. NP_001036008.1, SEQ ID NO: 29), encoded by the GLRA3 gene (GenBank Accession No. NM_001042543.3): [ka]

[0232] In some aspects, the engineered receptor is a chimeric LGIC receptor. In some embodiments, the chimeric receptor comprises a ligand binding domain sequence from at least a first LGIC and an ion pore conducting domain sequence from at least a second LGIC, or more simply, an "ion pore domain sequence". In some embodiments, the derived amino acid sequence is identical to the corresponding region of the LGIC from which it is derived. In some embodiments, the derived amino acid sequence may contain an alteration at at least one amino acid position compared to the corresponding region of the LGIC from which it is derived. In some embodiments, the amino acid sequence derived from the LGIC sequence differs from the corresponding region of the original amino acid sequence by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the derived amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity (including all ranges and subranges therebetween) to the corresponding region of the LGIC amino acid sequence.

[0233] In some embodiments, the first and second LGICs are Cys loop receptors. The ligand binding domain sequence and ion pore domain sequence of Cys loop receptors are well known in the art and can be easily identified from the literature by using publicly available software such as PubMed, Genbank, Uniprot, etc. In some embodiments, the ligand binding domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ligand binding domain of the first LGIC. In some embodiments, the ion pore domain of the chimeric receptor has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to the ion pore domain of the second LGIC. In the above sequences, the ligand binding domain is shown in bold and the ion pore domain is shown underlined.

[0234] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1 (SEQ ID NO:26). In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 29-235 of GlyRα1, e.g., amino acids 29-235, amino acids 29-240, amino acids 29-246, amino acids 29-248, amino acids 29-250, or amino acids 29-252 of SEQ ID NO:26. In certain such embodiments, the ligand binding domain consists essentially of amino acids 29-235 of SEQ ID NO: 26, consists essentially of amino acids 29-240 of SEQ ID NO: 26, consists essentially of amino acids 29-246 of SEQ ID NO: 26, consists essentially of amino acids 29-248 of SEQ ID NO: 26, consists essentially of amino acids 29-250 of SEQ ID NO: 26, or consists essentially of amino acids 29-252 of SEQ ID NO: 26. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than human GlyRα1.

[0235] In some embodiments, the ligand binding domain of the chimeric receptor comprises a ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises around amino acids 23-220 of the human α7-nAChR (SEQ ID NO:25), for example, amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO:25. In some embodiments, the ligand binding domain consists essentially of amino acids 23-220, amino acids 23-221, amino acids 23-222, amino acids 23-223, amino acids 23-224, amino acids 23-225, amino acids 23-226, amino acids 23-227, amino acids 23-228, amino acids 23-229, amino acids 23-230, or amino acids 23-231 of SEQ ID NO: 25. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0236] In some embodiments, the ligand binding domain of the chimeric receptor is derived from the ligand binding domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 23-220 of human α7-nAChR (SEQ ID NO:25), such as amino acids 23-220, 23-221, 23-222, 23-223, 23-224, 23-225, 23-226, 23-227, 23-228, 23-229, 23-230, or 23-231 of SEQ ID NO:25. In some embodiments, the ion pore domain sequence is derived from a Cys loop receptor other than the human α7-nAChR.

[0237] In some embodiments, the ion pore domain of the engineered receptor is derived from the ion pore domain sequence of the human glycine receptor. In some embodiments, the human glycine receptor is human GlyRα1. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 245-457 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 240-457, amino acids 245-457, amino acids 248-457, amino acids 249-457, amino acids 250-457, amino acids 255-457, or amino acids 260-457 of SEQ ID NO:26. In some embodiments, the ion pore domain consists essentially of amino acids 245-457 of SEQ ID NO:26, consists essentially of amino acids 248-457 of SEQ ID NO:26, consists essentially of amino acids 249-457 of SEQ ID NO:26, or consists essentially of amino acids 250-457 of SEQ ID NO:26.

[0238] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα2 (SEQ ID NO:27). In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO:27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 254-452 of SEQ ID NO: 27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 258-452 of SEQ ID NO: 27. In some embodiments, the ion pore domain sequence of human GlyRα2 comprises, consists essentially of, or consists of amino acids 260-452 of SEQ ID NO: 27.

[0239] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform L (SEQ ID NO:28). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 253-464 of SEQ ID NO: 28. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 257-464 of SEQ ID NO: 28. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform L comprises, consists essentially of, or consists of amino acids 259-464 of SEQ ID NO: 28.

[0240] In some embodiments, the ion pore domain of the chimeric receptor comprises an ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence derived from the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain of the chimeric receptor comprises, consists essentially of, or consists of an amino acid sequence identical to the ion pore domain sequence of human GlyRα3 isoform K (SEQ ID NO:29). In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 253-449 of SEQ ID NO: 29. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 257-449 of SEQ ID NO: 29. In some embodiments, the ion pore domain sequence of human GlyRα3 isoform K comprises, consists essentially of, or consists of amino acids 259-449 of SEQ ID NO: 29.

[0241] In some embodiments, the ion pore domain is derived from an ion pore domain sequence of a human nicotinic cholinergic receptor. In some embodiments, the human nicotinic cholinergic receptor is a human α7-nAChR. In some embodiments, the ion pore domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to amino acids 230-502, e.g., amino acids 227-502, amino acids 230-502, amino acids 231-502, amino acids 232-502, or amino acids 235-502 of α7-nAChR (SEQ ID NO:25). In certain such embodiments, the ion pore domain consists essentially of amino acids 227-502 of SEQ ID NO:25, consists essentially of amino acids 230-502 of SEQ ID NO:25, consists essentially of amino acids 231-502 of SEQ ID NO:25, consists essentially of amino acids 232-502 of SEQ ID NO:25, or consists essentially of amino acids 235-502 of SEQ ID NO:25.

[0242] In some embodiments, the ion pore domain of the chimeric ligand-gated ion channel of interest comprises an M2-M3 linker domain that is heterologous to the M2-M3 linker domain of the ion pore domain. By "M2-M3 linker domain" or "M2-M3 linker" is meant a sequence within the ion pore domain of the LGIC that is flanked at the amino (N)-terminus by the C-terminus of the transmembrane domain 2 (M2) of the receptor and at the carboxy (C)-terminus by the N-terminus of the transmembrane domain 3 (M3) of the receptor. The M2-M3 linker of the LGIC can be easily determined in the art and / or by using any publicly available protein analysis tool, such as Expasy, uniProt, etc. In some embodiments, when the ion pore domain of the chimeric receptor comprises a heterologous M2-M3 linker, the M2-M3 linker is derived from the same receptor as the ligand binding domain of the chimeric receptor. For example, where a ligand-gated ion channel of interest comprises a ligand binding domain from AChR and an ion pore domain from GlyR, the ion pore domain sequence may comprise an M2-M3 linker sequence from AChR. In some embodiments, the ion pore domain is from GlyRα1 and the M2-M3 linker is from α7-nAChR. In some embodiments, the native M2-M3 linker sequence removed from the ion pore domain corresponds to around amino acids 293-313 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 304-310, 293-306, 298-310, 305-311, 302-313, etc. In some such embodiments, the inserted M2-M3 linker is derived from around amino acids 281-295 of the α7-nAChR (SEQ ID NO:25), e.g., amino acids 290-295, 281-290, 281-295, 283-295, 287-292, etc., or a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to amino acids 281-295 or 283-295 of the α7-nAChR (SEQ ID NO:25).

[0243] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a Cys loop domain sequence that is heterologous to the Cys loop sequence of the ligand-binding domain. By "Cys loop domain sequence" or "Cys loop sequence" is meant a domain in the ligand-binding domain of the Cys loop LGIC that forms a loop structure flanked by cysteines at the N-terminus and C-terminus. Without wishing to be bound by theory, upon binding of a ligand to the ligand-binding domain, the Cys loop is structurally shifted to be close to the M2-M3 loop, and this shift is believed to mediate the biophysical translation of ligand binding in the extracellular domain to signal transduction in the ion pore domain (reviewed in Miller and Smart, Trends in Pharmacological Sci 2009:31(4)). The replacement of endogenous Cys loop sequence with a heterologous Cys loop sequence can increase the conductivity of LGIC by 1.5-fold or more, for example, at least 2-fold, 3-fold or 4-fold, in some cases at least 5-fold or 6-fold, and at least 7-fold, 8-fold, 9-fold or 10-fold at a particular dose. The Cys loop domain of a Cys loop receptor can be easily determined by using the art and / or any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand binding domain of a chimeric receptor comprises a heterologous Cys loop sequence, the Cys loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when a chimeric ligand-gated ion channel of interest comprises a ligand binding domain derived from AChR and an ion pore domain derived from GlyR, the ligand-gated ion channel of interest may comprise a ligand binding domain sequence derived from AChR, except for the sequence of the Cys loop domain derived from GlyR. In some embodiments, the ligand binding domain is derived from an α7-nAChR and the Cys loop sequence is derived from a GLyR.In some embodiments, the Cys loop sequence removed from the ligand binding domain corresponds to around amino acids 150-164 of α7-nAChR (SEQ ID NO:25), e.g., amino acids 150-157 of α7-nAChR. In some embodiments, the Cys loop sequence inserted is from around amino acids 166-180 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 166-172 of GlyRα1, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 166-180 of GlyRα1. In some embodiments, the Cys loop sequence inserted is from around amino acids 172-186 of GlyRα2 (SEQ ID NO:27), e.g., amino acids 172-178 of GlyRα2, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 172-186 of GlyRα2. In some embodiments, the inserted Cys loop sequence is derived from around amino acids 171-185 of GlyRα3 (sequence number 28 or 29), e.g., amino acids 171-177 of GlyRα3, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to amino acids 171-185 of GlyRα3.

[0244] In some embodiments, the ligand-binding domain of the subject chimeric ligand-gated ion channel comprises a β1-2 loop domain sequence that is heterologous to the β1-2 loop domain sequence of the ligand-binding domain. By "β1-2 loop domain sequence" or "β1-2 loop or β1-β2 loop" is meant a domain in the ligand-binding domain of the Cys loop LGIC that is flanked at its N-terminus by the C-terminus of the β1 sheet and at its C-terminus by the N-terminus of the β2 sheet. Without wishing to be bound by theory, it is believed that the β1-2 loop helps mediate the biophysical translation of ligand binding in the extracellular domain to the ion pore domain and subsequent signal transduction (i.e., chloride influx in the case of GlyR). Upon ligand binding, it is believed that the β1-2 loop, together with the Cys loop, comes close to the M2-M3 loop and mediates the biophysical translation of ligand binding in the extracellular domain to signal transduction in the ion pore domain where the M2-M3 loop resides (reviewed in Miller and Smart, supra). The replacement of the endogenous β1-2 loop sequence with a heterologous β1-2 loop sequence can increase the conductivity of LGIC by 1.5-fold or more, for example, at least 2-fold, 3-fold or 4-fold, in some cases at least 5-fold or 6-fold, and at least 7-fold, 8-fold, 9-fold or 10-fold at certain doses. The β1-2 loop of a Cys-loop receptor can be easily determined by using the art and / or any publicly available protein analysis tool, such as Expasy, uniProt, etc. Typically, when the ligand-binding domain of a chimeric receptor comprises a heterologous β1-2 loop sequence, the β1-2 loop sequence is derived from the same receptor as the ion pore domain of the chimeric receptor. For example, when the chimeric ligand-gated ion channel of interest comprises a ligand-binding domain derived from AChR and an ion pore domain derived from GlyR, the sequence of the β1-2 loop domain of the ligand-binding domain may be derived from GlyR. In some embodiments, the ligand-binding domain is derived from α7-nAChR.In some embodiments, the β1-2 loop sequence removed from the ligand binding domain corresponds to around amino acids 64-72 or 67-70 of α7-nAChR (SEQ ID NO:25), e.g., amino acids 67-70, 66-71, or 64-72 of α7-nAChR. In some embodiments, the β1-2 loop sequence inserted is around amino acids 79-85 of GlyRα1 (SEQ ID NO:26), e.g., amino acids 80-85, 81-84, 79-85, or 81-84 of GlyRα1, and includes at most three, at most two, at most one, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα2, and the β1-2 loop inserted corresponds to around amino acids 86-91 of GlyRα2 (SEQ ID NO:27), and includes at most three, at most two, at most one, or no amino acid mutations. In some embodiments, the ion pore domain is derived from GlyRα3 and the β1-2 loop inserted corresponds to about amino acids 85-90 of GlyRα3 (SEQ ID NO:28 or 29) and contains at most 3, at most 2, at most 1, or no amino acid mutations. In some embodiments, the mutations are amino acid substitutions.

[0245] In some embodiments, the present disclosure provides a chimeric LGIC receptor comprising a ligand binding domain derived from human α7-nAChR, the ligand binding domain comprising one or more amino acid substitutions of the present disclosure, and an ion pore domain derived from human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3. In some embodiments, the ligand binding domain comprises a Cys loop domain derived from human glycine receptor. In some embodiments, the ligand binding domain comprises a β1-2 loop domain derived from human glycine receptor.

[0246] Non-limiting examples of sequences of chimeric LGIC receptors of the present disclosure include the sequences disclosed herein as SEQ ID NOs: 30-31 and 33. In some embodiments, the chimeric LGIC receptor has 85% or more sequence identity to the sequences provided herein in SEQ ID NOs: 30-31 and 33, e.g., 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the sequences provided herein in SEQ ID NOs: 30-31 and 33. In the sequences, the signal peptide is shown in italics, the ligand binding domain is shown in bold, and the ion pore domain is shown underlined.

[0247] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that comprises a ligand binding domain (bold) that includes the human α7-nAChR signal peptide (italics) and the GlyRα1 Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined). In some embodiments, the chimeric LGIC receptor comprises an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 33: [ka]

[0248] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera that contains the human α7-nAChR signal peptide (italics) and a ligand binding domain (bold) that includes the GlyRα1 β1-2 loop sequence (lowercase) and the Cys loop sequence (lowercase) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0249] In some embodiments, the chimeric LGIC receptor is a CHRNA7 / GLRA1 chimera (R229 attachment point) that contains the human α7-nAChR signal peptide (italics) and ligand binding domain (bold) fused to the human GlyRα1 ion pore domain (underlined): [ka]

[0250] Amino acid mutations in ligand-gated ion channels As discussed above, in some aspects, the engineered receptor of interest comprises at least one amino acid mutation that changes the potency of ligand to engineered receptor compared to its potency to non-mutated parent receptor.In other words, one or more amino acid mutations, such as loss-of-function mutations or gain-of-function mutations, shift the potency of engineered receptor to ligand compared to that of non-mutated parent receptor.In some embodiments, the mutation is an amino acid substitution.In some embodiments, the one or more mutations are in the ligand-binding domain of engineered receptor. In some embodiments, when the ligand-binding domain of engineered receptor is a Cys-loop receptor protein, one or more amino acid mutations are substitutions with the residues corresponding to the residues of α7-nAChR (SEQ ID NO: 25) selected from the group consisting of W77, Y94, R101, W108, Y115, T128, N129, V130, L131, Q139, L141, Y151, S170, W171, S172, S188, Y190, Y210, C212, C213 and Y217.In some embodiments, one residue is substituted.In some embodiments, 2, 3, 4, or 5 or more residues are substituted, for example, 6, 7, 8, 9 or 10 residues are substituted. In certain embodiments, the residue corresponds to a residue of α7-nAChR (SEQ ID NO: 25) selected from the group consisting of W77, R101, Y115, N129, L131, S170, S172, and S188. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence.

[0251] In some embodiments, the one or more substitutions reduce the potency of the engineered receptor for acetylcholine and non-natural ligands, e.g., 2-fold or less, 3-fold or less, 4-fold or less, 5-fold or less, 10-fold or less, 20-fold or less, 30-fold or less, 50-fold or less, or 100-fold. In certain embodiments, the one or more substitutions correspond to R101I, R101S, R101D, Y115L, Y115M, Y115D, Y115T, T128M, T128R, T128I, N129I, N129V, N129P, N129W, N129T, N129D, N129E, L131E, L131P, L131T, L131D, L131S, L141S, L141R, W171F, W171H, S172F, S172Y, S172R, S172D, C212A, C212L, or C213P of α7-nAChR. In other examples, the one or more substitutions selectively reduce the potency of acetylcholine to the engineered receptor. In other words, the one or more substitutions reduce the potency of the engineered receptor for acetylcholine while essentially maintaining the potency for the non-natural ligand, or reduce the potency of the engineered receptor for acetylcholine by 2 or less, for example, 3, 4, 5 or less, and in some cases, 10, 20, 50, or 100 or less, than it would otherwise reduce the potency of the engineered receptor for the non-natural ligand. In some embodiments, the substitution corresponds to L131E, L131S, L131T, L131D, or S172D of α7-nAChR. In yet other embodiments, the one or more substitutions selectively reduce the potency of the non-natural ligand for the engineered receptor.In other words, the one or more substitutions reduce the potency of the engineered receptor against a non-natural ligand while essentially maintaining the potency against acetylcholine, or reduce the potency of the engineered receptor against a non-natural ligand by 2-fold or less, for example, 3-fold, 5-fold or less, and in some cases, 10-fold, 20-fold or 50-fold or less than it would otherwise reduce the potency of the engineered receptor against acetylcholine. In some embodiments, the substitution corresponds to W77M, Y115W, S172T, or S172C of α7-nAChR. In certain embodiments, the one or more substitutions are within the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC6987, ABT-126, and facinicline / RG3487.

[0252] In other embodiments, the one or more substitutions increase the potency of the engineered receptor for acetylcholine and / or non-natural ligands, e.g., 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, or 100-fold. In some embodiments, the substitutions correspond to L131N, L141W, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S172C, S188I, S188V, S188F, S188M, S188Q, S188T, S188P, or S188W. In some embodiments, the one or more substitutions increase the potency of both acetylcholine and non-natural ligand. In some embodiments, the substitutions correspond to L131N, S170G, S170A, S170L, S170I, S170V, S170P, S170F, S170M, S170T, S170C, S172T, S188I, S188V, S188F, S188M, S188Q or S188T of α7-nAChR. In other examples, the one or more substitutions selectively increase the potency of acetylcholine for the engineered receptor. In other words, the one or more substitutions increase the potency of the engineered receptor for acetylcholine by 2-fold or more, for example, 3-fold, 4-fold, or 5-fold or more, and in some examples, 10-fold, 20-fold, 50-fold, or 100-fold, more than it increases the potency of the engineered receptor for non-natural ligand. In some embodiments, the substitution corresponds to L141W, S172T, S172C, S188P or S188W of α7-nAChR. In certain embodiments, the one or more substitutions are in the α7-nAChR sequence. In certain embodiments, the non-natural ligand is selected from AZD-0328, TC-5619, TC6987, ABT-126 and facinicline / RG3487. In yet other examples, the one or more substitutions selectively increase the potency of the non-natural ligand against the engineered receptor.In other words, the one or more substitutions increase the potency of the engineered receptor for a non-natural ligand by 2-fold or more, e.g., 3-fold, 5-fold or more, and in some cases, 10-fold, 20-fold or 50-fold or more, more than it increases the potency of the engineered receptor for acetylcholine.

[0253] In some embodiments, the amino acid residue mutated in the engineered receptor of interest is not an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of wild-type a7 nAChR (SEQ ID NO: 25). In some embodiments, the mutation is an amino acid substitution. In some embodiments, the amino acid residue mutated in the engineered receptor of interest is an amino acid corresponding to R27, E41, Q79, Q139, L141, G175, Y210, P216, Y217, or D219 of wild-type a7 nAChR (SEQ ID NO: 25). In some embodiments, the substitution is not a substitution corresponding to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in a wild-type α7 nAChR. In some embodiments, the substitution corresponds to W77F, W77Y, W77M, Q79A, Q79Q, Q79S, Q79G, Y115F, L131A, L131G, L131M, L131N, L131Q, L131V, L131F, Q139G, Q139L, G175K, G175A, G175F, G175H, G175M, G175R, G175S, G175V, Y210F, P216I, Y217F, or D219A in wild-type α7 nAChR. In some embodiments, when such a substitution is present in an engineered receptor, it is present in combination with one or more amino acid mutations described herein.

[0254] In some embodiments, residues Y94, Y115, Y151, and Y190 of α7-nAChR (SEQ ID NO: 25) mediate the binding of natural ligand acetylcholine. In some embodiments, mutations at these residues reduce the binding of acetylcholine and can therefore be considered loss-of-function mutations. In some embodiments, residues W77, Y115, N129, V130, L131, Q139, L141, S170, Y210, C212, C213, and Y217 of α7-nAChR mediate the binding of non-natural ligand AZD0328 to this receptor, and mutations at these residues increase the affinity of AZD0328 and / or other ligands to this receptor and can therefore be considered gain-of-function mutations. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues in the ligand binding domain region of the α7-nAChR (SEQ ID NO: 25) or a chimeric receptor comprising the ligand binding domain region of the α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217. In some embodiments, the mutation is an amino acid substitution. In certain embodiments, the mutation in one or more amino acid residues in the ligand binding domain region of α7-nAChR (sequence number 25) or the ligand binding domain of a chimeric receptor comprising the ligand binding domain region of α7-nAChR is a substitution with one or more amino acid residues selected from the group consisting of W77, Y94, Y115, N129, V130, L131, Q139, L141, Y151, S170, Y190, Y210, C212, C213 and Y217.

[0255] In some embodiments, residues Y115, L131, L141, S170, W171, S172, C212, and Y217 of α7-nAChR (SEQ ID NO: 25) mediate the binding of acetylcholine and / or nicotine, and mutations at one or more of these residues can reduce the binding of acetylcholine and / or nicotine. In some embodiments, R101, Y115, L131, L141, W171, S172, S188, Y210, and Y217 of α7-nAChR mediate the binding of non-natural ligand ABT126, and mutations at one or more of these residues can increase the affinity of ABT126 and / or other ligands to α7-nAChR. In some embodiments, the mutation is an amino acid substitution. In some embodiments, R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR mediate binding of the non-natural ligand TC6987, and mutation of one or more of these residues can increase the affinity of TC6987 and / or other ligands for the α7-nAChR. In some embodiments, R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR mediate binding of the non-natural ligand facinicline / RG3487, and mutation of one or more of these residues can increase the affinity of facinicline / RG3487 and / or other ligands for the α7-nAChR. In some embodiments, the engineered receptor of interest comprises a mutation in one or more amino acid residues in the ligand binding domain region of an α7-nAChR or a ligand binding domain of a chimeric receptor comprising the ligand binding domain region of an α7-nAChR, wherein the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N120, N129, L131, L141, S170, W171, S172, S188, Y210, C212, C213 and Y217.In some embodiments, one or more amino acid residues alter the binding of acetylcholine and / or nicotine to α7-nAChR, wherein the amino acid is selected from the group consisting of Y115, L131, L141, S170, W171, S172, C212 and Y217 of α7-nAChR. In certain such embodiments, the amino acid is selected from C212 and S170. In some embodiments, the mutation in one or more amino acid residues alters the binding of ABT126 to α7-nAChR, wherein 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 one or more amino acid residues alters the binding of TC6987 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, Y115, T128, N129, L131, L141, W171, S172, Y210, C212, C213, and Y217 of the α7-nAChR. In certain such embodiments, the amino acid is selected from R101, T128, N129, Y210, and C213. In some embodiments, the mutation in one or more amino acid residues alters the binding of fasiniline / RG3487 to the α7-nAChR, where the one or more amino acid residues are selected from the group consisting of R101, N120, L131, L141, S170, W171, S172, Y210, and Y217 of the α7-nAChR. In certain such embodiments, the amino acids are selected from Y210, R101, and N129.

[0256] The present disclosure provides engineered receptors having two or more mutations, such as amino acid substitutions, compared to the parent receptor. In some embodiments, the parent receptor comprises a ligand binding domain derived from the human α7 nicotinic acetylcholine receptor (α7-nAChR). In some embodiments, the parent receptor is a chimeric receptor. In some embodiments, the parent receptor comprises an ion pore domain derived from the human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1, human glycine receptor α2, or human glycine receptor α3. In some embodiments, the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from the human glycine receptor. In some embodiments, the parent receptor comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the engineered receptor comprises two amino acid substitutions compared to the parent receptor comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the ligand binding domain of the engineered receptor comprises a β1-2 loop domain derived from the human glycine receptor α1 subunit.

[0257] 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).

[0258] In some embodiments, the two amino acid substitutions are at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of L131 and S172, Y115 and S170, and Y115 and L131. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution at S172D. In some embodiments, the ligand binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution at Y115D. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of L131S and S172D, L131T and S172D, L131D and S172D, Y115D and S170T, Y115D and L131Q, and Y115D and L131E. In some embodiments, the ligand binding domain comprises an amino acid substitution of L131E.

[0259] In some embodiments, the ligand binding domain comprises one or more amino acid substitutions at amino acid residues selected from the group consisting of Y140, R101, L131, Y115, and Y210, where the amino acid residues correspond to amino acid residues of α7-nAChR. In some embodiments, the ligand binding domain comprises an amino acid substitution of R101W and / or Y210V. In some embodiments, the ligand binding domain comprises two or more amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at amino acid residues selected from the group consisting of R101, L131, Y115, Y210, and Y140. In some embodiments, the ligand binding domain comprises two amino acid substitutions at a pair of amino acid residues selected from the group consisting of R101 and L131, Y115 and Y210, and R101 and Y210. In some embodiments, the ligand binding domain comprises a pair of amino acid substitutions selected from the group consisting of R101F and L131G, R101F and L131D, Y115E and Y210W, R101W and Y210V, R101F and Y210V, R101F and Y210F, R101M and L131A, and R101M and L131F. In some embodiments, the ligand binding domain comprises three amino acid substitutions at amino acid residues R101, Y115, and Y210. In some embodiments, the ligand binding domain comprises amino acid substitutions R101W, Y115E, and Y210W, or amino acid substitutions R101F, Y115E, and Y210W.

[0260] In some embodiments, the ligand-binding domain comprises an amino acid substitution at residue L131 and an amino acid substitution at R101F or R101M. In some embodiments, the amino acid substitution at residue L131 is L131G, L131D, L131A, L131F, or L131N.

[0261] In some embodiments, the ligand-binding domain comprises a hydrophobic amino acid substitution at residue Y210 and an amino acid substitution of R101W or R101F. In some embodiments, the amino acid substitution at residue Y210 is Y210V, Y210F, or Y210W.

[0262] Those skilled in the art will easily recognize suitable control receptors for comparison with the engineered receptors of the present disclosure. In some embodiments, the control receptor is identical in sequence to the engineered receptor, except for one or more identifying amino acid mutations (e.g., substitutions). In all cases, reference to the control receptor is meant to indicate that the described change in properties (e.g., potency against ligands) is the result of the amino acid mutations of the engineered receptor of the present disclosure.

[0263] The present disclosure provides an engineered receptor, which is a chimeric ligand-gated ion channel (LGIC) receptor, comprising: (a) a ligand-binding domain derived from human α7 nicotinic acetylcholine receptor (α7-nAChR) and comprising a Cys loop domain derived from human glycine receptor α1 subunit; and (b) an ion pore domain derived from human glycine receptor α1 subunit. In some embodiments, the engineered receptor is derived from a parent engineered receptor comprising or consisting of the amino acid sequence of SEQ ID NO: 33, and further comprises one or more amino acid substitutions based on the parent engineered receptor.

[0264] In some embodiments, the potency of the engineered receptor for acetylcholine is lower than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is at least about 1.5 times lower (e.g., about 2 times lower, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) than that of human α7 nicotinic acetylcholine receptor (α7-nAChR) for acetylcholine. In some embodiments, the potency of the engineered receptor for acetylcholine is evaluated by its EC50 for acetylcholine based on a cell reporter assay using YFP fluorescence quenching. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 100 μM, at least 200 μM, at least 300 μM, at least 500 μM, at least 700 μM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 1 mM. In some embodiments, the EC50 of the engineered receptor for acetylcholine is at least 3 mM. In some embodiments, having a higher EC50 for acetylcholine allows for a higher expression level of the engineered receptor in or on the cell surface in the presence of physiological concentrations of acetylcholine without passing a significant amount of current through the cell.

[0265] In some embodiments, the potency of the engineered receptor for the non-natural ligand is approximately the same as the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is higher than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, the potency of the engineered receptor for the non-natural ligand is at least about 1.5 times higher (e.g., about 2 times lower, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) than the potency of the human α7 nicotinic acetylcholine receptor (α7-nAChR) for the non-natural ligand. In some embodiments, determining potency involves determining the EC50 based on a cellular reporter assay using YFP fluorescence quenching as described in Example 2 of the present disclosure. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 15 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 150 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1 μM, less than 2 μM, less than 3 μM, less than 4 μM, less than 5 μM, less than 6 μM, less than 7 μM, less than 8 μM, less than 9 μM, or less than 10 μM. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 10 nM. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 100 nM. In some embodiments, the EC50 of the engineered receptor for the non-natural ligand is less than 1 μM.

[0266] In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, the efficacy of the engineered receptor in the presence of the non-natural ligand is at least about 1.5 times (e.g., about 2 times less, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or about 100 times (including all subranges and values ​​therebetween)) greater than the efficacy of the human α7 nicotinic acetylcholine receptor (α7-nAChR) in the presence of the non-natural ligand. In some embodiments, determining efficacy includes determining the amount of current passing through the engineered receptor in vitro in the presence of the non-natural ligand.

[0267] In some aspects, the ligand-gated ion channel of interest comprises one or more non-desensitizing mutations. In some embodiments, the mutations are amino acid substitutions. When used in the context of a ligand-gated ion channel, "desensitization" refers to a progressive reduction in ion influx in the prolonged presence of an agonist. This results in a progressive loss of efficacy of the neuron to the ligand. A non-desensitizing mutation refers to an amino acid mutation that prevents the neuron from becoming less responsive or unresponsive to the ligand by preventing the LGIC from becoming desensitized to the ligand. A non-desensitizing mutation can be easily identified by introducing a LGIC carrying the mutation into a neuron and analyzing the current flux over time during prolonged exposure to the ligand. If the LGIC does not contain a non-desensitizing mutation, the current will recover from the peak to a plateau during prolonged exposure, whereas if the LGIC contains a non-desensitizing mutation, the current will remain at the peak flux for the duration of exposure to the ligand. Exemplary amino acid mutations that result in desensitization include the V322L mutation in human GlyRα1 (post-processing of the proprotein V294L 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.

[0268] In some aspects, the ligand-gated ion channel of interest comprises one or more converting mutations. In some embodiments, the mutation is an amino acid substitution. By converting mutation is meant a mutation that changes the permeability of the ion pore domain of the LGIC such that it becomes permissive to the conduction of non-native ions, i.e., ions that cannot be naturally passed. In some cases, a mutation, e.g., replacement of amino acid residues 260-281 in human α7-nAChR (CHRNA7) (EKISLGITVLLSLTVFMLLVAE, SEQ ID NO: 34) or the corresponding amino acid in another cation-permeable LGIC with the peptide sequence PAKIGLGITVLLSLTTFMSGVAN (SEQ ID NO: 35), converts the permeability from cations to anions. In some cases, 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 glutamic acid (E) (which, as an A293E substitution in GLRA1, converts the LGIC from anion tolerant to calcium tolerant), or deletion of amino acid residue 278 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, substitution of amino acid residue 279 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, with glutamic acid (E), and substitution of amino acid residue 293 of GLRA1, or the corresponding amino acid in another anion-permeable LGIC, with valine (V) (which, as an P278Δ, A279E, T293V in GLRA1, converts the LGIC from anion tolerant to cation tolerant).

[0269] Further engineered receptors beyond those described herein can be identified by in vitro screening and validation methods. In some embodiments, a library of parent receptor mutants is generated from a limited number of parent receptors. The parent receptors can be mutated using methods known in the art, including error-prone PCR. In some embodiments, the library of parent receptor mutants is then transfected into yeast or mammalian cells and screened at high efficiency to identify functional receptors (e.g., to identify parent receptor mutants that can signal in response to a ligand). In some embodiments, the functional parent receptor mutants identified in this primary screen are then expressed in mammalian cells and screened for potency against ligands, for example, by plate reader and / or electrophysiological assays as described herein. Parent receptor mutants that show increased binding affinity to agonist ligands or allow the use of antagonist or modulator ligands as agonists in secondary screens can then be selected and further in vitro and / or in vivo validation and characterization assays can be performed.Such screening assays are known in the art and are described, for example, in Armbruster, BN et al. (2007) PNAS, 104, 5163-5168; Nichols, CD and Roth, BL (2009) Front. Mol. Neurosci. 2, 16; Dong, S. et al. (2010) Nat. Protoc. 5, 561-573; Alexander, GM et al. (2009) Neuron 63, 27-39; Guettier, JM et al. (2009) PNAS 106, 19197-19202; Ellefson JW et al. (2014) Nat Biotechnol. 32 (1): 97-101; Maranhao AC and Ellington AD. (2017) ACS Synth Biol. 20; 6 (1): 108-119; Talwar S et al. (2013) PLoS One; 8 (3): e58479; Gilbert DF et al. (2009) Front Mol Neurosci. 30; 2: 17; Lynagh and Lynch, (2010), Biol Chem. 14: 285 (20), 14890-14897; Islam R. et al. (2016) ACS Chem Neurosci. 21; 7 (12): 1647-1657; and Myers et al. (2008) Neuron. 8: 58 (3): 362-373.

[0270] A summary of these exemplary engineered receptors is provided below in Table 3. Each of these receptors comprises or consists of the amino acid sequence of SEQ ID NO:33, except for the indicated amino acid mutations. [Table 3-1] [Table 3-2]

[0271] Ligand In some embodiments, the ligand of the present disclosure refers to an exogenous drug or compound that has a known mechanism of action on mammalian cells (e.g., known to act as an agonist, antagonist, or modulator of a receptor). Such ligands can also be referred to as "binding agents." The ligands of the present disclosure may include proteins, lipids, nucleic acids, and / or small molecules. In some embodiments, the ligands include drugs or compounds approved by the U.S. Food and Drug Administration (FDA) for clinical use in treating a particular disease (e.g., a neurological disease). In some embodiments, the ligands include drugs or compounds that have not been approved by the FDA for clinical use but have been tested in one or more clinical trials, are currently being tested in one or more clinical trials, and / or are expected to be tested in one or more clinical trials. In some embodiments, the ligands include drugs or compounds that have not been approved by the FDA for clinical use but are routinely used in laboratory research. In some embodiments, the ligands are analogs of one of the above ligands. In some embodiments, the ligand is selected from the group consisting of AZD0328, ABT-126, AQW-051, cannabidiol, cilansetron, PH-399733, facinicline / RG3487 / MEM-3454, TC-6987, and TC-5619 / AT-101. In some embodiments, the ligand is selected from the group consisting of ABT-126, AZD-0328, RG3487, TC-6987, TC-6683, varenicline, and TC-5619. In some embodiments, the ligand is TC-5619.

[0272] In certain embodiments, the ligand is an analog of cilansetron, for example, as described by one of the following compound formulas 2-7 in either its R or S enantiomer. [ka]

[0273] 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.

[0274] In some embodiments, the ligand has the formula: [ka] The compound is AZD-0328, which is described in

[0275] In some embodiments, the ligand has the formula: [ka] The compound is TC-6987 described in

[0276] In some embodiments, the ligand has the formula: [ka] The compound is ABT-126, described in

[0277] In some embodiments, the ligand has the formula: [ka] and TC-5619 / Braddaniclin, as described in .

[0278] In some embodiments, the ligand has the formula: [ka] The compound is TC-6683 described in

[0279] In some embodiments, the ligand has the formula: [ka] and varenicline, as described in

[0280] In some embodiments, the ligand has the formula: [ka] and fascinicline / RG3487 as described in

[0281] Exemplary combinations of engineered receptors and non-natural ligands described herein are provided in Table 4 below. Each engineered receptor in Table 4 may exist as a protein, a polynucleotide encoding a protein, or a vector comprising a polynucleotide encoding a protein. In some embodiments, the engineered receptor comprises a ligand binding domain derived from human α7-nAChR. In some embodiments, the engineered receptor comprises an ion pore domain derived from human glycine receptor. In some embodiments, the human glycine receptor is human glycine receptor α1. In some embodiments, the engineered receptor comprises a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 33, except for the indicated mutations in Table 4. [Table 4]

[0282] composition The present disclosure also provides compositions (e.g., pharmaceutical compositions). In some embodiments, the composition or pharmaceutical composition comprises an AAV vector comprising an AAV capsid polypeptide of the present disclosure. In some embodiments, the composition or pharmaceutical composition comprises a nucleic acid comprising a polynucleotide sequence encoding an engineered receptor (e.g., a ligand-gated ion channel) of the present disclosure. In some embodiments, the composition comprises an AAV vector comprising an AAV capsid polypeptide of the present disclosure and further comprising a polynucleotide sequence encoding an engineered receptor of the present disclosure. In some embodiments, the polynucleotide encodes SEQ ID NO: 33. In some embodiments, the composition or pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, excipient, and / or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, and / or buffer is suitable for use in humans.

[0283] Such excipients, carriers, diluents, and / or buffers (collectively referred to as "pharmaceutical vehicles") include any pharmaceutical agent that may be administered without undue toxicity. They may be approved by federal or state regulatory agencies for use in mammals, such as humans, or may be listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. Such pharmaceutical vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical vehicles may be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary, stabilizing, thickening, lubricating, and coloring agents may be used. When administered to a mammal, the compounds and compositions of the present disclosure and the pharmaceutical acceptable vehicles, excipients, or diluents may be sterile. In some instances, aqueous media such as water, saline, and aqueous dextrose and glycerol solutions are used as vehicles when a compound of the present disclosure is administered intravenously.

[0284] The pharmaceutical composition may take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained release formulations thereof, or any other form suitable for administration to mammals.In some cases, the pharmaceutical composition is formulated for administration by routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans.Examples of suitable pharmaceutical vehicles and methods for their formulation are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, which are incorporated herein by reference.

[0285] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetic acid, propionic acid, malonic acid, benzoic acid, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may be present in such vehicles. A wide variety of pharma-ceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are fully described in a variety of publications, including, for example, A. Gennaro, (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[0286] In some embodiments, the pharmaceutical composition comprises a liquid that comprises an AAV capsid polypeptide or an AAV vector that comprises an AAV capsid polypeptide 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 aqueous composition that can gel in situ, such as an aqueous solution that can gel in situ.

[0287] Non-limiting examples of compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption enhancers or retarders that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharma-ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Additional active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) can also be incorporated into the composition.

[0288] In some embodiments, pharmaceutical compositions are formulated to be compatible with a particular route of administration or delivery. Thus, pharmaceutical compositions contain carriers, diluents, and / or excipients suitable for administration by various routes.

[0289] The choice of excipient will be determined in part by the particular vector, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present disclosure.

[0290] For example, the vectors can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, together with conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives, as necessary.

[0291] Suitable compositions for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound. Preparations are typically sterile and may be isotonic with the blood of the intended recipient. Non-limiting examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.

[0292] Cosolvents and adjuvants may be added to the formulation.Non-limiting examples of cosolvents include alcohols with hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0293] In some embodiments, the pharmaceutical compositions of the present disclosure are suitable for parenteral administration, for example, as aqueous and non-aqueous, isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, and as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be provided in unit-dose or multi-dose sealed containers such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, which only requires the addition of a sterile liquid excipient, for example, water, for injection immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type previously described.

[0294] As another example, the compound can be formulated into preparations suitable for oral administration, including: (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as a solid or granules; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings, and pharmacologically compatible excipients. Lozenge forms may include flavored tablets containing the active ingredient in a flavored, usually sucrose and acacia or tragacanth, and an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like, containing, in addition to the active ingredient, such excipients as described herein.

[0295] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of the compound of the present disclosure calculated in an amount sufficient to produce a desired effect, together with a pharma- ceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0296] method In some aspects of the present disclosure, the compositions and methods disclosed herein can be used to treat neurological diseases or disorders in subjects in need of treatment. In some aspects, the compositions and methods disclosed herein can be used to transduce neurons. In some aspects, the compositions and methods disclosed herein can be used to deliver heterologous nucleic acid to neurons. In some aspects, the compositions and methods disclosed herein can be used in assays. In some aspects, the compositions and methods disclosed herein can be used as research tools. In some aspects, the compositions and methods disclosed herein can be used as nucleic acid delivery tools.

[0297] In some aspects of the present disclosure, a method for treating a neurological disease or disorder in a subject is provided, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector comprising the capsid polypeptide of the present disclosure. In some embodiments, the AAV vector transduces hippocampal neurons. In some embodiments, the AAV vector transduces dorsal root ganglion neurons or trigeminal ganglion neurons. In some embodiments, the AAV vector comprises heterologous nucleic acid. In some embodiments, the heterologous nucleic acid encodes an engineered LGIC. In some embodiments, the method further comprises activating the engineered LGIC and then administering a small molecule ligand / drug / agonist that modulates the activity of the target neuron, thereby treating the neurological disease or disorder (e.g., pain or epilepsy) in the subject.

[0298] In some aspects of the present disclosure, the AAV vector is capable of transducing embryonic hippocampal neurons in vitro. In some embodiments, the method includes selecting the AAV vector as an AAV vector capable of transducing embryonic hippocampal neurons in vitro.

[0299] In some aspects of the present disclosure, a method for treating focal epilepsy in a subject is provided, comprising administering an effective amount of nucleic acid to the subject, wherein the nucleic acid comprises the polynucleotide sequence encoding the engineered LGIC receptor of the present disclosure.In some embodiments, the nucleic acid is delivered into hippocampal neurons.In some embodiments, the method further comprises activating engineered LGIC, and then administering a small molecule ligand that modulates the activity of target neurons, thereby treating focal epilepsy in the subject.

[0300] In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder associated with hippocampal neuronal dysregulation, such as focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, and fragile X syndrome. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat focal epilepsy. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat schizophrenia. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat autism spectrum disorder. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat Alzheimer's disease. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat Rett syndrome. In some embodiments, the vectors, nucleic acids, or compositions disclosed herein are used to treat fragile X syndrome.

[0301] The present disclosure, in part, contemplates compositions and methods for controlling, managing, preventing or treating epilepsy in a subject.In some embodiments, epilepsy is focal epilepsy.In some embodiments, focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0302] In some embodiments, the compositions and methods herein can be used to improve the level of epileptic seizures in subjects.In some embodiments, the compositions and methods herein can be used to prevent or control the level of epileptic seizures in subjects.Epileptic seizures can be classified as tonic-clonic, tonic, clonic, myoclonus, absence or atonic seizures.

[0303] In some embodiments, the compositions and methods herein can reduce the number or frequency of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% (including all ranges and subranges therebetween). In some embodiments, the compositions and methods herein can reduce the number or frequency of epileptic seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% (including all ranges and subranges therebetween).

[0304] In some embodiments, the compositions and methods herein can reduce the level and / or duration of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% (including all ranges and subranges therebetween). In some embodiments, the compositions and methods herein can reduce the level and / or duration of epileptic seizures experienced by a subject by at least 5%, at least 10%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% (including all ranges and subranges therebetween).

[0305] 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 an AAV vector and / or nucleic acid encoding an engineered LGIC of the present disclosure. Without wishing to be bound by any particular theory, the present disclosure contemplates using vectors and / or nucleic acids encoding the engineered LGICs disclosed herein to modulate neuronal activity to alleviate epilepsy in a subject.

[0306] In various embodiments, a vector and / or nucleic acid encoding an engineered LGIC that encodes 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 the ligand, the neuronal cells expressing the engineered receptor are activated, reducing their sensitivity to epilepsy.

[0307] In some embodiments, the epilepsy is focal epilepsy (focal epileptic seizures). Focal epilepsy is a neurological condition whose primary symptom is recurrent seizures affecting one hemisphere (half) of the brain.

[0308] There are four categories of focal epilepsy: (a) focal awareness-preserving seizures, where the subject is aware of what is happening during the seizure; (b) focal awareness-impaired seizures, where the subject is confused or does not know or remember what is happening during the seizure; (c) focal motor seizures, where the subject moves to some degree - anything from twitching to convulsing to rubbing hands to walking around; and (d) focal non-motor seizures, where the subject does not twitch or make any other movement during the seizure. Instead, it causes a change in how the subject feels or thinks (e.g., intense emotions, strange sensations, or symptoms such as a racing heartbeat, goosebumps, or feeling hot or cold).

[0309] Focal epilepsy is characterized by seizures originating in a specific part (lobe) of the brain. Focal epilepsy includes idiopathic localization-related epilepsy (ILRE), frontal lobe epilepsy, temporal lobe epilepsy, parietal lobe epilepsy, and occipital lobe epilepsy.

[0310] Idiopathic localization-related epilepsy (ILRE) is caused by unknown factors.

[0311] Frontal lobe epilepsy is a term for recurrent seizures that begin in the frontal lobe, the area of ​​the brain behind the forehead.

[0312] Temporal lobe epilepsy is a term for recurrent seizures that originate in the temporal lobe, a part of the brain located on the side of the head behind the temples and cheekbones. The temporal lobe is the area of ​​the brain that most commonly produces seizures. The midline (middle) part of both temporal lobes is very important in epilepsy - it is a frequent origin of seizures and prone to damage or scarring. Mesial temporal lobe epilepsy is the most common form of human epilepsy. Often, its pathophysiological substrate is hippocampal sclerosis.

[0313] Parietal Lobe Epilepsy. The parietal lobe is the part of the brain that lies on the top and sides of the head. Known as the "association cortex," the parietal lobe is responsible for attaching meaning to the brain's functions. It is here that the brain creates visual images, sounds are recognized as words, and touch is associated with specific objects. In a sense, the parietal lobe is where perceptions are matched up with physical reality.

[0314] Occipital lobe epilepsy. Occipital lobe epilepsy is the term for recurrent seizures that begin in the occipital lobe, the part of the brain at the back of the head that is primarily responsible for vision.

[0315] In some aspects of the present disclosure, the AAV vector is capable of transducing iPSC-derived neurons in vitro. In some embodiments, the method includes selecting the AAV vector as an AAV vector capable of transducing iPSC-derived neurons in vitro.

[0316] In some aspects of the present disclosure, a method for treating peripheral neuropathy and neuropathic pain such as trigeminal neuralgia in a subject is provided, comprising administering an effective amount of nucleic acid to the subject, wherein the nucleic acid comprises the polynucleotide sequence encoding the engineered LGIC receptor of the present disclosure.In some embodiments, the nucleic acid is delivered into DRG or TGG neuron.In some embodiments, the method further comprises activating engineered LGIC, and then administering a small molecule ligand that modulates the activity of target neuron, thereby treating peripheral neuropathy and neuropathic pain such as trigeminal neuralgia in a subject.

[0317] In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder. In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a neurological disease or disorder associated with DRG / TGG neuron dysregulation, such as neuropathic pain. In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used in the manufacture of a medicament for treating a spinal cord-related disease, such as spasticity, spinal cord injury, and avulsion injury. In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used to treat spasticity. In some embodiments, the treatment of spasticity comprises transducing neurons in the spinal column. In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used to treat spinal cord injury. In some aspects, the vectors, nucleic acids, or compositions disclosed herein are used to treat avulsion injury. In some embodiments, the treatment of spinal cord injury or avulsion injury comprises transducing neurons in the dorsal horn.

[0318] The present disclosure contemplates, in part, compositions and methods for controlling, managing, preventing, or treating pain in a subject. In some embodiments, the pain is neuropathic pain. "Pain" refers to discomfort and / or strange sensations in the subject's body. The sensation of pain can range from mild and occasional to severe and constant. Pain can be classified as acute pain or chronic pain. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain, or psychogenic pain. In some cases, pain is caused or associated with disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods herein include neuropathic pain, including peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, neuropathy associated with cancer, neuropathy associated with HIV / AIDS, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, Parkinson's disease, pain associated with epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and pain associated with central nervous system trauma, strain / sprain, and burn; nociceptive pain, including myocardial infarction, acute pancreatitis, post-operative pain, post-traumatic pain, renal colic, pain associated with cancer, pain associated with fibromyalgia, pain associated with carpal tunnel syndrome, and back pain.

[0319] The compositions and methods herein can be used to improve the level of pain in a subject.In some cases, the level of pain in a subject is improved by at least 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 100% (including all ranges and subranges therebetween).The level of pain in a subject can be evaluated by various methods.In some cases, the level of pain is evaluated by self-report (i.e., the human subject expresses a verbal report of the level of pain he / she is experiencing).In some cases, the level of pain is evaluated by behavioral indicators of pain, such as facial expressions, limb movements, vocalization, anxiety and guarding. These types of assessments can be useful, for example, when a subject is unable to self-report (e.g., young children, unconscious subjects, non-human subjects). Pain levels can be assessed after treatment with a composition of the present disclosure, compared to the level of pain the subject experienced prior to treatment with the composition.

[0320] In various embodiments, the method for controlling, managing, preventing, or treating pain in a subject comprises administering to the subject an effective amount of an AAV vector and / or nucleic acid encoding an engineered LGIC of the present disclosure. Without wishing to be bound by any particular theory, the present disclosure contemplates using vectors and / or nucleic acids encoding the engineered LGICs disclosed herein to modulate neuronal activity to reduce pain in a subject.

[0321] In various embodiments, a vector encoding an engineered receptor and / or a nucleic acid encoding an engineered LGIC that activates or depolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells, e.g., inhibitory interneurons, that are to reduce pain sensation. In the presence of the ligand, the neuronal cells expressing the engineered receptor are activated, reducing their sensitivity to pain and enhancing the analgesic effect of stimulating these neuronal cells.

[0322] In various embodiments, a vector encoding an engineered receptor that inactivates or hyperpolarizes a neuronal cell is administered to (or introduced into) one or more neuronal cells that are to have increased sensitivity to nociception or pain, such as nociceptors, peripheral sensory neurons, C fibers, Aδ fibers, Aβ fibers, DRG neurons, TGG neurons, etc. In the presence of the ligand, the neuronal cells expressing the engineered receptor are inactivated, decreasing sensitivity to pain and enhancing the analgesic effect.

[0323] In some embodiments, the compositions and methods of the present disclosure are effective in reducing pain.The examples of pain suitable for treatment by the vectors, compositions and methods of the present disclosure include, but are not limited to, acute pain, chronic pain, neuropathic pain, nociceptive pain, allodynia, inflammatory pain, inflammatory hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-associated neuropathy, nerve injury, rheumatoid arthritis pain, osteoarthritis pain, burn, back pain, eye pain, visceral pain, cancer pain (e.g. bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, post-herpetic neuralgia, post-operative pain, post-stroke pain and menstrual pain.

[0324] Pain can be classified as acute or chronic. "Acute pain" refers to pain that begins suddenly and is usually sharp in quality. Acute pain can be mild and last for a moment, or severe and last for weeks or months. In many cases, acute pain does not last longer than three months, and it disappears when the underlying cause of the pain is treated or cured. However, unrelieved acute pain can result in chronic pain. "Chronic pain" refers to ongoing or recurrent pain that lasts beyond the normal course of an acute illness or injury, or that lasts longer than three to six months and adversely affects the well-being of an individual. In some embodiments, the term "chronic pain" refers to pain that continues when it should not. Chronic pain can be nociceptive pain or neuropathic pain.

[0325] In some embodiments, the pain is predicted or expected to occur in association with or as a result of an injury, infection, or medical intervention. In some embodiments, the infection causes nerve damage. In some embodiments, the medical intervention is surgery, such as surgery to the central core of the body. In some embodiments, the medical intervention is surgery to remove part or all of one or more tissues, tumors, or organs of the body. In some embodiments, the medical intervention is an amputation. In some embodiments, the compositions and methods of the present disclosure are effective in reducing acute pain. In some embodiments, the compositions and methods of the present disclosure are effective in reducing chronic pain.

[0326] Chronic pain exists when discomfort and abnormal sensitivity are characteristic of the patient's symptoms. Individuals may be present with a variety of pain symptoms. Such symptoms include: 1) spontaneous pain, which may be dull, burning, or stabbing; 2) exaggerated pain response to noxious stimuli (hyperalgesia); and 3) pain caused by normally innocuous stimuli (allodynia-Meyer et al., 1994, Textbook of Pain, 13-44). Patients suffering from various forms of acute and chronic pain may have similar symptoms, but the underlying mechanisms may be different and therefore may require different treatment strategies. Thus, pain can also be divided into several different subtypes according to different pathophysiology, such as nociceptive pain, inflammatory pain, and neuropathic pain.

[0327] 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.

[0328] Nociceptive pain is induced by tissue injury or by strong stimuli that may cause injury. Moderate to severe acute nociceptive pain is the main feature of pain from central nervous system trauma, strain / sprain, burn, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumor-related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer treatment (e.g. post-chemotherapy syndrome, chronic postoperative pain syndrome or post-radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiation therapy. Back pain may be due to herniated or ruptured intervertebral disc or abnormality of lumbar facet joints, sacroiliac joints, paraspinal muscles or posterior longitudinal ligament. Back pain may resolve naturally, but in some patients where it lasts for more than 12 weeks, it can become a chronic condition and be particularly debilitating.

[0329] Neuropathic pain can be defined as the pain that is initiated or caused by primary lesion or dysfunction in the nervous system.The etiology of neuropathic pain includes, for example, peripheral neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, and the pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiency.

[0330] Neuropathic pain can be associated with pain disorders, a term that refers to diseases, disorders or conditions associated with or caused by pain. Examples of pain disorders include arthritis, allodynia, typical trigeminal neuralgia, trigeminal neuralgia, somatoform disorders, hypoesthesia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthesia, causalgia, sciatica disorders, osteoarthritis, fibromyalgia, visceral diseases, chronic pain disorders, migraine / headache, chronic fatigue syndrome, complex regional pain syndrome, neurodystrophy, plantar fasciitis or pain associated with cancer.

[0331] In some embodiments, the neuropathic pain is peripheral neuropathy. Peripheral neuropathy refers to a condition that occurs when the nerves that carry messages from the rest of the body to the brain and spinal cord and from the brain and spinal cord to the rest of the body are damaged or diseased. The various types of peripheral neuropathy range from carpal tunnel syndrome (a common traumatic injury after chronic repetitive use of the hand and wrist, such as with computer use) to nerve damage associated with diabetes. In general, peripheral neuropathy can be classified into mononeuropathy and polyneuropathy. Mononeuropathy includes carpal tunnel syndrome, ulnar nerve palsy, radial nerve palsy, and peroneal nerve palsy. Polyneuropathy occurs when multiple peripheral nerves in the body malfunction at the same time. Polyneuropathy can have a wide variety of causes, including exposure to certain toxins, such as alcohol abuse, nutritional deficiencies (especially vitamin B deficiency), and complications from diseases such as cancer or kidney failure. One of the most common forms of chronic polyneuropathy is diabetic neuropathy, a condition that occurs in people with diabetes. It is more severe in people whose blood sugar levels are poorly controlled. Less commonly, diabetes can also cause mononeuropathy. One of the most severe polyneuropathy is Guillain-Barre syndrome, a rare disease that strikes suddenly when the body's immune system attacks the nerves inside the body as they leave the spinal cord. Symptoms tend to appear quickly and get worse quickly, and can result in paralysis. Early symptoms include weakness and tingling, which may eventually spread upwards towards the arms. In more severe cases, blood pressure problems, heart rate problems, and difficulty breathing can occur. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic form of Guillain-Barre where symptoms continue for months or even years. Early diagnosis and treatment are important for CIDP patients, who have a 30% risk of eventually becoming wheelchair bound.

[0332] In some embodiments, the neuropathic pain is trigeminal neuralgia. Trigeminal neuralgia (TN), also called tic, is a chronic pain condition that affects the trigeminal nerve or the fifth cranial nerve, one of the most widely distributed nerves in the head. The trigeminal nerve is a set of cranial nerves in the head. It is the nerve responsible for providing sensation to the face. One trigeminal nerve runs on the right side of the head, while the other runs on the left side. Each of these nerves has three different branches. After the trigeminal nerve leaves the brain and travels inside the skull, it splits into three smaller branches that control sensation throughout the face: Ophthalmic nerve (V1): The first branch controls sensation in a person's eyes, upper eyelids, and forehead. Maxillary nerve (V2): The second branch controls sensation in the lower eyelid, cheek, nostrils, upper lip, and upper gums. Mandibular nerve (V3): The third branch controls sensation in the jaw, lower lip, lower gums, and some of the muscles used for chewing.

[0333] TN is a form of neuropathic pain. The typical or "classic" form of the disorder (called "Type 1" or TN1) causes extreme, scattered, sudden, burning, or shock-like facial pain lasting anywhere from a few seconds to as long as two minutes per episode. These attacks occur in rapid succession and can last up to two hours in rapid succession. The "atypical" form of the disorder (called "Type 2" or TN2) is characterized by a constant aching, burning, stabbing pain of somewhat lower intensity than Type 1. Both forms of pain can occur in the same person, sometimes at the same time. The intensity of the pain can be physically and mentally incapacitating. TN is associated with a variety of conditions. TN can be caused by blood vessels compressing the trigeminal nerve as it leaves the brainstem. This compression causes erosion or damage to the protective coating (myelin sheath) around the nerve. Symptoms of TN can also occur in people with multiple sclerosis, a disease that causes deterioration of the myelin sheath of the trigeminal nerve. Rarely, TN symptoms can be caused by nerve compression from a tumor, or a tangle of arteries and veins called an arteriovenous malformation. Injury to the trigeminal nerve (perhaps the result of sinus surgery, oral surgery, stroke, or facial trauma) can also result in neuropathic facial pain.

[0334] The inflammatory process is a complex series of biochemical and cellular events that are activated in response to tissue injury or the presence of foreign substances, resulting in swelling and pain. Joint pain is a common form of inflammatory pain.

[0335] Other types of pain amenable to treatment with the vectors, compositions, and methods of the present disclosure include, but are not limited to, pain resulting from musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatic) arthropathy, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis, and pyomyositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredema, and skeletal muscle ischemia; headaches, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache, mixed headache, and headaches associated with vascular disorders; and orofacial pain, including toothache, earache, burning mouth syndrome, and temporomandibular myofascial pain.

[0336] The effective amount of the disclosed compositions and methods for reducing the amount of pain experienced by human subjects can be determined using various pain scales. Patient self-report can be used to evaluate whether pain is reduced; see, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231. Alternatively, observational pain scales can be used. LANSS pain scale can be used to evaluate whether pain is reduced; see, for example, Bennett (2001) Pain 92:147. Visual analog pain scale can be used; see, for example, Schmader (2002) Clin. J. Pain 18:350. Likert pain scale can be used; for example, 0 is no pain, 5 is moderate pain, and 10 is the worst possible pain. Self-report pain scales for children include, for example, facial pain scale; Wong-Baker facial pain rating scale; and color analog scale. Self-report pain scales for adults include, for example, visual analog scales; verbal numerical rating scales; verbal language scales; and the Brief Pain Questionnaire.Examples of pain measurement scales include the Alder Hey Triage pain score (Stewart et al. (2004) Arch. Dis. Child. 89:625); behavioral pain scales (Payen et al. (2001) Critical Care Medicine 29:2258); the Brief Pain Questionnaire (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23: 129); the Non-Verbal Pain Indicator Checklist (Feldt (2000) Pain Manag. Nurs. 1: 13); the Critical Care Pain Observation Tool (Gelinas et al. (2006) Am. J. Crit. Care 15:420); the COMFORT scale (Ambuel et al. (1992) J. Pediatric Psychol. 17:95); the Dallas Pain Questionnaire (Ozguler et al. (2002) Spine 27:1783); the Dolorimeter Pain Index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); the Facial Pain Scale-Revised (Hicks et al. (2001) Pain 93:173); the FLACC (Face Legs Activity Cry Consolability) scale; the McGill Pain Questionnaire (Melzack (1975) Pain 1 :277); the descriptive differential scale (Gracely and Kwilosz (1988) Pain 35:279); the numeric 11-point box (Jensen et al. (1989) Clin. J. Pain 5: 153); the numeric rating scale (Hartrick et al. (2003) Pain Pract. 3:310); the Wong-Baker FACES pain rating scale; and the visual analog scale (Huskisson (1982) J. Rheumatol. 9:768).

[0337] In some embodiments, a method of alleviating pain in a subject is provided, comprising administering an AAV vector comprising a capsid polypeptide of the present disclosure, which introduces an engineered LGIC encoded by the AAV vector into a neuronal cell, and providing an effective amount of a ligand that activates the engineered receptor to control the activity of the cell, thereby alleviating pain in the subject. The method provides significant analgesia for pain without off-target effects such as general central nervous system depression. In certain embodiments, the method provides at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including all ranges and subranges therebetween) reduction in neuropathic pain in the subject, compared to untreated subjects. In some embodiments, the method includes measuring pain in the subject before and after administration of the ligand, and pain in the subject is reduced by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including all ranges and subranges therebetween). In such examples, the measurement may be made 4 hours or more after administration of the ligand, for example, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, or 4 days or more after administration of the ligand.

[0338] In some cases, the compositions and methods are used to treat post-traumatic stress disorder (PTSD), gastroesophageal reflux disease (GERD), addiction (e.g., alcohol, drugs), anxiety, depression, memory loss, dementia, sleep apnea, stroke, urinary incontinence, narcolepsy, essential tremor, movement disorders, atrial fibrillation, cancer (e.g., brain tumors), Parkinson's disease, or Alzheimer's disease. Other non-limiting examples of neurological diseases or disorders that can be treated by the compositions and methods herein include apraxia, agraphia, alcoholism, dyslexia, aneurysm, amaurosis fugax, amnesia, amyotrophic lateral sclerosis (ALS), Angelman syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, Asperger's syndrome, ataxia, ataxia telangiectasia, attention deficit hyperactivity disorder, auditory processing disorder, autism spectrum disorder, bipolar disorder, Bell's palsy, brachial plexus injury, brain tumors, and neurodegenerative disorders. Lesion, brain injury, brain tumor, Canavan disease, Capgras delusion, carpal tunnel syndrome, burning pain, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, craniopathy, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), cerebral gigantism, cerebral palsy, cerebral vasculitis, cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, coffin syndrome Lowry syndrome, coma, complex regional pain syndrome, compressive neuropathy, congenital facial paraplegia, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cyclothymic disorder, cytomegalic inclusion body disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, developmental Sexual coordination disorder, diabetic neuropathy, diffuse sclerosis, diplopia, Down syndrome, Dravet syndrome, Duchenne muscular dystrophy, speech disorder, autonomic neuropathy, dyscalculia, dysgraphia, dyskinesia, reading disorder, dystonia, empty sella syndrome, encephalitis, cerebral herniation, trigeminal nerve region hemangiomatosis, fecal incontinence, enuresis, epilepsy, intellectual disability in women, Erb's palsy, erythromelalgia, exploding head syndrome, Fabry disease, Fahr's syndrome, syncope, familial convulsive paralysis, febrile seizures,Fisher syndrome, Friedreich's ataxia, fibromyalgia, Foville syndrome, fetal alcohol syndrome, fragile X syndrome, fragile X-associated tremor / ataxia syndrome (FXTAS), Gaucher disease, generalized epilepsy febrile seizures plus, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, heterotopic grey matter, Guillain-Barré syndrome, generalized anxiety disorder, HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasms, hereditary spastic paraplegia, hereditary polyneuropathic ataxia, herpes zoster oticus, herpes zoster, Hirayama syndrome group, Hirschsprung's disease, Holmes-Addie syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hyperadrenocorticism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile Refsum's disease, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, isodicentric 15, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kinsbone syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Krabbe disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral medulla (Warren's syndrome) Berg syndrome, learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, cerebral leukodystrophy, leukoencephalopathy with disappearance of white matter, dementia with Lewy bodies, lissencephaly, locked-in syndrome, intervertebral disc disease, lumbar spinal stenosis, Lyme disease - neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, macropsia, mal de débarquement, macrocephalic leukoencephalopathy with subcortical cysts, megalencephaly, Merkelson-Rosenthal syndrome, Meniere's disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, micropsia, migraine, Miller-Fitch syndrome Sher syndrome, Ministroke (Transient Ischemic Attack), Sound Aversion Syndrome, Mitochondrial Myopathy, Moebius syndrome, Monolimbic muscular atrophy, Motor Skills Disorder, Moyamoya Disease, Mucopolysaccharidosis, Multiinfarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Muscular Dystrophy, Myalgic Encephalomyelitis, Myasthenia Gravis, Myelinolytic Diffuse Sclerosis, Infantile Myoclonic Encephalopathy, Myoclonus, Myopathy, Myotubular Myopathy, Myotonia Congenita, Narcolepsy, Neuro-Behçet's Disease, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Signs of AIDS,Neurological sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, neuropathy, neuropathy, Nyman-Pick disease, non-24-hour sleep-wake disorder, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, latent spinal malunion, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, otosclerosis, overuse syndrome, recurrent vision, paresthesia, Parkinson's disease, congenital paramyotonia, paraneoplastic disorders, seizures, Parry-Romberg syndrome, PANDAS, Pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, pervasive developmental disorder, photic sneeze reflex, phytanic acid storage disease, Pick's disease, compressed nerve, pituitary tumor, P MG, polyneuropathy, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, post-herpetic neuralgia (PHN), orthostatic hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive facial hemifacial atrophy, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, prosopagnosia, pseudotumor cerebri, quadrant hemianopsia, quadriplegia, rabies, radiculopathy, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Ramsay Hunt syndrome type III, Rasmussen's encephalitis, reflex neurovascular dystrophy, Refsum's disease, REM sleep behavior disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rhythmic movement disorder, Romberg's syndrome, chorea (Saint Vitus dance), Sandhoff disease, Schilder's disease, schizencephaly, sensory processing disorder, septo-optic dysplasia, shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal tumor, spinal muscular atrophy, spinal-bulbar muscular atrophy, spinocerebellar ataxia, split brain, Steele-Richardson-Oller syndrome Zewski syndrome, stiff-person syndrome, stroke, Sturge-Weber syndrome, stuttering, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial hemosiderosis, Sydenham's chorea, fainting, synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, tardive dysphrenia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, temporal lobe epilepsy, tetanus, tethered spinal cord syndrome, Thomsen's disease,Thoracic outlet syndrome, painful tics, Todd's paralysis, Tourette's syndrome, toxic encephalopathy, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trichotillomania, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, Unverricht-Lundborg disease, von Hippel-Lindau disease (VHL), Biliusk encephalomyelitis (VE), Wallenberg syndrome, West syndrome, cervical spasticity, William's syndrome, Wilson's disease, or Zellweger syndrome.

[0339] The subject treated by the methods and compositions disclosed herein may be a human or a non-human animal. In some embodiments, the subject is a human. In some embodiments, the human is an adult (18 years of age or older). Non-limiting examples of non-human animals include non-human primates, livestock, household pets, and laboratory animals. For example, the non-human animal may be an ape (e.g., chimpanzee, baboon, gorilla, or orangutan), an Old World monkey (e.g., rhesus monkey), a New World monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a llama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal. The compositions and methods described herein are suitable for the treatment of veterinary animals. Veterinary animals may include, but are not limited to, dogs, cats, horses, cows, sheep, mice, rats, guinea pigs, hamsters, rabbits, snakes, turtles, and lizards. In some aspects, contacting a tissue or cell population with a composition comprises administering the composition to the cell population or subject. In some embodiments, administration is performed in vitro, for example, by adding the composition to a cell culture system. In some aspects, administration is performed in vivo, for example, by administration by a specific route. When administering more than one composition, the compositions may be administered by the same route at the same time (e.g., on the same day) or by the same route at different times. Alternatively, the compositions may be administered by different routes at the same time (e.g., on the same day) or by different routes at different times.

[0340] Administration The present disclosure provides a dosing regimen for administering the AAV of the present disclosure or the pharmaceutical composition of the present disclosure.In some embodiments, the pharmaceutical composition comprises the nucleic acid sequence of the ligand-gated ion channel of the present disclosure.In some embodiments, the pharmaceutical composition comprises the AAV of the present disclosure, which comprises the nucleic acid sequence of the ligand-gated ion channel of the present disclosure.

[0341] In some embodiments, the vectors and / or nucleic acids of the disclosure are administered or introduced into one or more neuronal cells, which may be of the same type or a mixed population of neuronal cells of different types.

[0342] In some embodiments, the neuronal cell is a hippocampal neuron. In some embodiments, the neuronal cell is an excitatory neuron. In some embodiments, the vector of the present disclosure is administered or introduced into one or more hippocampal neuronal cells.

[0343] In some embodiments, the neuronal cell is a nociceptive neuron or a peripheral sensory neuron. Examples of sensory neurons include, but are not limited to, dorsal root ganglion (DRG) neurons and trigeminal ganglion (TGG) neurons. In some embodiments, the neuronal cell is an inhibitory interneuron involved in neuronal pain circuits. In some embodiments, the vector and / or nucleic acid of the present disclosure is administered or introduced into one or more DRG neuronal cells. In some embodiments, the vector and / or nucleic acid of the present disclosure is administered or introduced into one or more TGG neuronal cells.

[0344] Non-limiting examples of administration methods include subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, infusion, intracranial, intrathecal, intranasal, intraganglionic, intraspinal, cisternal and intraneuronal administration. In some embodiments, administration may include injection of a liquid formulation of the vector and / or nucleic acid. In some embodiments, administration may include oral delivery of a solid formulation of the vector and / or nucleic acid. In some embodiments, oral formulations can be administered with food. In some embodiments, the vector and / or nucleic acid of the present disclosure is administered parenterally, intravenously, intramuscularly, intraperitoneally, intrathecally, intraneuronally, intraganglionally, intraspinal or intraventricularly to a subject to introduce the vector and / or nucleic acid into one or more neuronal cells. In some embodiments, the vector is AAV.

[0345] In some embodiments, the AAV and / or nucleic acid of the disclosure are administered to neurons intracranially, intrathecally (IT), intracerebrally, intracerebroventricularly, or by direct injection into the epileptic focus of the hippocampus.

[0346] In some embodiments, the AAV and / or nucleic acid of the disclosure is administered to a sensory neuron or nociceptor, e.g., a DRG neuron, a TGG neuron, etc., by intrathecal (IT) or intraganglionic (IG) administration.

[0347] Intrathecal (IT) administration includes delivery via the spinal column or cisterna magna. The IT route delivers AAV and / or nucleic acid to the cerebrospinal fluid (CSF). This administration route may be suitable, for example, for the treatment of chronic pain or other peripheral nervous system (PNS) or central nervous system (CNS) indications. In animals, IT administration has been achieved by inserting an IT catheter via the cisterna magna and advancing it caudally to the lumbar level. In humans, IT delivery can be easily performed by lumbar puncture (LP), a routine clinical procedure that shows an excellent safety profile.

[0348] In some embodiments, the AAV and / or nucleic acid of the present disclosure are administered to a subject by intraganglionic administration. Intraganglionic administration may include direct injection into one or more ganglia. The IG route can deliver the AAV and / or nucleic acid directly to the DRG or TGG parenchyma. In some embodiments, IG administration to the DRG is performed by open neurosurgery. In some embodiments, open neurosurgery is invasive and undesirable in humans. In some embodiments, minimally invasive, CT imaging guided techniques for safely targeting the DRG can be used, for example, for human subjects. AAV and / or nucleic acid can be delivered into the DRG parenchyma using a custom-made needle assembly for convection-enhanced drug delivery (CED). In a non-limiting example, the vector and / or nucleic acid of the present disclosure can be delivered to one or more dorsal root ganglia and / or trigeminal ganglia for the treatment of chronic pain. In another non-limiting example, the vector and / or nucleic acid of the present disclosure can be delivered to the nodose ganglion (vagus nerve) for the treatment of epilepsy.

[0349] In some embodiments, the vector and / or nucleic acid of the present disclosure is administered to the subject by intracranial administration (i.e., directly to the brain). In a non-limiting example of intracranial administration, the vector and / or nucleic acid of the present disclosure can be delivered to the cortex of the brain, for example, to treat epileptic seizure focus, to the paraventricular hypothalamus, for example, to treat satiety disorder, or to the central amygdala, for example, to treat satiety disorder. In another particular case, the vector and / or nucleic acid can be administered to the subject by intraneural injection (i.e., directly to the nerve). The nerve can be selected based on the indication to be treated, for example, injection into the sciatic nerve to treat chronic pain, or injection into the vagus nerve to treat epilepsy or satiety disorder. In some embodiments, the vector and / or nucleic acid can be administered to the subject by injection into a sensory nerve terminal, for example, to treat chronic pain. In some embodiments, the vector and / or nucleic acid of the present disclosure is administered to the subject by direct injection into the epileptic focus of the hippocampus.

[0350] Dosages may vary and may depend on whether the treatment is preventive or therapeutic, the type, onset, progression, severity, frequency, duration, or probability of disease treatment for the desired clinical endpoint, previous or concurrent treatment, the general health, age, sex, race, or immunocompetence of the subject, and other factors that would be understood by a person skilled in the art. The amount, number, frequency, or duration of the dose may be increased or decreased proportionately as indicated by adverse side effects of the treatment or therapy, complications, or other risk factors, and the condition of the subject. Those skilled in the art will understand the factors that may affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0351] Vector dose can be expressed as the number of vector genome units delivered to a subject. The size of an individual vector genome will generally depend on the type of viral vector used. The vector genome of the present disclosure can be about 1.0 kilobase, 1.5 kilobase, 2.0 kilobase, 2.5 kilobase, 3.0 kilobase, 3.5 kilobase, 4.0 kilobase, 4.5 kilobase to 5.0 kilobase, or more than 5.0 kilobase (including all ranges and subranges therebetween).

[0352] In some embodiments, the vector dose administered is about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , about 9×10 6 , about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×107 , about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , about 1×10 9 , about 2×10 9 , about 3×10 9 , about 4×10 9 , about 5×10 9 , about 6×10 9 , about 7×10 9 , about 8×10 9 , about 9×10 9 , about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 , about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 6×10 13 , about 7×10 13, about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 6×10 14 , about 7×10 14 , about 8×10 14 , about 9×10 14 , about 1×10 15 , about 2×10 15 , about 3×10 15 , about 4×10 15 , about 5×10 15 , about 6×10 15 , about 7×10 15 , about 8×10 15 , about 9×10 15 , about 1×10 16 , about 2×10 16 , about 3×10 16 , about 4×10 16 , about 5×10 16 , about 6×10 16 , about 7×10 16 , about 8×10 16 , about 9×10 16 , about 1×10 17 or more vector genome units (including all ranges and subranges therebetween).

[0353] In some embodiments, the vector dose administered is at least 1×10 6 , at least 2 × 10 6 , at least 3 × 10 6 , at least 4 × 10 6 , at least 5 × 10 6 , at least 6 × 10 6 , at least 7 × 10 6 , at least 8 × 10 6 , at least 9×10 6 , at least 1 × 10 7 , at least 2 × 10 7 , at least 3 × 10 7 , at least 4 × 10 7 , at least 5 × 10 7, at least 6 × 10 7 , at least 7 × 10 7 , at least 8 × 10 7 , at least 9×10 7 , at least 1 × 10 8 , at least 2 × 10 8 , at least 3 × 10 8 , at least 4 × 10 8 , at least 5 × 10 8 , at least 6 × 10 8 , at least 7 × 10 8 , at least 8 × 10 8 , at least 9×10 8 , at least 1 × 10 9 , at least 2 × 10 9 , at least 3 × 10 9 , at least 4 × 10 9 , at least 5 × 10 9 , at least 6 × 10 9 , at least 7 × 10 9 , at least 8 × 10 9 , at least 9×10 9 , at least 1 × 10 10 , at least 2 × 10 10 , at least 3 × 10 10 , at least 4 × 10 10 , at least 5 × 10 10 , at least 6 × 10 10 , at least 7 × 10 10 , at least 8 × 10 10 , at least 9×10 10 , at least 1 × 10 11 , at least 2 × 10 11 , at least 3 × 10 11 , at least 4 × 10 11 , at least 5 × 10 11 , at least 6 × 10 11 , at least 7 × 10 11 , at least 8 × 10 11 , at least 9×10 11 , at least 1 × 10 12 , at least 2 × 10 12 , at least 3 × 10 12 , at least 4 × 1012 , at least 5 × 10 12 , at least 6 × 10 12 , at least 7 × 10 12 , at least 8 × 10 12 , at least 9×10 12 , at least 1 × 10 13 , at least 2 × 10 13 , at least 3 × 10 13 , at least 4 × 10 13 , at least 5 × 10 13 , at least 6 × 10 13 , at least 7 × 10 13 , at least 8 × 10 13 , at least 9×10 13 , at least 1 × 10 14 , at least 2 × 10 14 , at least 3 × 10 14 , at least 4 × 10 14 , at least 5 × 10 14 , at least 6 × 10 14 , at least 7 × 10 14 , at least 8 × 10 14 , at least 9×10 14 , at least 1 × 10 15 , at least 2 × 10 15 , at least 3 × 10 15 , at least 4 × 10 15 , at least 5 × 10 15 , at least 6 × 10 15 , at least 7 × 10 15 , at least 8 × 10 15 , at least 9×10 15 , at least 1 × 10 16 , at least 2 × 10 16 , at least 3 × 10 16 , at least 4 × 10 16 , at least 5 × 10 16 , at least 6 × 10 16 , at least 7 × 10 16 , at least 8 × 10 16 , at least 9×10 16 Or at least 1×10 17of vector genome units (including all ranges and subranges therebetween).

[0354] In some embodiments, the vector dose administered is 1×10 6 Below, 2 x 10 6 Below, 3 x 10 6 Below, 4 x 10 6 Below, 5 x 10 6 Below, 6 x 10 6 Below, 7 x 10 6 Below, 8 x 10 6 Below, 9 x 10 6 Below, 1×10 7 Below, 2 x 10 7 Below, 3 x 10 7 Below, 4 x 10 7 Below, 5 x 10 7 Below, 6 x 10 7 Below, 7 x 10 7 Below, 8 x 10 7 Below, 9 x 10 7 Below, 1×10 8 Below, 2 x 10 8 Below, 3 x 10 8 Below, 4 x 10 8 Below, 5 x 10 8 Below, 6 x 10 8 Below, 7 x 10 8 Below, 8 x 10 8 Below, 9 x 10 8 Below, 1×10 9 Below, 2 x 10 9 Below, 3 x 10 9 Below, 4 x 10 9 Below, 5 x 10 9 Below, 6 x 10 9 Below, 7 x 10 9 Below, 8 x 10 9 Below, 9 x 10 9 Below, 1×10 10 Below, 2 x 10 10 Below, 3 x 10 10 Below, 4 x 10 10 Below, 5 x 10 10 Below, 6 x 10 10 Below, 7 x 10 10 Below, 8 x 10 10 Below, 9 x 10 10 Below, 1×1011 Below, 2×10 11 Below, 3×10 11 Below, 4×10 11 Below, 5×10 11 Below, 6×10 11 Below, 7×10 11 Below, 8×10 11 Below, 9×10 11 Below, 1×10 12 Below, 2×10 12 Below, 3×10 12 Below, 4×10 12 Below, 5×10 12 Below, 6×10 12 Below, 7×10 12 Below, 8×10 12 Below, 9×10 12 Below, 1×10 13 Below, 2×10 13 Below, 3×10 13 Below, 4×10 13 Below, 5×10 13 Below, 6×10 13 Below, 7×10 13 Below, 8×10 13 Below, 9×10 13 Below, 1×10 14 Below, 2×10 14 Below, 3×10 14 Below, 4×10 14 Below, 5×10 14 Below, 6×10 14 Below, 7×10 14 Below, 8×10 14 Below, 9×10 14 Below, 1×10 15 Below, 2×10 15 Below, 3×10 15 Below, 4×10 15 Below, 5×10 15 Below, 6×10 15 Below, 7×10 15 Below, 8×10 15 Below, 9×10 15 Below, 1×10 16 Below, 2×10 16 Below, 3×10 16 Below, 4×10 16 Below, 5×10 16 Below, 6×1016 Below, 7 x 10 16 Below, 8 x 10 16 Below, 9 x 10 16 or less, or 1×10 17 The vector genome units include the following (including all ranges and subranges therebetween):

[0355] In some embodiments, the vector dose is expressed as vector genome units per kilogram of subject body weight (vg / kg). In some embodiments, the vector dose numbers above are based on a subject weighing 50 kg, and the doses can be translated accordingly into vg / kg and applied to different subjects based on the subject's body weight. For example, about 5×10 14 The vector genome unit dose is approximately 1 × 10 per kilogram. 13 This can be translated into vector genome units (vg / kg).

[0356] In some embodiments, the vector dose is expressed according to the concentration or potency of the vector administered to the subject. In some embodiments, the vector dose can be expressed as a volume multiple of the number of units per volume (e.g., genome units / volume).

[0357] 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 fluid of about 0.01 mL, about 0.02 mL, about 0.03 mL, about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08 mL, about 0.09 mL, about 0.1 mL, about 0.15 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 2.0 mL, about 3. 0 mL, about 4.0 mL, about 5.0 mL, about 6.0 mL, about 7.0 mL, about 8.0 mL, about 9.0 mL, about 10.0 mL, about 11.0 mL, about 12.0 mL, about 13.0 mL, about 14.0 mL, about 15.0 mL, about 16.0 mL, about 17.0 mL, about 18.0 mL, about 19.0 mL, about 20.0 mL, about 25.0 mL or a volume of greater than 25.0 mL (including all ranges and subranges therebetween). In some embodiments, the vector is at least 0.01 mL, at least 0.02 mL, at least 0.03 mL, at least 0.04 mL, at least 0.05 mL, at least 0.06 mL, at least 0.07 mL, at least 0.08 mL, at least 0.09 mL, at least 0.1 mL, at least 0.15 mL, at least 0.2 mL, at least 0.25 mL, at least 0.3 mL, at least 0.4 mL, at least 0.5 mL, at least 0.6 mL, at least 0.7 mL, at least 0.8 mL, at least 0.9 mL, at least 1.0 mL, at least and / or at least 2.0 mL, at least 3.0 mL, at least 4.0 mL, at least 5.0 mL, at least 6.0 mL, at least 7.0 mL, at least 8.0 mL, at least 9.0 mL, at least 10.0 mL, at least 11.0 mL, at least 12.0 mL, at least 13.0 mL, at least 14.0 mL, at least 15.0 mL, at least 16.0 mL, at least 17.0 mL, at least 18.0 mL, at least 19.0 mL, at least 20.0 mL, or at least 25.0 mL (including all ranges and subranges therebetween).In some embodiments, the vector is 0.01 mL or less, 0.02 mL or less, 0.03 mL or less, 0.04 mL or less, 0.05 mL or less, 0.06 mL or less, 0.07 mL or less, 0.08 mL or less, 0.09 mL or less, 0.1 mL or less, 0.15 mL or less, 0.2 mL or less, 0.25 mL or less, 0.3 mL or less, 0.4 mL or less, 0.5 mL or less, 0.6 mL or less, 0.7 mL or less, 0.8 mL or less, 0.9 mL or less, 1.0 mL or less, 2.0 mL or less Administered in a volume of 3.0 mL or less, 4.0 mL or less, 5.0 mL or less, 6.0 mL or less, 7.0 mL or less, 8.0 mL or less, 9.0 mL or less, 10.0 mL or less, 11.0 mL or less, 12.0 mL or less, 13.0 mL or less, 14.0 mL or less, 15.0 mL or less, 16.0 mL or less, 17.0 mL or less, 18.0 mL or less, 19.0 mL or less, 20.0 mL or less, or 25.0 mL or less (including all ranges and subranges therebetween).

[0358] In some embodiments, the vectors contemplated herein are at least 1×10 9 Genomic units / mL, at least 1 × 10 10 Genomic units / mL, at least 5 × 10 10 Genomic units / mL, at least 1 × 10 11 Genomic units / mL, at least 5 × 10 11 Genomic units / mL, at least 1 × 10 12 Genomic units / mL, at least 5 × 10 12 Genomic units / mL, at least 6 × 10 12 Genomic units / mL, at least 7 × 10 12 Genomic units / mL, at least 8 × 10 12 Genomic units / mL, at least 9 × 10 12 Genomic units / mL, at least 10 × 10 12 Genomic units / mL, at least 15 × 10 12 Genomic units / mL, at least 20 × 10 12 Genomic units / mL, at least 25 × 10 12 Genomic units / mL, at least 50 × 10 12 Genomic units / mL, or at least 100 × 10 12In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of about 1×10 genome units / mL, including all ranges and subranges therebetween. 9 Genome units / mL, approximately 1 x 10 10 Genomic units / mL, approximately 5 × 10 10 Genome units / mL, approximately 1 x 10 11 Genomic units / mL, approximately 5 × 10 11 Genome units / mL, approximately 1 x 10 12 Genomic units / mL, approximately 5 × 10 12 Genomic units / mL, approximately 6 x 10 12 Genome units / mL, approximately 7 x 10 12 Genomic units / mL, approximately 8 x 10 12 Genomic units / mL, approximately 9 × 10 12 Genomic units / mL, approximately 10 x 10 12 Genomic units / mL, approximately 15 x 10 12 Genomic units / mL, approximately 20 x 10 12 Genomic units / mL, approximately 25 x 10 12 Genomic units / mL, approximately 50 x 10 12 Genomic units / mL, or approximately 100 x 10 12 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of 1×10 genome units / mL, including all ranges and subranges therebetween. 9 Genomic units / mL or less, 1 x 10 10 Genomic units / mL or less, 5 x 10 10 Genomic units / mL or less, 1 x 10 11 Genomic units / mL or less, 5 x 10 11 Genomic units / mL or less, 1 x 10 12 Genomic units / mL or less, 5 x 10 12 Genomic units / mL or less, 6 x 10 12 Genomic units / mL or less, 7 x 10 12 Genomic units / mL or less, 8 x 10 12 Genomic units / mL or less, 9 x 10 12 Genomic units / mL or less, 10 x 10 12 Genomic units / mL or less, 15 x 10 12 Genomic units / mL or less, 20 x 10 12 Genomic units / mL or less, 25 x 10 12Genomic units / mL or less, 50 x 10 12 Genomic units / mL or less, or 100 x 10 12 It is administered to subjects at a titer of genomic units / mL or less (including all ranges and subranges therebetween).

[0359] In some embodiments, the vectors contemplated herein are about 5×10 9 Infectious units / mL, approximately 6 × 10 9 Infectious units / mL, approximately 7 × 10 9 Infectious units / mL, approximately 8 × 10 9 Infectious units / mL, approximately 9 × 10 9 Infectious units / mL, approximately 1 × 10 10 Infectious units / mL, approximately 1.5 × 10 10 Infectious units / mL, approximately 2 × 10 10 Infectious units / mL, approximately 2.5 × 10 10 Infectious units / mL, approximately 5 × 10 10 Infectious units / mL, approximately 1 × 10 11 Infectious units / mL, approximately 2.5 × 10 11 Infectious units / mL, approximately 5 × 10 11 Infectious units / mL, approximately 1 × 10 12 Infectious units / mL, approximately 2.5 × 10 12 Infectious units / mL, approximately 5 × 10 12 Infectious units / mL, approximately 1 × 10 13 Infectious units / mL, approximately 5 × 10 13 infectious units / mL, or approximately 1 × 10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of at least 5×10 infectious units / mL (including all ranges and subranges therebetween). 9 Infectious units / mL, at least 6 × 10 9 Infectious units / mL, at least 7 × 10 9 Infectious units / mL, at least 8 × 10 9 Infectious units / mL, at least 9 × 10 9 Infectious units / mL, at least 1 × 10 10 Infectious units / mL, at least 1.5 × 10 10 Infectious units / mL, at least 2 × 10 10 Infectious units / mL, at least 2.5 × 10 10Infectious units / mL, at least 5 × 10 10 Infectious units / mL, at least 1 × 10 11 Infectious units / mL, at least 2.5 × 10 11 Infectious units / mL, at least 5 × 10 11 Infectious units / mL, at least 1 × 10 12 Infectious units / mL, at least 2.5 × 10 12 Infectious units / mL, at least 5 × 10 12 Infectious units / mL, at least 1 × 10 13 Infectious units / mL, at least 5 × 10 13 infectious units / mL, or at least 1 × 10 14 In some embodiments, the vectors contemplated herein are administered to a subject at a titer of 5×10 infectious units / mL, including all ranges and subranges therebetween. 9 Infectious units / mL or less, 6 x 10 9 Infectious units / mL or less, 7 x 10 9 Infectious units / mL or less, 8 x 10 9 Infectious units / mL or less, 9 x 10 9 Infectious units / mL or less, 1 x 10 10 Infectious units / mL or less, 1.5 x 10 10 Infectious units / mL or less, 2 x 10 10 Infectious units / mL or less, 2.5 x 10 10 Infectious units / mL or less, 5 x 10 10 Infectious units / mL or less, 1 x 10 11 Infectious units / mL or less, 2.5 x 10 11 Infectious units / mL or less, 5 x 10 11 Infectious units / mL or less, 1 x 10 12 Infectious units / mL or less, 2.5 x 10 12 Infectious units / mL or less, 5 x 10 12 Infectious units / mL or less, 1 x 10 13 Infectious units / mL or less, 5 x 10 13 Infectious units / mL or less, or 1 × 10 14 It is administered to subjects at a titer of infectious units / mL or less (including all ranges and subranges therebetween).

[0360] In some embodiments, the vector of the present disclosure comprises about 5×1010 Transducing units / mL, approximately 1 x 10 11 Transducing units / mL, approximately 2.5 x 10 11 Transducing units / mL, approximately 5 x 10 11 Transducing units / mL, approximately 1 x 10 12 Transducing units / mL, approximately 2.5 x 10 12 Transducing units / mL, approximately 5 x 10 12 Transducing units / mL, approximately 1 x 10 13 Transducing units / mL, approximately 5 x 10 13 transducing units / mL, or approximately 1 x 10 14 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of at least 5×10 transducing units / mL, including all ranges and subranges therebetween. 10 Transducing units / mL, at least 1 x 10 11 Transducing units / mL, at least 2.5 x 10 11 Transducing units / mL, at least 5 x 10 11 Transducing units / mL, at least 1 x 10 12 Transducing units / mL, at least 2.5 x 10 12 Transducing units / mL, at least 5 x 10 12 Transducing units / mL, at least 1 x 10 13 Transducing units / mL, at least 5 x 10 13 Transducing units / mL, or at least 1 x 10 14 In some embodiments, the vectors of the present disclosure are administered to a subject at a titer of 5×10 transducing units / mL, including all ranges and subranges therebetween. 10 Transducing units / mL or less, 1 x 10 11 Transducing units / mL or less, 2.5 x 10 11 Transducing units / mL or less, 5 x 10 11 Transducing units / mL or less, 1 x 10 12 Transducing units / mL or less, 2.5 x 10 12 Transducing units / mL or less, 5 x 10 12 Transducing units / mL or less, 1 x 10 13 Transducing units / mL or less, 5 x 10 13 Transducing units / mL or less, or 1 x 10 14It is administered to subjects at a titer of no more than 100 transducing units / mL (including all ranges and subranges therebetween).

[0361] In some embodiments, the vector dose is determined by the route of administration. In some embodiments, intraganglionic injections are administered at about 1×10 in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intrathecal injection contains about 1×10 vector genome in a volume of about 1.0 mL to about 12.0 mL. 10 ~Approx. 1×10 15 In some embodiments, the intracranial injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intraneural injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the intraspinal injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 In some embodiments, the cisternal infusion contains about 5×10 vector genomes in a volume of about 0.5 mL to about 5.0 mL. 9 ~Approx. 5×10 13 In some embodiments, the subcutaneous injection contains about 1×10 vector genome in a volume of about 0.1 mL to about 1.0 mL. 9 ~Approx. 1×10 13 It may include a vector genome.

[0362] In some embodiments, the vector dose is the total dose of a single administration. In some embodiments, the vector dose is the total dose over a period of time. In some embodiments, the period of time is about 3 hours, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 12 months, or more than 12 months. In some embodiments, the period of time is within 3 hours, within 6 hours, within 12 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, within 3 months, within 4 months, within 5 months, within 6 months, or within 12 months.

[0363] In some embodiments, the vector and / or nucleic acid of the present disclosure is delivered to a subject by infusion. The dose delivered to a subject by infusion can be measured as the infusion rate. Non-limiting examples of infusion rates include 1-10 μL / min for intraganglionic, intraspinal, intracranial or intraneural administration; and 10-1000 μL / min for intrathecal or cisternal administration. In some cases, the vector and / or nucleic acid is delivered to a subject by MRI-guided convection-enhanced drug delivery (CED). This technique allows for increased viral diffusion and transduction distributed throughout a large volume of the brain and reduces backflow of the vector and / or nucleic acid along the needle track.

[0364] The present disclosure also provides dosing regimens for administering a ligand (e.g., a small molecule LGIC agonist) when the administered AAV vector and / or nucleic acid encodes an engineered LGIC.

[0365] The therapeutically effective amount of the ligand can be administered once or more than once, daily or for a longer period of time. In some cases, the therapeutically effective amount of the ligand is administered as needed (e.g., when pain relief or epilepsy control is required). The ligand can be administered continuously (e.g., daily, without a break for the duration of the treatment regimen). In some cases, the treatment regimen can be less than one week, one week, two weeks, three weeks, one month, or longer than one month. In some cases, the therapeutically effective amount of the ligand is administered for one day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or for more than ten consecutive days. In certain cases, the therapeutically effective amount of the ligand is administered for three consecutive days. In some cases, a therapeutically effective amount of the ligand can be administered once per week, twice per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per week, nine times per week, ten times per week, eleven times per week, twelve times per week, thirteen times per week, fourteen times per week, fifteen times per week, sixteen times per week, seventeen times per week, eighteen times per week, nineteen times per week, twenty times per week, twenty-five times per day, thirty-five times per day, thirty-five times per day, forty times per day, or more than forty times per day. In some cases, a therapeutically effective amount of the ligand can be administered once per day, twice per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, ten times per day, or more than ten times per day. In some cases, a therapeutically effective amount of the ligand is administered at least every hour, at least every 2 hours, at least every 3 hours, at least every 4 hours, at least every 5 hours, at least every 6 hours, at least every 7 hours, at least every 8 hours, at least every 9 hours, at least every 10 hours, at least every 11 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.Doses of the ligand can be administered to the subject continuously, or 1, 2, 3, 4, or 5 times a day; 1, 2, 3, 4, 5, 6, or 7 times a week, 1, 2, 3, or 4 times a month, once every 2, 3, 4, 5, or 6 months, or once a year, or even at longer intervals. The duration of treatment can last for 1 day, 1, 2, or 3 weeks, 1, 2, 3, 4, 5, 7, 8, 9, 10, or 11 months, 1, 2, 3, 4, 5 or more years, or longer.

[0366] The number of times that the composition is administered to the subject in need of it depends on the medical professional's discretion, the disorder, the severity of the disorder, and the subject's response to the formulation.In some embodiments, the composition is administered at least once.In some embodiments, the administration is more than once, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in a given period.The dosage and / or frequency of each administration can be adjusted as necessary based on the patient's condition and physiological response.

[0367] Methods for generating AAV virions In one aspect, the present disclosure provides a method for producing the AAV virion of the present disclosure.Generally, the method comprises inserting or transducing the AAV vector of the present disclosure into a host cell that can package the AAV vector into AAV virion.Exemplary methods are described and referenced below; however, any method known to those skilled in the art can be used to produce the AAV virion of the present disclosure.

[0368] AAV vectors that contain heterologous nucleic acid and are used to generate AAV virions can be constructed using methods well known in the art.See, for example, Koerber et al. (2009) Mol. Ther., 17:2088;Koerber et al. (2008) Mol Ther., 16:1703-1709;and U.S. Patent Nos. 7,439,065, 6,951,758, and 6,491,907.For example, heterologous sequences can be directly inserted into the AAV genome with the main AAV open reading frame ("ORF") excised therefrom.Other parts of the AAV genome can be deleted, as long as a sufficient portion of the ITR remains and allows replication and packaging functions.

[0369] To produce AAV virions, AAV vectors are introduced into suitable host cells using known techniques such as transfection. Suitable host cells for producing AAV virions include any species and / or type of cells that can be used or have been used as recipients of heterologous AAV DNA molecules and can support the expression of necessary AAV production cofactors from helper viruses. Such host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be used or have been used as recipients of heterologous DNA molecules. The term includes the descendants of the original transfected cells. Thus, as used herein, "host cells" generally refers to cells that are transfected with exogenous DNA sequences. Cells derived from stable human cell lines, HEK293 (e.g., readily available through the American Type Culture Collection under accession number ATCC CRL1573) can be used.

[0370] Methods for producing AAV virions in insect cells are known in the art and can be used to produce the AAV virions of interest. See, for example, U.S. Patent Publication No. 2009 / 0203071; U.S. Patent No. 7,271,002; and Chen (2008) Mol. Ther. 16:924.

[0371] kit In one aspect, the present disclosure provides a kit comprising a vector comprising a polynucleotide encoding an engineered receptor of the present disclosure. In one aspect, the present disclosure provides a kit comprising an engineered receptor of the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV of the present disclosure. In some embodiments, the kit comprises a non-natural ligand of the present disclosure.

[0372] In some embodiments, the kit comprises: (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide; and (b) instructions for administering the AAV vector to transduce dorsal root ganglion neurons or trigeminal ganglion neurons. In some embodiments, the kit comprises a device adapted for administering the AAV vector. In some embodiments, the device is adapted for intrathecal (IT) or intraganglionic (IG) administration.

[0373] In some embodiments, the kit comprises: (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide; and (b) instructions for administering the AAV vector to transduce hippocampal neurons. In some embodiments, the kit comprises a device adapted for administering the AAV vector. In some embodiments, the device is adapted for intracranial administration.

[0374] In some embodiments, the kit further comprises packaging material and one or more components therein. The kit typically comprises a label or attached insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. The kit may contain a collection of such components, e.g., AAV capsid polypeptides, AAV vectors, or AAV virions, and optionally a second active component, such as another compound, agent, drug, or composition. In some embodiments, the AAV vector genome encodes a LGIC (e.g., an engineered LGIC), and the second active agent is a ligand for the LGIC (e.g., a small molecule drug).

[0375] A kit refers to a physical structure that houses one or more components of the kit. The packaging material is capable of maintaining the components sterile and may be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0376] The label or insert may include identifying information of the active ingredient's clinical pharmacology, including the amount of dose, mechanism of action, pharmacokinetics and pharmacodynamics therein. The label or insert may include information identifying the manufacturer, serial number, place and date of manufacture, expiration date. The label or insert may include information identifying the manufacturer information, serial number, place and date of manufacture. The label or insert may include information regarding the disease for which the kit components can be used. The label or insert may include instructions for a physician or subject to use one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0377] The label or insert may include information about any benefit that the component may provide, such as preventive or therapeutic benefit. The label or insert may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or physician about situations in which it is not appropriate to use a particular composition. Adverse side effects or complications may also occur if the subject has taken, will take, or is currently taking one or more other drugs that may be incompatible with the composition, or if the subject has taken, will take, or is currently taking another incompatible treatment protocol or therapeutic regimen, and therefore the instructions may include information about such incompatibilities.

[0378] Further Numbered Embodiments Further numbered embodiments of the present disclosure are provided as follows:

[0379] Embodiment 1. A method of treating neuropathic pain in a subject in need of such treatment, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces a neuron in the subject, and the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron.

[0380] Embodiment 1.1. The method of embodiment 1, wherein the neuropathic pain is a peripheral neuropathy.

[0381] Embodiment 1.2. The method of embodiment 1, wherein the neuropathic pain is trigeminal neuralgia.

[0382] Embodiment 2. A method of treating a neurological disease or disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces neurons in the subject.

[0383] Embodiment 3. The method of embodiment 2, wherein the neurological disease or disorder is neuropathic pain, spasticity, spinal cord injury, or avulsion injury.

[0384]

[0023] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the AAV vector is an AAV9-TV vector comprising a capsid polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9.

[0385] Embodiment 5. A method of transducing a neuron, comprising contacting said neuron with an adeno-associated virus (AAV) vector, wherein said neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron.

[0386]

[0023] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the AAV vector comprises a heterologous nucleic acid.

[0387] Embodiment 7. A method of delivering a heterologous nucleic acid to a neuron, comprising contacting said neuron with an adeno-associated virus (AAV) vector comprising a capsid polypeptide, wherein said neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron.

[0388]

[0023] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the AAV vector is an AAV vector capable of transducing iPSC-derived neurons in vitro.

[0389]

[0021] Embodiment 9. The method of any one of embodiments 1 to 8, comprising selecting the AAV vector as an AAV vector capable of transducing iPSC-derived neurons in vitro.

[0390]

[0023] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the AAV vector is an AAV2, AAV2.5, AAV2.5-TV, AAV2.5-2YF, AAV2.5-TV2YF, AAV5, AAV6, AAV9, AAV9-TV, AAV9-2YF, AAV9-TV2YF, or AAV-PHP.S vector.

[0391]

[0023] Embodiment 11. The method of any one of embodiments 1 to 9, wherein the AAV vector is an AAV9-TV vector.

[0392]

[0023] Embodiment 12. The method of any one of embodiments 1 to 9, wherein the AAV vector is an AAV6 vector.

[0393] Embodiment 13. The method of any one of embodiments 1 to 9, wherein the AAV vector is an AAV5 vector.

[0394] Embodiment 14. The method of any one of embodiments 1 to 9, wherein the AAV vector is an AAV2.5-TV2YF vector.

[0395] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the AAV vector comprises a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12.

[0396]

[0023] Embodiment 16. The method of any one of embodiments 1 to 14, wherein the AAV vector comprises a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3 and 5-9.

[0397]

[0023] Embodiment 17. The method of any one of embodiments 1 to 14, wherein the AAV vector comprises a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 5-7 and 9.

[0398]

[0023] Embodiment 18. The method of any one of embodiments 1 to 14, wherein the AAV vector comprises a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the capsid polypeptide comprises a non-threonine mutation at a position corresponding to T492 in SEQ ID NO:8.

[0399] Embodiment 19. The method of embodiment 18, wherein the non-threonine mutation is a valine, isoleucine, or leucine substitution.

[0400] Embodiment 20. The method of embodiment 18, wherein the non-threonine mutation is a valine substitution.

[0401] Embodiment 21. The method of any one of embodiments 1 to 20, wherein the neuron is a dorsal root ganglion neuron.

[0402]

[0023] Embodiment 22. The method of any one of embodiments 1 to 20, wherein the neuron is a trigeminal ganglion neuron.

[0403]

[0023] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the neurons comprise isolectin B4 (IB4) positive nerve fibers.

[0404]

[0023] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the neurons comprise NF200-positive nerve fibers.

[0405] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the neurons comprise CGRP-positive nerve fibers.

[0406]

[0023] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the neurons comprise C-fibers.

[0407]

[0023] Embodiment 27. The method of any one of embodiments 1 to 25, wherein said neurons comprise A-delta fibers.

[0408]

[0031] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the AAV vector is administered by intrathecal (IT) or intraganglionic (IG) administration.

[0409] Embodiment 29. The method of embodiment 28, wherein the AAV vector is administered directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

[0410] Embodiment 30. A method of treating focal epilepsy in a subject in need thereof, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces neurons in the subject, and the neurons are hippocampal neurons.

[0411] Embodiment 30.1. A method of treating a neurological disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an adeno-associated virus (AAV) vector, wherein the AAV vector transduces hippocampal neurons in the subject.

[0412] Embodiment 30.2. The method of embodiment 30.1, wherein the neurological disease or disorder is focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome.

[0413] Embodiment 31. A method of transducing a neuron, comprising contacting said neuron with an adeno-associated virus (AAV) vector, wherein said neuron is a hippocampal neuron.

[0414]

[0036] Embodiment 32. The method of any one of embodiments 30 or 31, wherein the AAV vector comprises a heterologous nucleic acid.

[0415] Embodiment 33. A method of delivering a heterologous nucleic acid to a neuron, comprising contacting said neuron with an adeno-associated virus (AAV) vector comprising a capsid polypeptide, wherein said neuron is a hippocampal neuron.

[0416] Embodiment 34. The method of any one of embodiments 30 to 33, wherein the AAV vector is an AAV vector capable of transducing embryonic hippocampal neurons in vitro.

[0417]

[0036] Embodiment 35. The method of any one of embodiments 30 to 34, comprising selecting the AAV vector as an AAV vector capable of transducing embryonic hippocampal neurons in vitro.

[0418] Embodiment 36. The method of any one of embodiments 30 to 35, wherein the AAV vector is an AAV2, AAV2.5, AAV2.5-TV, AAV2.5-2YF, AAV2.5-TV2YF, AAV5, AAV6, AAV9, AAV9-TV, AAV9-2YF, AAV9-TV2YF, or AAV-PHP.S vector.

[0419] Embodiment 37. The method of any one of embodiments 30 to 36, wherein the AAV vector is an AAV9 vector.

[0420] Embodiment 38. The method of any one of embodiments 30 to 36, wherein the AAV vector is an AAV9-TV vector.

[0421] Embodiment 39. The method of any one of embodiments 30 to 36, wherein the AAV vector is an AAV6 vector.

[0422] Embodiment 40. The method of any one of embodiments 30 to 36, wherein the AAV vector is an AAV5 vector.

[0423]

[0036] Embodiment 41. The method of any one of embodiments 30 to 40, wherein the AAV vector comprises a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12.

[0424] Embodiment 42. The method of any one of embodiments 30 to 40, wherein the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3 and 5-9.

[0425] Embodiment 43. The method of any one of embodiments 30 to 40, wherein the capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6-9.

[0426] Embodiment 44. The method of any one of embodiments 30 to 40, wherein said capsid polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

[0427]

[0036] Embodiment 45. The method of any one of embodiments 30 to 44, wherein the neuron is an excitatory neuron.

[0428]

[0036] Embodiment 46. The method of embodiment 45, wherein the neuron is a CAMK2 positive neuron.

[0429] Embodiment 47. The method of any one of embodiments 30 to 44, wherein the neuron is an inhibitory neuron.

[0430] Embodiment 48. The method of embodiment 47, wherein the neuron is a GABAergic neuron.

[0431] Embodiment 49. The method of any one of embodiments 30 to 48, wherein the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0432] Embodiment 50. The method of any one of embodiments 30 to 49, wherein the AAV vector is administered intracranially, intrathecally (spinal), intrathecally (cisternal), intracerebrally, intraventricularly, or by direct injection into the epileptic focus of the hippocampus.

[0433] Embodiment 51. The method of embodiment 50, wherein the AAV vector is administered by direct injection into the epileptic focus of the hippocampus.

[0434]

[0023] Embodiment 52. The method of any one of embodiments 6 to 29 and 32 to 51, wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0435] Embodiment 53. The method of embodiment 52, wherein the ligand-gated ion channel comprises a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor.

[0436] Embodiment 54. The method of embodiment 52 or 53, wherein the ligand binding domain comprises an amino acid sequence having at least 85% identity to amino acid residues 23 to 220 of SEQ ID NO:25.

[0437] Embodiment 55. The method of embodiment 54, wherein said ligand-binding domain comprises an amino acid mutation at a residue selected from those corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172 and Y210 of SEQ ID NO:25.

[0438] Embodiment 56. The method of embodiment 54 or 55, wherein the ligand binding domain comprises one or more amino acid mutations listed in Table 3.

[0439] Embodiment 57. The method of embodiment 56, wherein the ligand-binding domain comprises mutations corresponding to R101W, Y115E and Y210W in SEQ ID NO:33.

[0440] Embodiment 58. The method of embodiment 56, wherein the ligand-binding domain comprises mutations corresponding to R101W and Y210V in SEQ ID NO:33.

[0441] Embodiment 59. The method of embodiment 56, wherein the ligand-binding domain comprises mutations corresponding to R101M and L131F in SEQ ID NO:33.

[0442] Embodiment 60. The method of any one of embodiments 53 to 59, wherein the human glycine receptor is human glycine receptor alpha 1, human glycine receptor alpha 2, or human glycine receptor alpha 3.

[0443] Embodiment 61. The method of embodiment 60, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity to amino acids 255 to 457 of SEQ ID NO:26, 260 to 452 of SEQ ID NO:27, amino acids 259 to 464 of SEQ ID NO:28, or amino acids 259 to 449 of SEQ ID NO:29.

[0444] Embodiment 62. The method of any one of embodiments 53 to 61, wherein the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from the human glycine receptor.

[0445]

[0023] Embodiment 63. The method of embodiment 62, wherein the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:26.

[0446]

[0023] Embodiment 64. The method of embodiment 62, wherein the Cys loop domain comprises amino acids 166-180 of SEQ ID NO:26.

[0447] Embodiment 65. The method of any one of embodiments 53 to 64, wherein said ligand binding domain of the engineered receptor comprises the β1-2 loop domain derived from the human glycine receptor α1 subunit.

[0448]

[0023] Embodiment 66. The method of embodiment 65, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:26.

[0449] Embodiment 67. The method of any one of embodiments 53 to 66, wherein said human glycine receptor is human glycine receptor alpha 1 and said ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

[0450] Embodiment 68. The method of any one of embodiments 52 to 67, comprising administering a ligand for the ligand-gated ion channel.

[0451] Embodiment 69. The method of embodiment 68, wherein the ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487.

[0452] Embodiment 70. The method of embodiment 68, wherein the ligand is TC-5619.

[0453] Embodiment 70.1. The method of embodiment 68, wherein the ligand is ABT-126.

[0454] Embodiment 71. The method of any one of embodiments 1 to 70.1, wherein the subject is a primate.

[0455] Embodiment 72. The method of embodiment 71, wherein the subject is a human, optionally an adult.

[0456] Embodiment 73. (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; (b) instructions for administering the AAV vector to transduce dorsal root ganglion neurons or trigeminal ganglion neurons. Kit including:

[0457] Embodiment 74. The kit of embodiment 73, further comprising a device adapted for administration of said AAV vector by intrathecal (IT) or intraganglionic (IG) administration.

[0458] Embodiment 75. The kit of embodiment 74, wherein the device is adapted for administration directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

[0459] Embodiment 76. (a) an adeno-associated virus (AAV) vector comprising a capsid polypeptide comprising, or consisting of, an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; (b) instructions for administering the AAV vector to transduce hippocampal neurons. Including the kit.

[0460] Embodiment 77. The kit of embodiment 76, further comprising a device adapted for administration of said AAV vector by intracranial administration, intrathecal (spinal), intrathecal (cisternal), intracerebral, intraventricular administration, or direct injection into the epileptic focus of the hippocampus.

[0461] Embodiment 78. The kit of embodiment 77, wherein the device is adapted for administration by direct injection into the epileptic focus of the hippocampus.

[0462] Embodiment 79. The kit of any one of embodiments 73 to 78, wherein the AAV vector comprises a heterologous nucleic acid encoding a ligand-gated ion channel.

[0463] Embodiment 80. The kit of embodiment 79, wherein the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.

[0464] Embodiment 81. The kit of embodiment 79 or 80, comprising a ligand for said ligand-gated ion channel.

[0465] Embodiment 82. The kit of embodiment 81, wherein the ligand is TC-5619.

[0466] Embodiment 82.1. The kit of embodiment 81, wherein the ligand is ABT-126.

[0467] Embodiment 83. A method of treating neuropathic pain in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to a dorsal root ganglion neuron or a trigeminal ganglion neuron of the subject, and the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0468] Embodiment 83.1. The method of embodiment 83, wherein the neuropathic pain is a peripheral neuropathy.

[0469] Embodiment 83.2. The method of embodiment 83, wherein the neuropathic pain is trigeminal neuralgia.

[0470] Embodiment 84. A method of treating a neurological disease or disorder in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0471] Embodiment 85. The method of embodiment 84, wherein the neurological disease or disorder is neuropathic pain, spasticity, spinal cord injury, or avulsion injury.

[0472] Embodiment 86. The method of any one of embodiments 83 to 85, wherein the heterologous nucleic acid is administered by intrathecal (IT) or intraganglionic (IG) administration.

[0473] Embodiment 87. The method of embodiment 86, wherein the heterologous nucleic acid is administered directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

[0474]

[0046] Embodiment 88. The method of any one of embodiments 83 to 87, wherein the heterologous nucleic acid is contained within a vector.

[0475] Embodiment 89. The method of embodiment 88, wherein the vector is a viral vector, and the viral vector transduces the dorsal root ganglion neurons or trigeminal ganglion neurons.

[0476]

[0046] Embodiment 90. The method of any one of embodiments 83 to 89, wherein the neurons comprise isolectin B4 (IB4) positive nerve fibers.

[0477]

[0046] Embodiment 91. The method of any one of embodiments 83 to 90, wherein the neurons comprise NF200-positive nerve fibers.

[0478] Embodiment 92. The method of any one of embodiments 83 to 91, wherein the neurons comprise CGRP-positive nerve fibers.

[0479]

[0041] Embodiment 93. The method of any one of embodiments 83 to 92, wherein the neurons comprise C-fibers.

[0480]

[0046] Embodiment 94. The method of any one of embodiments 83 to 92, wherein the neurons comprise A-delta fibers.

[0481] Embodiment 95. A method of treating focal epilepsy in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to hippocampal neurons of the subject, and the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0482] Embodiment 95.1. A method of treating a neurological disease or disorder in a subject in need thereof, comprising administering an effective amount of a heterologous nucleic acid, wherein the heterologous nucleic acid is delivered to hippocampal neurons of the subject, and wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

[0483] Embodiment 95.2. The method of embodiment 95.1, wherein the neurological disease or disorder is focal epilepsy, schizophrenia, autism spectrum disorder, Alzheimer's disease, Rett syndrome, or fragile X syndrome.

[0484] Embodiment 96. The method of any one of embodiments 95 to 95.2, wherein the heterologous nucleic acid is administered by intracranial administration, intrathecal (spinal), intrathecal (cisternal), intracerebral, intraventricular administration, or direct injection into the epileptic focus of the hippocampus.

[0485] Embodiment 97. The method of embodiment 96, wherein the heterologous nucleic acid is administered by direct injection into the epileptic focus of the hippocampus.

[0486]

[0046] Embodiment 98. The method of any one of embodiments 95 to 97, wherein the heterologous nucleic acid is contained within a vector.

[0487] Embodiment 99. The method of embodiment 98, wherein the vector is a viral vector, and the viral vector transduces the hippocampal neurons.

[0488]

[0023] Embodiment 100. The method of any one of embodiments 95 to 99, wherein the neuron is an excitatory neuron.

[0489]

[0031] Embodiment 101. The method of embodiment 100, wherein the neuron is a CAMK2 positive neuron.

[0490]

[0031] Embodiment 102. The method of any one of embodiments 95 to 99, wherein the neuron is an inhibitory neuron.

[0491] Embodiment 103. The method of embodiment 102, wherein the neuron is a GABAergic neuron.

[0492] Embodiment 104. The method of any one of embodiments 95 to 103, wherein the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

[0493] Embodiment 105. The method of any one of embodiments 83 to 104, wherein the ligand-gated ion channel comprises a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor.

[0494] Embodiment 106. The method of embodiment 105, wherein the ligand binding domain comprises an amino acid sequence having at least 85% identity to amino acid residues 23 to 220 of SEQ ID NO:25.

[0495] Embodiment 107. The method of embodiment 106, wherein said ligand-binding domain comprises an amino acid mutation at a residue selected from those corresponding to W77, R101, Y115, L131, Q139, Y140, S170, S172 and Y210 of SEQ ID NO:25.

[0496] Embodiment 108. The method of embodiment 105 or 106, wherein said ligand binding domain comprises one or more amino acid mutations listed in Table 3.

[0497] Embodiment 109. The method of any one of embodiments 105 to 108, wherein the human glycine receptor is human glycine receptor alpha 1, human glycine receptor alpha 2, or human glycine receptor alpha 3.

[0498] Embodiment 110. The method of embodiment 109, wherein the ion pore domain comprises an amino acid sequence having at least 85% identity to amino acids 255 to 457 of SEQ ID NO:26, 260 to 452 of SEQ ID NO:27, amino acids 259 to 464 of SEQ ID NO:28, or amino acids 259 to 449 of SEQ ID NO:29.

[0499] Embodiment 111. The method of any one of embodiments 105 to 110, wherein the ligand binding domain of the engineered receptor comprises a Cys loop domain derived from the human glycine receptor.

[0500]

[0032] Embodiment 112. The method of embodiment 111, wherein the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:26.

[0501]

[0046] Embodiment 113. The method of embodiment 111, wherein the Cys loop domain comprises amino acids 166-180 of SEQ ID NO:26.

[0502] Embodiment 114. The method of any one of embodiments 105 to 113, wherein said ligand binding domain of the engineered receptor comprises the β1-2 loop domain derived from the human glycine receptor α1 subunit.

[0503]

[0046] Embodiment 115. The method of embodiment 114, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:26.

[0504] Embodiment 116. The method of any one of embodiments 105 to 115, wherein said human glycine receptor is human glycine receptor alpha 1 and said ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33.

[0505]

[0046] Embodiment 117. The method of any one of embodiments 83 to 116, comprising administering a ligand for the ligand-gated ion channel.

[0506] Embodiment 118. The method of embodiment 117, wherein the ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487.

[0507] Embodiment 119. The method of embodiment 117, wherein the ligand is TC-5619.

[0508] Embodiment 119.1. The method of embodiment 117, wherein the ligand is ABT-126.

[0509] Embodiment 120. The method of any one of embodiments 83 to 119.1, wherein the subject is a primate.

[0510] Embodiment 121. The method of embodiment 120, wherein the subject is a human, optionally an adult. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14] [Table 5-15] [Table 5-16] EXAMPLES

[0511] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now conceive numerous variations, changes and substitutions without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used in implementing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0512] Example 1 Selection of AAV capsids using human iPSC-derived sensory neuron progenitors Culture and AAV transduction of iPSC-derived sensory neurons: Axol Bio's human iPSC sensory neuron progenitors were derived from integration-free iPSCs (induced pluripotent stem cells) and differentiated into neurons using small molecules. The cells were further cultured, enriched for neurons, and differentiated in our laboratory for 5 days according to the manufacturer's protocol. At day 5 in culture, larger and longer neurites appeared and cell bodies became more arranged. Cells were transduced with various AAVs containing the indicated capsids and encoding a GFP transgene under the CMV promoter at an MOI (multiplicity of infection) of 3e5. Cells were imaged 3 and 5 days after transduction and harvested for RNA and protein expression analysis at day 5. The same lysed mix was used for the two assays.

[0513] ELISA evaluation: Samples were lysed in Abcam GFP ELISA kit (ab171581) cell extraction buffer. Total protein concentration per sample was determined using Pierce Micro BCA Protein Assay Kit (23235) according to the manufacturer's protocol. Concurrently, GFP concentration per sample was determined using Abcam GFP ELISA kit (ab171581) according to the manufacturer's protocol. Overall, the amount of GFP (ng) per total protein...

Claims

1. A composition for use in a method of treating a neurological disease or disorder in a subject in need of such treatment, the composition comprising an adeno-associated virus (AAV) vector, the method comprising administering to the subject an effective amount of the AAV vector, wherein the AAV vector transduces neurons in the subject.

2. The composition described in claim 1, wherein the neurological disease or disorder is neuropathic pain, the neuron is a dorsal root ganglion neuron or a trigeminal ganglion neuron, and, optionally, the neuropathic pain is a peripheral neuropathy or trigeminal neuralgia.

3. The composition described in claim 1, wherein the neurological disease or disorder is spasticity.

4. The composition described in claim 1, wherein the neurological disease or disorder is focal epilepsy and the neuron is a hippocampal neuron.

5. 1. An in vitro method of transducing a neuron, comprising contacting the neuron with an adeno-associated virus (AAV) vector, wherein the neuron is a dorsal root ganglion neuron, a trigeminal ganglion neuron, or a hippocampal neuron.

6. The composition of any one of claims 1 to 4 or the method of claim 5, wherein the AAV vector comprises a heterologous nucleic acid.

7. 1. An in vitro method of delivering a heterologous nucleic acid to a neuron, comprising contacting the neuron with an adeno-associated virus (AAV) vector comprising a capsid polypeptide, wherein the neuron is a dorsal root ganglion neuron, a trigeminal ganglion neuron, or a hippocampal neuron.

8. 8. The composition of any one of claims 1 to 4 or the method of claims 5 or 7, wherein the AAV vector is an AAV vector capable of transducing iPSC-derived neurons in vitro or embryonic hippocampal neurons in vitro.

9. The composition described in claim 6 or the method described in any one of claims 5 to 7, wherein the method includes selecting the AAV vector as an AAV vector capable of transducing iPSC-derived neurons in vitro or embryonic hippocampal neurons in vitro.

10. 8. The composition of any one of claims 1 to 4 or the method of claims 5 or 7, wherein the AAV vector is an AAV2, AAV2.5, AAV2.5-TV, AAV2.5-2YF, AAV2.5-TV2YF, AAV5, AAV6, AAV9, AAV9-TV, AAV9-2YF, AAV9-TV2YF, or AAV-PHP.S vector.

11. A composition described in any one of claims 1 to 4 or a method described in claims 5 or 7, wherein the AAV vector comprises a capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 9, 10, and 11.

12. The composition or method of claim 11, wherein the capsid polypeptide comprises a non-threonine mutation at a position corresponding to T492 in SEQ ID NO:

8.

13. 13. The composition or method of claim 12, wherein the non-threonine mutation is a valine, isoleucine, or leucine substitution.

14. The composition of any one of claims 1, 2, 12, and 13, or the method of any one of claims 5, 7, 12, and 13, wherein the neurons comprise isolectin B4 (IB4)-positive nerve fibers, NF200-positive nerve fibers, CGRP-positive nerve fibers, C fibers, or A-delta fibers.

15. A composition described in any one of claims 1, 2, 12, and 13 or a method described in claim 12 or 13, characterized in that the neurological disease or disorder is neuropathic pain and the AAV vector is administered by intrathecal (IT) or intraganglionic (IG) administration.

16. 16. The composition or method of claim 15, wherein the AAV vector is administered directly to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

17. The composition of any one of claims 4, 12, and 13 or the method of any one of claims 5, 7, 12, and 13, wherein the neuron is an excitatory neuron.

18. 18. The composition or method of claim 17, wherein the neuron is a CAMK2 positive neuron.

19. The composition of any one of claims 4, 12, and 13 or the method of any one of claims 5, 7, 12, and 13, wherein the neuron is an inhibitory neuron.

20. 20. The composition or method of claim 19, wherein the neuron is a GABAergic neuron.

21. 21. The composition of any one of claims 4, 12, 13, 18, and 20 or the method of any one of claims 12, 13, 18, and 20, wherein the focal epilepsy is mesial temporal lobe epilepsy (mTLE).

22. The composition of any one of claims 4, 12, 13, 18, and 20 or the method of any one of claims 12, 13, 18, and 20, wherein the neurological disease or disorder is focal epilepsy and the AAV vector is administered intracranially, intrathecal (spinal), intrathecal (cisternal), intracerebral, intraventricular, or by direct injection into the epileptic focus of the hippocampus.

23. 21. The composition of any one of claims 12, 13, 16, 18, and 20, or the method of any one of claims 7, 12, 13, 16, 18, and 20, wherein the heterologous nucleic acid comprises a polynucleotide sequence encoding a ligand-gated ion channel.

24. 24. The composition or method of claim 23, wherein the ligand-gated ion channel comprises a ligand binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor.

25. The composition or method of claim 24, wherein the ligand-binding domain comprises an amino acid sequence having at least 85% sequence identity to amino acid residues 23 to 220 of SEQ ID NO:

25.

26. 26. The composition or method of claim 25, wherein the ligand-binding domain comprises an amino acid mutation corresponding to at least one of W77, Q79, R101, Y115, L131, Q139, Y140, Y151, S170, S172, Y210, or Y217 of SEQ ID NO:

25.

27. 27. The composition or method of claim 25 or 26, wherein the ligand binding domain comprises one or more amino acid mutations listed in Table 3.

28. 28. The composition or method of claim 27, wherein the ligand-binding domain comprises mutations corresponding to (i) R101W, Y115E, and Y210W in SEQ ID NO: 33, (ii) R101W and Y210V in SEQ ID NO: 33, or (iii) R101M and L131F in SEQ ID NO:

33.

29. 29. The composition or method of any one of claims 24 to 26 and 28, wherein the human glycine receptor is human glycine receptor alpha 1, human glycine receptor alpha 2, or human glycine receptor alpha 3.

30. 30. The composition or method of claim 29, wherein the ion pore domain comprises an amino acid sequence having at least 85% sequence identity 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.

31. 31. The composition or method of any one of claims 24-26, 28, and 30, wherein the ligand binding domain of an engineered receptor comprises a Cys loop domain derived from the human glycine receptor.

32. 32. The composition or method of claim 31 , wherein the Cys loop domain comprises amino acids 166-172 of SEQ ID NO:26 or amino acids 166-180 of SEQ ID NO:

26.

33. 33. The composition or method of any one of claims 24 to 26, 28, 30 and 32, wherein the ligand binding domain of an engineered receptor comprises the β1-2 loop domain derived from the α1 subunit of the human glycine receptor.

34. The composition or method of claim 33, wherein the β1-2 loop domain comprises amino acids 81-84 of SEQ ID NO:

26.

35. 35. The composition or method of any one of claims 24 to 26, 28, 30, 32, and 34, wherein the human glycine receptor is human glycine receptor alpha 1 and the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

33.

36. The composition or method of any one of claims 24 to 26, 28, 30, 32, and 34, wherein the subject is administered a non-natural ligand of the ligand-gated ion channel or the neuron is contacted with a non-natural ligand of the ligand-gated ion channel, wherein the non-natural ligand is selected from the group consisting of AZD-0328, TC-6987, ABT-126, TC-5619, TC-6683, varenicline, and facinicline / RG3487.

37. The composition of any one of claims 1 to 4, 6, and 8 to 36 or the method of any one of claims 5 to 36, wherein the subject is a primate.

38. 38. The composition or method of claim 37, wherein the subject is a human, optionally an adult.

39. (a) an adeno-associated virus (AAV) vector comprising (i) a capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 9, 10, and 11, and (ii) a polynucleotide sequence encoding a ligand-gated ion channel, the ligand-gated ion channel comprising a ligand-binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor; (b) instructions for administering the AAV vector to transduce dorsal root ganglion neurons or trigeminal ganglion neurons. Kit including:

40. 40. The kit of claim 39, further comprising a device adapted for administration of the AAV vector by intrathecal (IT) or intraganglionic (IG) administration.

41. 41. The kit of claim 40, wherein the device is adapted for direct administration to the dorsal root ganglion or trigeminal ganglion by intraganglionic (IG) administration.

42. (a) an adeno-associated virus (AAV) vector comprising (i) a capsid polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6, 8, 9, 10, and 11, and (ii) a polynucleotide sequence encoding a ligand-gated ion channel, the ligand-gated ion channel comprising a ligand-binding domain derived from a human α7 nicotinic acetylcholine receptor (α7-nAChR) and an ion pore domain derived from a human glycine receptor; (b) A kit comprising instructions for administering the AAV vector to transduce hippocampal neurons.

43. 43. The kit of claim 42, further comprising a device adapted for administration of the AAV vector by intracranial administration, intrathecal (spinal), intrathecal (cisternal), intracerebral, intraventricular administration, or direct injection into the epileptic focus of the hippocampus.

44. 44. The kit of claim 43, wherein the device is adapted for administration by direct injection into the epileptic focus of the hippocampus.

45. 45. The kit of any one of claims 39 to 44, wherein the AAV vector comprises a heterologous nucleic acid encoding a ligand-gated ion channel.

46. 46. ​​The kit of claim 45, wherein the ligand-gated ion channel comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

33.

47. 47. The kit of claim 46, comprising a non-natural ligand for the ligand-gated ion channel.

48. The kit of claim 47, wherein the non-natural ligand is TC-5619 or ABT-126.

49. A composition for use in a method of transducing a neuron, the composition comprising an adeno-associated virus (AAV) vector, the method comprising contacting the neuron with the adeno-associated virus (AAV) vector, and the neuron being a dorsal root ganglion neuron, a trigeminal ganglion neuron, or a hippocampal neuron.

50. A composition for use in a method of delivering heterologous nucleic acid to a neuron, the composition comprising an adeno-associated virus (AAV) vector comprising a capsid polypeptide, the method comprising contacting the neuron with the adeno-associated virus (AAV) vector, the neuron being a dorsal root ganglion neuron, a trigeminal ganglion neuron, or a hippocampal neuron.

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