Method for treating obsessive-compulsive disorder

Abstract

The present application relates to a method for treating obsessive-compulsive disorder, comprising: a) identifying one or more specific brain regions exhibiting abnormal activation in a subject; and b) targeting the one or more specific brain regions, inhibiting or disrupting neuronal activity in the brain region(s), or interrupting or blocking neuronal signals in the brain region(s) of the subject.

Description

A method for treating obsessive-compulsive disorder Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for treating obsessive-compulsive disorder. Background Technology

[0002] Currently, obsessive-compulsive disorder (OCD) is defined as a mental disorder characterized by persistent (>6 months) and recurrent obsessive thoughts (or compulsive behaviors). OCD is characterized by patients experiencing repetitive, intrusive thoughts and / or performing repetitive behaviors or rituals (compulsive behaviors) in an attempt to alleviate the resulting anxiety or fear. These obsessive thoughts and behaviors severely interfere with the patient's daily life, work, and social functioning.

[0003] Existing treatment methods for OCD include psychotherapy, drug therapy, and physical therapy.

[0004] Psychotherapy: Cognitive behavioral therapy (CBT) and mindfulness therapy are commonly used psychotherapies that help patients identify and change irrational obsessive thoughts and behavioral patterns, and improve self-control. However, the effectiveness of psychotherapy varies from person to person and requires a high degree of cooperation and long-term adherence from the patient.

[0005] Drug treatment: Selective serotonin reuptake inhibitors (SSRIs) are the first-line drug treatment for OCD, relieving symptoms by increasing serotonin levels in the brain. However, some patients do not respond well to SSRIs and experience side effects such as nausea and insomnia. For patients with poor insight, nerve blocks are commonly used clinically, but the effective rate is only 40%–55%.

[0006] Physical therapy includes non-invasive physical therapy (such as repetitive transcranial magnetic stimulation (rTMS), deep transcranial magnetic stimulation (dTMS), and transcranial direct current stimulation (tDCS)) and invasive physical therapy (such as deep brain stimulation (DBS). These physical therapies work by acting on specific brain regions, altering neural activity in the dysfunctional areas, thereby restoring normal functional levels. However, the efficacy and safety of physical therapy still require further research and validation.

[0007] The reported lifetime prevalence of obsessive-compulsive disorder (OCD) worldwide is 0.8%–3.0%. Domestically, the reported point prevalence of OCD is 0.1%–0.3%, and the lifetime prevalence is 0.26%–0.32%, with 30–60% of OCD patients not responding adequately to medication or cognitive behavioral therapy (CBT).

[0008] The pathogenesis of obsessive-compulsive disorder (OCD) is complex, involving abnormalities in multiple brain regions. In recent years, numerous studies have shown that abnormalities in the CSTC circuit (cortico-striatal-thalamo-cortical) are closely related to the pathological mechanisms of OCD. The CSTC circuit is a complex neural network encompassing multiple brain regions, including the cortex, striatum, and thalamus, which are interconnected by nerve fibers to form a closed loop. Under normal circumstances, the CSTC circuit is responsible for coordinating and controlling an individual's behavior and thought processes. However, in OCD patients, abnormalities occur in this circuit, leading to obsessive thoughts and behaviors.

[0009] Specifically, anomalies in the CSTC loop may involve the following aspects:

[0010] Neurotransmitter Imbalance: In patients with OCD, the levels of neurotransmitters (such as dopamine and serotonin) within the CSTC circuit may be imbalanced. This imbalance may lead to abnormal nerve signal transmission, thereby triggering obsessive thoughts and behaviors.

[0011] Brain region dysfunction: Various brain regions within the CSTC circuit may exhibit dysfunction in OCD patients. For example, the nucleus accumbens, cingulate gyrus, striatum, and orbitofrontal cortex may be overactive, leading to an overreaction to obsessive thoughts and behaviors. For instance, the thalamus may fail to properly process and filter neural signals from the cortex, allowing obsessive thoughts and behaviors to persist.

[0012] Abnormal structural connectivity: Abnormalities may also occur in the nerve fiber connections within the CSTC circuit. This abnormality can lead to impaired nerve signal transmission, preventing the CSTC circuit from functioning properly and thus triggering OCD symptoms.

[0013] Further research has shown that specific brain regions within the CSTC circuit (such as the dorsal anterior cingulate cortex and thalamus) exhibit significant structural and metabolic abnormalities in OCD patients. These abnormalities are closely related to the clinical symptoms of OCD, providing new clues for understanding the pathogenesis of OCD. Summary of the Invention

[0014] On one hand, this application provides a method for treating obsessive-compulsive disorder, comprising a) identifying one or more specific brain regions exhibiting abnormal activation in a subject; b) inhibiting or disrupting neuronal activity in the one or more specific brain regions, or interrupting or blocking neuronal signals in the subject's brain regions.

[0015] In some implementations, the subject is a patient with obsessive-compulsive disorder.

[0016] In some implementations, identifying one or more specific brain regions exhibiting abnormal activation in a subject further includes: identifying one or more specific brain regions exhibiting abnormal activation in a subject by neuroimaging.

[0017] In some embodiments, the neuroimaging includes: measurement of neuronal firing signals (Spikes), measurement of cortical potentials (ECoG), PET scan imaging, electroencephalography (EEG) with electromagnetic signal detection, magnetoencephalography (MEG), functional magnetic resonance imaging (fMRI), and near-infrared spectroscopy (NIRS).

[0018] In some embodiments, the PET scanning imaging includes: Aβ-PET scanning imaging and / or tau-PET.

[0019] In some embodiments, the method further includes: obtaining brain region detection results of the subject through neuroimaging, and determining one or more specific brain regions of abnormal activation in the subject by comparing the brain region detection results of the subject with reference values.

[0020] In some implementations, the reference value is the brain region detection result of a healthy control group obtained through the neuroimaging.

[0021] In some embodiments, the method further includes: 1) under specific task stimulation, the BOLD signal intensity in certain brain regions of the subject is significantly higher than that of the healthy control group, or the activation pattern of certain brain regions is significantly different from that of the healthy control group, such as the range, intensity, or timing of the activated brain regions, and such difference is determined to be statistically significant by appropriate statistical tests; 2) in the resting state, the spontaneous neural activity in certain brain regions of the subject is also significantly higher than that of the healthy control group, or the activation pattern of certain brain regions is significantly different from that of the healthy control group, such as the range, intensity, or timing of the activated brain regions, and such difference is determined to be statistically significant by appropriate statistical tests.

[0022] In some embodiments, the brain region detection results of the subjects and / or brain region detection results and / or reference values ​​of the control groups are generated based on brain oxygen metabolism rate.

[0023] In some embodiments, the one or more specific brain regions include: the medial prefrontal cortex (mPFC), the orbitofrontal cortex (OFC), the anterior cingulate cortex (ACC), the nucleus accumbens (NAc), the ventral striatum (VS), the caudate nucleus (Cd), the bed nucleus of the stria terminalis (BNST), the subthalamus nucleus (STN), and the globus pallidus internus (GPi).

[0024] In some implementations, inhibiting or disrupting neuronal activity in specific brain regions includes severing fiber bundles of the inferior thalamic peduncles (ITP), the medial forebrain bundle (MFB), and the anterior limb of the internal capsule (ALIC).

[0025] On the other hand, this application provides a method for treating obsessive-compulsive disorder, including inhibiting or destroying neuronal activity in a specific brain region, or interrupting or blocking neuronal signals in a specific brain region of the subject.

[0026] In some implementations, the specific brain regions include mPFC, OFC, ACC, NAc, VS, Cd, BNST, STN, and GPi.

[0027] In some implementations, blocking neuronal signaling in specific brain regions of a subject involves expressing exogenous receptors to the subject.

[0028] In some implementations, blocking neuronal signals in a specific brain region of the subject includes expressing exogenous receptors in that specific brain region of the subject.

[0029] In some implementations, blocking neuronal signals in specific brain regions of a subject further includes administering an exogenous ligand to the subject.

[0030] In some embodiments, the exogenous receptor includes a G protein-coupled receptor (GPCR) or an ion channel protein.

[0031] In some embodiments, the G protein-coupled receptor is a designer receptor DREADD specifically activated by the designer drug.

[0032] In some implementations, the DREADD includes Rq (R165L), hM1Dq, hM5Dq, rM3D, hM2Di, M4Di and its variants, hM3Dq, AlstR, or KORD.

[0033] In some embodiments, when the exogenous receptor is hM4Di or its variants or hM3Dq, the exogenous ligand includes clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160, and compound 21 (C21).

[0034] In some embodiments, the exogenous receptor includes a photosensitive GPCR.

[0035] In some embodiments, the photosensitive GPCR includes Lamplight (Lamprey Parapinopsin), rod opsin, cone opsin, Mu opioid receptor-rod opsin chimera, and Mu opioid receptor.

[0036] In some embodiments, the ion channel protein is a ligand-gated ion channel (LGIC) protein or a light-gated ion channel protein.

[0037] In some embodiments, the LGIC ligand-gated ion channels include GlyR-M, GluCl, PSAM-5HT3HC, PSAM-GlyR, PSAM-nAChR, PSAM4-5HT3, PSAM4-GlyR, TRPV1, or GABAA.

[0038] In some embodiments, when the exogenous receptor is an LGIC ligand-gated ion channel, the exogenous ligand includes ivermectin, selamectin, doramectin, emamectin, epramectin, abamedin, moxicillin, PSEM22S, PSEM89S, PSEM9S, varenicline, capsaicin, or zolpidem.

[0039] In some embodiments, the light-gated ion channel proteins include NpHR, eNpHR3.0, Arch, eArch 3.0, ArchT, eArchT 3.0, eBR, iC1C2, iChloC, GtACR1, GtACR2, SwiChRca, PsChR1, Phobos, Aurora, Jaws, Mac, eMac 3.0, ChR2, ChIEF, C1V1, ReaChR, ChrimsonR, and Chronos.

[0040] In some embodiments, it further includes delivering a nucleic acid molecule encoding an exogenous receptor to the subject prior to administration of the exogenous ligand.

[0041] In some embodiments, the nucleic acid molecule is delivered to the subject in a viral vector.

[0042] In some embodiments, the viral vector is an adeno-associated virus (AAV), a herpesvirus vector, a retroviral vector, a vaccinia virus vector, an adenovirus vector, or a lentiviral vector.

[0043] In some embodiments, the AAV carrier includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV Retro, AAV DJ, AAVrh10, Anc80, or variations of the above AAV carriers.

[0044] In some implementations, the nucleic acid encoding the modified exogenous receptor is operatively linked to the promoter.

[0045] In some implementations, the promoter is one of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0046] In some embodiments, the constitutive promoters include the immediate early promoter of cytomegalovirus (CMV), viral simian virus 40 (SV40), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus thymidine kinase (HSV-tk) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70 kDa 5 (HSPA5), heat shock protein 90 kDa β member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin (β-KIN), and human ROSA. One or more of the following promoters: 26, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, or β-actin promoter.

[0047] In some embodiments, the inducible promoter includes one of the following: tetracycline-responsive promoter, ecdysone-responsive promoter, cumate-responsive promoter, glucocorticoid-responsive promoter, estrogen-responsive promoter, PPAR-γ promoter, and RU-486-responsive promoter.

[0048] In some embodiments, the tissue-specific promoters include neuron-specific promoters, glial cell-specific promoters, heart-specific promoters, muscle-specific promoters, lung-specific promoters, liver-specific promoters, kidney-specific promoters, pancreas-specific promoters, adipose-specific promoters, endothelial-specific promoters, retinal-specific promoters, prostate-specific promoters, skin-specific promoters, and macrophage-specific promoters.

[0049] In some embodiments, the promoter is a neuron-specific promoter, which includes: human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, Advillin promoter, Drosophila single homologue 1 (SIM1) promoter, oxytocin (OXT) promoter, and spiky mouse-associated protein (AgRP) promoter. Promoters include protein kinase C-δ (PKC-δ), auxin-releasing peptide, glutamate decarboxylase (GAD1 / 2) promoter, choline acetyltransferase (ChAT) promoter, vesicle GABA transporter (VGAT) promoter, glutamate decarboxylase (GAD65) promoter, tyrosine hydroxylase (TH) promoter, and promoters without a distant homeobox (Dlx), cell activity-dependent promoters (c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter, or synthetic activity-dependent promoters such as PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), and FRAM (Fos Robust Activity Marker).

[0050] In some embodiments, the promoter is CaMKIIα.

[0051] In some embodiments, the glial cell-specific promoters include the astrocyte fibrillary acidic protein (GFAP) promoter, Gfabc1D promoter, ALDH1L1 promoter, Pirt promoter, Cst3 promoter, Cx30 promoter, myelin basal protein (MBP) promoter, oligodendrocyte myelin glycoprotein (MOG) promoter, CNP (NPPC) promoter, PLP promoter, Pdgfra promoter, olig2 promoter, NG2 promoter, CD11b promoter, Iba1 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, and Foxj1 promoter.

[0052] In some embodiments, the nucleic acid molecule is delivered to the subject using a non-viral method.

[0053] In some embodiments, the non-viral method is liposome transfection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection.

[0054] In some embodiments, the G protein-coupled receptor is Gi-coupled or Gq-coupled.

[0055] In some embodiments, the method of destroying neurons in a specific brain region includes surgically removing neurons in the specific brain region or severing fiber bundles of the inferior thalamic peduncles (ITP), the medial forebrain bundle (MFB), and the anterior limb of the internal capsule (ALIC).

[0056] In some embodiments, the method of destroying neurons in a specific brain region includes inducing apoptosis of neurons in that specific brain region.

[0057] In some implementations, methods for destroying neurons in specific brain regions include administering apoptosis proteins to the subject or administering nucleic acids encoding apoptosis proteins.

[0058] In some embodiments, the apoptosis protein is caspase 3 / 7.

[0059] In some implementations, methods involving neurons in specific brain regions include administering toxin receptor proteins and their ligands to the subject.

[0060] In some implementations, methods for damaging neurons in specific brain regions include administering to a subject the coding sequence of a toxin receptor protein and its ligand.

[0061] In some implementations, the toxin receptor protein is DTR (diphtheria toxin receptor).

[0062] In some embodiments, the method of interrupting or blocking neuronal signals in a specific brain region includes administering to the subject an inhibitor of neurotransmitters synthesized or secreted by neurons in the aforementioned brain region.

[0063] In some embodiments, the method of interrupting or blocking neuronal signals in a specific brain region includes administering to a subject an antagonist of a neurotransmitter synthesized or secreted by neurons in the aforementioned brain region.

[0064] In some embodiments, the method of interrupting or blocking neuronal signals in a specific brain region includes administering a neutralizer of neurotransmitters synthesized or secreted by neurons in the aforementioned brain region to the subject.

[0065] In some implementations, methods for interrupting or blocking neuronal signals in specific brain regions include administering TeNT (tetanus toxin) to the subject.

[0066] In some implementations, methods for interrupting or blocking neuronal signals in specific brain regions include inhibiting the release of neurotransmitters.

[0067] In some implementations, methods for interrupting or blocking neuronal signals in specific brain regions include inhibiting the synthesis of neurotransmitters.

[0068] In some embodiments, the administration includes oral, intrathecal, intraganglionic, intracranial, subcutaneous, intraspinal, intracisional, or local administration.

[0069] On the other hand, this application provides the use of the aforementioned exogenous receptor, exogenous ligand, nucleic acid molecule encoding exogenous receptor, apoptosis protein, nucleic acid encoding apoptosis protein, toxin receptor protein and its ligand, coding sequence of toxin receptor protein, inhibitor of neurotransmitters synthesized or secreted by neurons in a specific brain region, antagonist of neurotransmitters synthesized or secreted by neurons in a specific brain region, and neutralizer of neurotransmitters synthesized or secreted by neurons in a specific brain region for the preparation of a drug for treating obsessive-compulsive disorder.

[0070] On the other hand, this application provides the aforementioned exogenous receptor, exogenous ligand, nucleic acid molecule encoding exogenous receptor, apoptosis protein, nucleic acid encoding apoptosis protein, toxin receptor protein and its ligand, coding sequence of toxin receptor protein, and inhibitors of neurotransmitters synthesized or secreted by neurons in specific brain regions, antagonists of neurotransmitters synthesized or secreted by neurons in specific brain regions, and neutralizers of neurotransmitters synthesized or secreted by neurons in specific brain regions for the treatment of obsessive-compulsive disorder.

[0071] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0072] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0073] Figure 1 shows the chemogenetic strategy based on neuron-specific promoters described in this application for inhibiting ACC in the treatment of sapap3. - / - Proof-of-concept study of obsessive-compulsive disorder phenotype in KO transgenic mice: changes in grooming phenotype before and after drug administration. A, frequency of grooming episodes; B, duration of grooming behavior.

[0074] Figure 2 shows the proof-of-concept of the mouse model of 8-OH-DPAT obsessive-compulsive disorder, which is based on the neuron-specific promoter-based chemogenetic strategy described in this application to inhibit ACC. A, DCZ (0.3 mg / kg, ip); B, CNO (3 mg / kg, ip); C, Clozapine (2 mg / kg, po). Mean ± SEM; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0075] Figure 3 shows that bilateral ACC expression of kir2.3 improves sapap3. - / - The model's facial damage.

[0076] Figure 4 shows the immobilization behavior of bilateral ACC expression of Kir2.3 inhibiting 8-OH-DPAT-induced fixation. A, T-maze experiment flowchart; B, efficacy test. Mean ± SEM; *P<0.05; **P<0.01; ****P<0.0001.

[0077] Figure 5 shows the improvement of sapap3 expression by bilateral ACC-induced expression of kir2.3. - / - Behavioral phenotypes and facial lesions in the model. A, sapap3 - / - The model induces the expression of AAV9::cfos-kir2.3-T2A-mcherry virus; B, bilateral ACC induces Kir2.3 expression and inhibits sapap3. - / - The model exhibits excessive grooming behavior; C, bilateral ACC induces Kir2.3 expression and inhibits sapap3. - / - Facial damage caused by excessive grooming in the model.

[0078] Figure 6 shows the bilateral ACC-induced Kir2.3 expression inhibiting 8-OH-DPAT-induced immobilization behavior. A, 8-OH-DPAT-induced expression of AAV9::cfos-kir2.3-T2A-mcherry virus; B, T-maze assay flowchart; C, efficacy test. Mean ± SEM; **P<0.01; ***P<0.001. Detailed Implementation

[0079] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0080] Terminology Definition

[0081] The term "obsessive-compulsive disorder" is a mental disorder characterized by persistent (e.g., more than 6 months) recurring obsessive thoughts (or) compulsive behaviors.

[0082] In this application, the term "anterior cingulate cortex" or "ACC" refers to the anterior part of the cingulate cortex (or limbic lobe), a horseshoe-shaped structure located near the center of the brain. The ACC is part of the cerebral cortex, located below the frontal and parietal lobes, surrounding the head of the corpus callosum.

[0083] In this application, the term "medial prefrontal cortex" or "mPFC" refers to the medial portion of the prefrontal cortex of the brain, located on the medial surface of the prefrontal cortex of both hemispheres of the brain, close to the midline of the brain. It is an important region of the cerebral cortex, adjacent to the superior frontal sulcus and cingulate sulcus.

[0084] In this application, the term "orbitofrontal cortex" or "OFC" refers to the cortical region located in the anterior part of the prefrontal lobe of the brain, adjacent to the orbit. Anatomically, it covers a large area of ​​the ventral frontal lobe and is further subdivided into different parts by the olfactory groove, intraorbital groove, extraorbital groove, and transverse orbital groove, such as the rectus gyrus, intraorbital gyrus, preorbital gyrus, extraorbital gyrus, and posterior orbital gyrus.

[0085] In this application, the term "nucleus accumbens" or "NAc" refers to a key structure located in the basal ganglia region of the brain, which is part of the limbic system. It is located at the junction of the basal ganglia and the limbic system, inferior to the septum, inferior to the caudate putamen, anteriorly connected to the preolfactory nucleus, and posteriorly terminated in the striatum.

[0086] In this application, the term "ventral striatum" or "VS" refers to the ventral portion of the striatum, which includes structures such as the nucleus accumbens and nearby olfactory tubercles. The ventral striatum has extensive connections with the limbic system, the fronto-orbital cortex, and the granular cortex.

[0087] In this application, the term "caudate nucleus" or "Cd" refers to a C-shaped structure located in the center of the cerebral hemisphere, which is divided into three parts: head, body, and tail. The head and body of the caudate nucleus form the lateral wall of the frontal horn and the body of the lateral ventricle, respectively, which laterally surround the thalamus.

[0088] In this application, the term "bed nucleus of the stria terminalis" or "BNST" is a nucleus located at the base of the forebrain, between the caudate nucleus and the thalamus, and extending to the amygdala.

[0089] In this application, the term "cortical hypothalamic nucleus" or "STN" refers to a nucleus located in the hypothalamus, below the red nucleus and substantia nigra, whose fibers connect the cerebral cortex and basal ganglia.

[0090] In this application, the term "medial globus pallidus" or "GPi" refers to an important nucleus in the basal ganglia, located deep within the cerebral hemisphere, which together with the lateral globus pallidus externus (GPe) constitutes the globus pallidus.

[0091] In this application, the term "thalamic peduncle" or "ITP" refers to a bundle of nerve fibers that connect the thalamus and the brainstem.

[0092] In this application, the term "medial forebrain tract" or "MFB" refers to an important bundle of nerve fibers located at the base of the brain, which originates from the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain, projects anteromedially, and terminates in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and other limbic system structures.

[0093] In this application, the term "anterior limb of the internal capsule" or "ALIC" refers to the anterior region of the internal capsule, located in the white matter within the cerebral hemisphere. The internal capsule is one of the most important fiber bundles within the cerebral hemisphere, connecting the cerebral cortex with the brainstem, spinal cord, and various parts of the cerebral hemisphere.

[0094] In this application, the term "receptor" refers to a local site or gene expression receptor in a neural synapse that is responsible for receiving neurotransmitters and generating neural signals.

[0095] In this application, the term "ligand" refers to a polypeptide or small molecule that can specifically bind to a switch receptor to modulate the activity of excitable cells expressing the switch receptor.

[0096] In some implementations, the receptor is designed to specifically bind to an exogenous ligand. In some embodiments, the exogenous ligand specifically binds to the switch receptor to modulate the activity of excitable cells expressing the switch receptor, and detectably binds to naturally occurring receptors, but does not cause physiologically measurable changes when binding to naturally occurring receptors.

[0097] In this application, the term "exogenous" or "foreign" is used in this disclosure to refer to any molecule originating outside of an organism, including nucleic acids, proteins or peptides, small molecule compounds, etc. "Small molecule" refers to a composition with a molecular weight less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about 0.5 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptide mimics, peptide admixtures, carbohydrates, lipids, or other organic or inorganic molecules.

[0098] In this application, the term "neurotransmitter" refers to a chemical substance used by neurons to communicate with each other and with target tissues during synaptic transmission. If a neurotransmitter stimulates a target cell to produce an effect, then it is an excitatory neurotransmitter acting in an excitatory synapse. On the other hand, if it inhibits a target cell, it is an inhibitory neurotransmitter acting in an inhibitory synapse. As used herein, "excitatory neurotransmitter" or "inhibitory neurotransmitter" can be artificially synthesized and directly introduced into the target neuron.

[0099] In this application, the term "G protein-coupled receptor (GPCR)" refers to a receptor that, upon binding to a natural ligand and activation of the receptor, transmits G protein-mediated signals, thereby generating G protein-coupled cellular responses. G protein-coupled receptors are a large family of evolutionarily related proteins (see WO97 / 35478). GPCRs interact with complexes of isotriguanine nucleotide-binding proteins (G proteins) to regulate various intracellular signaling pathways, including ion channels.

[0100] In this application, the term "a designer receptor exclusively activated by a designer drug (DREADDs)" can refer to any GPCR activated by an exogenous ligand. Exogenous ligands have low affinity for wild-type receptors, resulting in low-responsive ligand-receptor interactions, but can have high affinity for switch receptors (e.g., DREADDs). In some cases, DREADDs are unresponsive or substantially unresponsive to endogenous ligands, and therefore are primarily activated by exogenous ligands. Examples of DREADDs include, but are not limited to, those designed for muscarinic acetylcholine receptors (e.g., hM1Ds) as described by Armbruster et al., PNAS, 2007. q hM2D i hM3D q M4D i and hM5D q Those on κ opioid receptors, such as those described by Vardy et al., Neuron, 2015, are referenced and incorporated herein by reference. In some cases, DREADD is made from clozapine-N-oxides (e.g., Rq(R165L), rM3D, hM1D). q hM2D i hM3D q M4D i and hM5D qReceptor activation. In some cases, the exogenous ligand for DREADD can be an N4'-alkyl-substituted CNO analogue, including compound 4b (3-chloro-6-(4-ethylpiperazin-1-yl)-5H-benzo[b][1,4]benzodiazepine); compound 6 (4-(8-chloro-5H-dibenzo[b,e][1,4]diazepine-11-yl)-1,1-dimethylpiperazin-1-onium iodide); compound 11 (3-chloro-6-(piperazin- Compounds 11-(1-(piperazin-1-yl)-5H-benzo[b][1,4]benzodiazepine); 13-(8-chloro-11-[4-(1,1-dideuterated ethyl)piperazin-1-yl]-5H-dibenzo[b,e][1,4]diazepine); and 21-(11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine; 11-(4-ethylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine). In some embodiments, DREADD is activated by sarsine B (e.g., KORD). It should be understood that any GPCR can be designed as DREADD.

[0101] Examples of DREADDs can include inhibitory DREADDs such as hM2Di, M4Di and their mutants (e.g., M4Di-399, M4Di-PD, M4Di-Endo, M4Di-PD-ENDO, shortened hM4Di (size-reduced hM4Di)), hM4Dnrxn, and KORD, as well as excitatory DREADDs such as hM3Dq, hM5Dq, Rq(R165L), and rhM3D. hM3Dq is a variant of the human M3 muscarinic (hM3) receptor. It can be activated by clozapine-N-oxide (CNO) and participates in the Gq signaling pathway. Gq signaling releases intracellular calcium reserves and enhances neuronal excitability. Therefore, the firing rate of neurons expressing hM3Dq treated with CNO is significantly increased. Besides CNO, other DREADD ligands, such as compound 21 (C21), DCZ, clozapine, olanzapine, desclozapine, perlapine, JHU 37152, and JHU37160, can also activate hM3Dq.

[0102] Rq(R165L) is a variant of the human M3 muscarinic receptor. It is coupled with β-arrestin and activates non-canonical GPCR signaling independent of G proteins.

[0103] hM3Ds are Gs-coupled DREADDs generated from hM3Dq. In the presence of DREADD ligands (e.g., CNO, C21, DCZ, olanzapine, desclozapine, perlapine, JHU 37152, JHU37160), hM3Ds increase cAMP production but do not increase IP3 or intracellular Ca2+. 2+ The generation of.

[0104] M4Di is an artificial receptor derived from the muscarinic acetylcholine receptor M4 (M4) by introducing two single-point mutations (Y3.33C and A5.46G) located near the ligand-binding pocket. This directly disrupts the structure of the tyrosine cap, leading to a loss of affinity for the endogenous ligand acetylcholine, but allowing pharmacologically inert compounds such as CNO, clozapine, and DCZ to activate the receptor. Upon stimulation by CNO, M4Di activates the Gi protein-coupled inward rectifying potassium channel GIRK, resulting in cell membrane hyperpolarization and thus inhibiting neuronal activity. We refer to the mutated receptor as M4Di. In this application, M4Di can be human M4Di (hM4Di), chicken (Gallus gallus) M4Di, macaque (Macaca mulatta) M4Di, guinea pig (Rattus norvegicus) M4Di, or rabbit (Oryctolagus cuniculus) M4Di.

[0105] The sequence of hM4Di is shown in SEQ ID NO:1, the M4Di of chicken (Gallus gallus) is shown in SEQ ID NO:6, the M4Di of macaque (Macaca mulatta) is shown in SEQ ID NO:11, the M4Di of guinea pig (Rattus norvegicus) is shown in SEQ ID NO:16, and the M4Di of rabbit (Oryctolagus cuniculus) is shown in SEQ ID NO:21.

[0106] M4Di-399 is a mutant of hM4Di, which, compared to M4Di, includes a mutation at the protein kinase A (PKA) phosphorylation site. The sequence of human M4Di-399 is shown in SEQ ID NO:2. The sequence of chicken M4Di-399 is shown in SEQ ID NO:7. The sequence of rhesus monkey M4Di-399 is shown in SEQ ID NO:12. The sequence of guinea pig M4Di-399 is shown in SEQ ID NO:17. The sequence of rabbit M4Di-399 is shown in SEQ ID NO:22.

[0107] M4Di-PD is a mutant of M4Di, characterized by mutations in the proximal and / or distal β-repressor binding domains compared to M4Di. The sequence of human M4Di-PD is shown in SEQ ID NO:3. The sequence of chicken M4Di-PD is shown in SEQ ID NO:8. The sequence of rhesus monkey M4Di-PD is shown in SEQ ID NO:13. The sequence of guinea pig M4Di-PD is shown in SEQ ID NO:18. The sequence of rabbit M4Di-PD is shown in SEQ ID NO:23.

[0108] M4Di-ENDO is a mutant of M4Di with a mutation in the SH3-binding domain of the endocytic protein, which is a proline-rich region. The sequence of human M4Di-ENDO is shown in SEQ ID NO:4. The sequence of chicken M4Di-ENDO is shown in SEQ ID NO:9. The sequence of rhesus monkey M4Di-ENDO is shown in SEQ ID NO:14. The sequence of guinea pig M4Di-ENDO is shown in SEQ ID NO:19. The sequence of rabbit M4Di-ENDO is shown in SEQ ID NO:24.

[0109] M4Di-PD-ENDO is a mutant of M4Di, possessing both the M4Di-PD and M4Di-ENDO mutations. The sequence of human M4Di-PD-ENDO is shown in SEQ ID NO:5. The sequence of chicken M4Di-PD-ENDO is shown in SEQ ID NO:10. The sequence of rhesus monkey M4Di-PD-ENDO is shown in SEQ ID NO:15. The sequence of guinea pig M4Di-PD-ENDO is shown in SEQ ID NO:20. The sequence of rabbit M4Di-PD-ENDO is shown in SEQ ID NO:25.

[0110] hM4Dnrxn is an axon-selective variant of hM4Di. hM4Dnrxn is constructed by adding intracellular amino acid sequences from neurexin-1 (aa1, 425-1, 479) to the C-terminus of hM4D.

[0111] The shortened hM4Di is a mutant of hM4Di, in which the third intracellular loop (ICL3) is replaced by a shorter peptide chain, such as QNTIS (SEQ ID: 26), compared to hM4Di.

[0112] KORD is a Gi-coupled variant of the human κ-opioid receptor, and Salvinorin B (SALB) is an inert ligand of KORD.

[0113] In this application, the term "photosensitive GPCR" refers to a GPCR that is activated or inhibited by some light signal. Illustrative examples of photosensitive GPCRs include visual pigments and their variants. Visual pigments use light-absorbing chromophores to receive light signals. Through the cis-trans isomerization of the chromophore, light energy is converted into chemical free energy, which is then used for conformational changes in the protein to activate the G protein.

[0114] In some implementations, the photosensitive GPCR is derived from the opsin of the rhodopsin protein. Exemplary photosensitive GPCRs include Lamplight (Lamprey Parapinopsin), rod opsin, cone opsin, Mu opioid receptor-rod opsin chimera, and Mu opioid receptor.

[0115] In this application, the term "ion channel protein" refers to a protein molecule that crosses the cell membrane, allowing ions to pass from one side of the membrane to the other. In some embodiments, ion channel proteins include voltage-gated ion channels, ligand-gated ion channels, light-gated ion channels, mechanosensitive ion channels, and thermosensitive ion channels (e.g., TRPV4). In some embodiments, ion channel proteins include cation (e.g., calcium, potassium, sodium, proton) channel proteins, anion (e.g., chloride) channel proteins, and non-selective ion channel proteins.

[0116] In this application, the term "ligand-gated ion channel (LGIC) protein" refers to a large class of transmembrane proteins that allow or inhibit the passage of ions when interacting with specific chemical substances (ligands). The binding of a ligand to an ion channel protein directly results in the opening or closing of the channel.

[0117] In this application, the term "light-gated ion channel" refers to a group of ion channel proteins whose opening or closing is in response to light. Light-gated ion channels used herein may be naturally occurring or synthetically produced. Illustrative examples of light-gated ion channels include channelorhodopsin and its variants.

[0118] In some embodiments, light-gated ion channel proteins are activated by light of a specific wavelength. In some embodiments, light-gated ion channel proteins are inhibited by light of a specific wavelength. In some embodiments, light-gated ion channel proteins are activated by light of a specific wavelength and inhibited by light of another wavelength. For example, combinations of light-gated ion channel proteins with specific wavelengths that inhibit neuronal activation include: iChloC, iC1C2, SwilChRca, GtACR2, Phobos at 460nm-480nm; Aurora at 517nm; PsChR1, Mac, eMac 3.0, Arch, ArchT, eArchT 3.0, eArch 3.0, eBR at 540-570nm; NpHR, eNpHR3.0 at 590nm; and Jaws at 630nm.

[0119] In this application, "specific brain region" refers to a brain region that is abnormally activated compared to a reference value (e.g., the corresponding brain region in a healthy person). When a certain brain region is activated, neurons in that region consume more oxygen and glucose, leading to increased local cerebral blood flow and a higher level of oxyhemoglobin than deoxyhemoglobin, which in turn causes an increase in the intensity of local cortical BOLD (Blood Oxygenation Level Dependent) signals. The aforementioned "compared to" can refer to comparing the neuroimaging results of obsessive-compulsive disorder (OCD) patients with those of healthy individuals. If the comparison reveals a significantly enhanced BOLD signal in a certain brain region in OCD patients, and this difference is confirmed to be statistically significant through appropriate statistical testing, then the brain region can be considered to have "abnormal activation," and thus considered a specific brain region. In this application, specific brain regions may include the media prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), ventral striatum (VS), caudate nucleus, bed nucleus of the stria terminalis (BNST), subthalamus nucleus (STN), and globus pallidus internus (GPi).

[0120] In this application, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" generally refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically defined, the term may include nucleic acids containing analogs of natural nucleotides, said nucleic acids having similar binding properties to a reference nucleic acid (e.g., sequence information shown) and being metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, the sequence of a nucleic acid may include variants modified in a conserved manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences.

[0121] In this application, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0122] In this application, the term “pharmaceutically acceptable” generally refers to those compounds, materials, compositions, and / or dosage forms that are commensurate with a reasonable benefit / risk ratio and suitable for use in human and animal tissue contact without excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of reasonable medical judgment.

[0123] In this application, the term "pharmaceutically acceptable carrier" generally refers to any of those carriers commonly used and is limited only by physicochemical considerations (such as solubility and lack of reactivity with active binders) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that would be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc.; their uses are well known in the art.

[0124] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients and preferably inert at the doses and concentrations used, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0125] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent is generally any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability resulting from the disease).

[0126] In this application, the terms "host cell" or "cell" generally refer to an individual cell, cell line, or cell culture that may contain or already contains a vector including the nucleic acid molecules isolated as described in this application, or that is capable of expressing the nucleic acid molecules isolated as described in this application. The host cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they need to be capable of expressing the nucleic acid molecules isolated as described in this application. The host cell can be obtained by in vitro transfection of cells using the vector described in this application. The host cell can be a prokaryotic cell (e.g., *E. coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or neuronal cells). For example, the host cell may be an *E. coli* cell. For example, the host cell may be a yeast cell. For example, the host cell may be a mammalian cell. For example, the mammalian cell may be an N2A cell.

[0127] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for DNA or RNA replication.

[0128] The vector is a vector for transcription and / or translation of expression. The vector also includes vectors having a variety of the functions described above. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.

[0129] In this application, the term "viral vector" is used broadly to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer. Nucleic acid molecules include virus-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically include various viral components and sometimes host cell components other than nucleic acids. A viral vector can refer to a virus or viral particle capable of transferring nucleic acids into cells, or the transferred nucleic acid itself.

[0130] In this application, the term "lentivirus" generally refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); viscena-maedivirus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0131] In this application, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus in which some functions are provided by co-infected helper viruses. Thirteen AAV serotypes have been characterized. General information and reviews of AAVs can be found, for example, in Carter, 1989, *Handbook of Parvoviruses*, Vol. 1, pp. 169–228, and Berns, 1990, *Virology*, pp. 1743–1764, Raven Press, (New York). However, it is entirely expected that these same principles will apply to other AAV serotypes, as the various serotypes are known to be very closely related in both structure and function, even at the genetic level. For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three associated capsid proteins, such as those expressed in AAV6. The correlation was further demonstrated by heteroduplex analysis, which revealed extensive cross-hybridization along the genome length between serotypes and the presence of similar self-annealing segments corresponding to the ends of inverted terminal repeats (ITRs). Similar infection patterns also indicated that replication function in each serotype is under similar regulatory control.

[0132] In this application, the term "AAV vector" generally refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by an AAV terminal repeat sequence (ITR). When present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products, such an AAV vector can be replicated and packaged into an infectious viral particle. The terms "AAV virion," "AAV viral particle," or "AAV vector particle" refer to a viral particle composed of at least one AAV capsid protein and a capsidated polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply "AAV vector." Therefore, the production of an AAV vector particle necessarily includes the production of an AAV vector such that the vector is contained within the AAV vector particle.

[0133] The AAV "rep" and "cap" genes refer to the genes encoding the replication protein and capsid protein, respectively. The AAV rep and cap genes have been identified in all AAV serotypes studied to date, and these genes are described in this paper and the cited references. In wild-type AAV, the rep and cap genes are generally adjacent to each other in the viral genome (i.e., they are "coupled" together to form adjacent or overlapping transcription units), and they are generally conserved among AAV serotypes. The AAV rep and cap genes can also be referred to individually or collectively as "AAV packaging genes." The AAV cap gene encodes the Cap protein, which, in the presence of rep and adenovirus helper functions, packages the AAV vector and binds to target cell receptors.

[0134] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. Promoters can be recognized by RNA polymerase, which initiates transcription to synthesize RNA. During RNA synthesis, promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). A promoter comprises a core promoter region and a regulatory region, located in the regulatory sequence controlling gene expression, upstream of the gene transcription start site (at the 5' direction of the DNA antisense strand), and does not itself have a coding function. Based on their mode of action and function, promoters are classified into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or developmentally specific), and inducible promoters (regulated by various biological, external chemical, or physical signals inside and outside the cell).

[0135] The term "tissue-specific promoter" refers to promoters that regulate and guide gene expression in a tissue-specific manner, such as in brain tissue, muscle tissue, liver tissue, and kidney tissue.

[0136] In this application, the term "operably linked" generally refers to placing a regulatory sequence necessary for the expression of a coding sequence in an appropriate position relative to the coding sequence in order to achieve the expression of the coding sequence. The term "operably linked" can also refer to the arrangement of the coding sequence and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition is sometimes also applied to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules in which hybrid nucleic acid molecules are generated.

[0137] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0138] In this application, the terms “polypeptide,” “peptide,” “protein,” and “protein protein” are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers containing modified amino acids. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeled component). The term “amino acid” includes natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0139] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0140] In this application, the term "treatment" generally refers to a clinical intervention used to alter the natural processes of an individual or cell in a clinicopathological process. It may include improving the disease state, eliminating lesions, or improving prognosis. In this application, "treatment" includes any beneficial or desired effect associated with pain relief and may even include minimal pain relief. Treatment may optionally include reducing or alleviating pain, or delaying the progression of pain. "Treatment" does not necessarily mean the complete eradication or cure of a disease or condition or its associated symptoms.

[0141] In this application, the term "relief" refers to reducing, shortening, or delaying a symptom, disease, condition, or phenotype. The symptom, disease, condition, or phenotype may include subjective perceptions of the subject, such as pain, dizziness, or other physiological disturbances, or medically detectable indicators, such as lesions detected through medical testing.

[0142] In this application, the term "prevention" generally refers to the preventive application of a combination to a healthy subject to prevent the occurrence of a disease or condition. It may also include the preventive application of a combination to a patient in the pre-treatment stage of an allergic disease to be treated. "Prevention" does not require the complete elimination of the likelihood of the disease or condition occurring; in other words, "prevention" generally means a reduction in the likelihood of the disease or condition occurring in the presence of said application combination. In this application, "prevention" refers to a method for preventing, suppressing, or reducing the likelihood of pain occurring or recurring. It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of pain symptoms. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of pain before its onset or recurrence.

[0143] In this application, the term "application" can refer to a composition of oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intra-articular, extra-gastric, intra-arterial, intradermal, intravenous, intraosseous, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intra-articular, intracavitary, intrathecal, intracranial, intracavitary, intrathecal, intrasheath, intraviral, intracerebral, intraventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheter, stent, or by implanted reservoir or other application device, actively or passively (e.g., by diffusion) to the perivascular space and adventitia.

[0144] Invention Details

[0145] On the one hand, this application provides a method for treating obsessive-compulsive disorder, including...

[0146] a) Identify one or more specific brain regions that are abnormally activated in the subject;

[0147] b) To inhibit or disrupt neuronal activity in one or more specific brain regions, or to interrupt or block neuronal signals in the subject’s brain region.

[0148] In some implementations, the subject is a patient with obsessive-compulsive disorder.

[0149] In some implementations, identifying one or more specific brain regions that are abnormally activated in a subject further includes: identifying one or more specific brain regions that are abnormally activated in a subject by neuroimaging.

[0150] In some embodiments, the neuroimaging includes: measurement of neuronal firing signals (Spikes), measurement of cortical potentials (ECoG), PET scan imaging, electroencephalography (EEG) with electromagnetic signal detection, magnetoencephalography (MEG), functional magnetic resonance imaging (fMRI), and near-infrared spectroscopy (NIRS).

[0151] In some embodiments, the PET scanning imaging includes: Aβ-PET scanning imaging and / or tau-PET.

[0152] In some embodiments, the method further includes: obtaining brain region detection results of the subject through neuroimaging, and determining one or more specific brain regions of abnormal activation in the subject by comparing the brain region detection results of the subject with reference values.

[0153] In some implementations, the reference value is the brain region detection result of a healthy control group obtained through the neuroimaging.

[0154] In some implementations, the method further includes: comparing the brain region detection results of the subject and the brain region detection results of the control group in terms of activation in different brain regions, wherein a p-value of less than 0.05 is used as the statistical significance criterion for judging abnormal activation of brain regions.

[0155] In some implementations, the brain region detection results of the subjects and / or brain region detection results and / or reference values ​​of the control groups are generated based on the brain oxygen metabolism rate.

[0156] In some implementations, the one or more specific brain regions include neurons in the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), ventral striatum (VS), caudate nucleus (Cd), bed nucleus of the stria terminalis (BNST), subthalamus nucleus (STN), and globus pallidus internus (GPi), or interruption or blocking of neuronal signals in the subject's brain regions.

[0157] On the one hand, this application provides a method for treating obsessive-compulsive disorder, including inhibiting or disrupting neuronal activity in specific brain regions, or interrupting or blocking neuronal signals in specific brain regions of a subject.

[0158] In some implementations, the specific brain regions include mPFC, OFC, ACC, NAc, VS, Cd, BNST, STN, and GPi.

[0159] On one hand, this application provides a method for treating obsessive-compulsive disorder, including inhibiting, disrupting, interrupting, or blocking neurons in brain regions such as the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), ventral striatum (VS), caudate nucleus, bed nucleus of the stria terminalis (BNST), subthalamus nucleus (STN), and globus pallidus internus (GPi), as well as fiber tracts of the thalamic peduncles (ITP), medial forebrain bundle (MFB), and anterior limb of the internal capsule (ALIC).

[0160] In some embodiments, all or part (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi, and fiber bundles of ITP, MFB, and ALIC are destroyed. In some embodiments, all or part (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of neurons in dACC are destroyed.

[0161] In some implementations, all signals from neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi are blocked or interrupted.

[0162] In some implementations, all or a portion (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi are inhibited.

[0163] In some embodiments, methods for inhibiting, disrupting, interrupting, or blocking neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include biological, chemical, and / or physical methods. In some embodiments, methods for inhibiting, disrupting, interrupting, or blocking neurons in the above brain regions utilize chemogenetic techniques, optogenetic techniques, electroencephalography, pharmacological modulation, or combinations thereof.

[0164] In some implementations, methods for destroying neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include surgically removing neurons in these brain regions and / or severing fiber bundles of ITP, MFB, and ALIC.

[0165] In some implementations, methods for destroying neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include inducing cell death of neurons in these brain regions.

[0166] In some implementations, cell death may include apoptosis, ferroptosis, autophagy, necrosis, pyroptosis, etc.

[0167] In some embodiments, methods for disrupting neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include inducing apoptosis in these neurons. In some embodiments, methods for disrupting neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include introducing apoptosis-inducing proteins into these neurons. In some embodiments, methods for disrupting neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include introducing an apoptosis-inducing agent into these neurons.

[0168] In some implementations, the apoptosis protein is Caspases 3 / 7.

[0169] In some implementations, the apoptosis inducer is FASL, DCC, or UNC5B.

[0170] In some embodiments, methods for disrupting ACC neurons include inducing ferroptosis in neurons of brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi. In some embodiments, methods for disrupting neurons of brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include inhibiting the GPX4, SLC7A11, HSPB1, and NRF2 genes in neurons of these brain regions. In some embodiments, methods for disrupting neurons of brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include introducing a ferroptosis inducer into neurons of these brain regions.

[0171] In some implementations, the cell ferroptosis inducer is Erastin, DPI2, BSO, SAS, lanperisone, SRS, RSL3, DPI7, DPI10, FIN56, sorafenib, or artemisinin.

[0172] In some embodiments, methods for disrupting neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include introducing toxin receptor proteins and their ligands into the neurons of these brain regions.

[0173] In some implementations, the toxin receptor protein is DTR (diphtheria toxin receptor).

[0174] In some implementations, the toxin receptor protein is expressed in neurons of brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi in the subject, and the toxin is administered to the subject.

[0175] In some implementations, the toxin is diphtheria toxin.

[0176] In some implementations, methods for interrupting or blocking signals from neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include introducing inhibitors of neurotransmitters synthesized or secreted by the corresponding neurons into the neurons of the aforementioned brain regions.

[0177] In some implementations, methods for interrupting or blocking signals from neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include introducing antagonists of neurotransmitters synthesized or secreted by the corresponding neurons into the neurons of the aforementioned brain regions.

[0178] In some implementations, methods for interrupting or blocking signals from neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include introducing a neutralizer of neurotransmitters synthesized or secreted by the corresponding neurons into the neurons of the aforementioned brain regions.

[0179] In some embodiments, methods for interrupting or blocking signaling in neurons of brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi include introducing TeNT (tetanus toxin) into ACC neurons. In some embodiments, methods for interrupting or blocking signaling in ACC neurons include inhibiting the release of neurotransmitters. In some embodiments, methods for interrupting or blocking signaling in dACC neurons include inhibiting the release of neurotransmitters.

[0180] In some embodiments, methods for interrupting or blocking signaling from neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include inhibiting the release of neurotransmitters. In some embodiments, methods for interrupting or blocking signaling from neurons in the above brain regions include inhibiting the synthesis of neurotransmitters.

[0181] In some implementations, methods for inhibiting neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi include electroencephalography (EEG). In some embodiments, the EEG stimulation is high-frequency stimulation or deep brain stimulation. In some embodiments, the electrical frequency used in the EEG stimulation is greater than 50 Hz.

[0182] In some implementations, blocking neurons in brain regions such as mPFC, OFC, ACC, NAc, VS / VC, Cd, BNST, STN, and GPi in a subject includes expressing exogenous receptors to the subject.

[0183] In some embodiments, the exogenous receptor includes a G protein-coupled receptor (GPCR) or an ion channel protein.

[0184] In some embodiments, GPCRs may include one or more of the following: CHRM1, GNRHR, GPR73, GPR45, PTHR1, CHRM2, GNRHR2, GPR73, GPR63, PTHR2, CHRM3, HRH1, GPR10, GPR83, SCTR, CHRM4, HRH2, F2R, PGR15, ADCYAP1R1, CHRM5, HRH3, F2RL1, PGR15L, VIPR1, ADORA1, HRH4, F2RL2, GPR103, VIPR2, ADORA2A, FSHR 93. F2RL3, GPR103L, BAI1, ADORA2B, LHCGR, P2RY1, GRCA, BAI2, ADORA3, TSHR, P2RY2, PGR1, BAI3, P2RY12, GPR54, P2RY4, HGPCR11, CD97, GPR105, LTB4R, P2RY6, SALPR, EMR 1. GPR86, LTB4R2, P2RY11, MAS1, EMR2, GPR87, MRGX1, LGR7, GPR90, EMR3, ADRA1A, MRG X2, LGR8, P2Y5, PGR16, ADRA1B, MRGX3, RGR, GPR23, LEC1, ADRA1D, MRGX4, HTR1A, P2Y10 275. LEC2, ADRA2A, MRGD, HTR1B, FKSG79, LEC3, ADRA2B, MrgA1, HTR1D, PGR2, CELSR1, ADRA2C, MrgA2, HTR1E, PGR3, CELSR2, ADRB1, MrgA3, HTR1F, AGR9, CELSR3, ADRB2 21. MrgA4, HTR2A, CMKLR1, GPR64, ADRB3, MrgA5, HTR2B, EBI2, PGR17, ADMR, MrgA6, HTR2C, GPC R150, DJ287G14, C3AR1, MrgA7, HTR4, GPR1, KIAA0758, C5R1, MrgA8, HTR5A, GPR15, PGR18, GPR 77. MrgA9, HTR5B, GPR17, PGR19, AGTR1, MrgA10, HTR6, GPR18, PGR20, AGTR2, MrgA11, HTR7, GP R19, ​​TEM5, AGTRL1, MrgA12, SSTR1, GPR20, KIAA1828, BRS3, MrgA13, SSTR2, GPR22, PGR21, GRPR 31. MrgA14, SSTR3, GPR25, ETL, NMBR, MrgA15, SSTR4, GPR30,FLJ14454、BDKRB1、MrgA16、SSTR5、GPR31、GPR56、BDKRB2、MrgA19、G2A、GPR 32、OA1、CNR1、MrgB1、GPR4、GPR33、PGR22、CNR2、MrgB2、GPR65、GPR34、PGR23 、CCR1、MrgB3、GPR68、GPR35、PGR24、CCR2、MrgB4、EDG1、GPR39、PGR25、CCR3 、MrgB5、EDG2、GPR40、PGR26、CCR4、MrgB6、EDG3、GPR44、PGR27、CCR541、MrgB 8、EDG4、GPR55、VLGR1、CCR6、MrgB10、EDG5、GPR61、CCR743、MrgB11、EDG6、G PR62、CCR8、MrgB13、EDG7、GPR75、CCR9、GPR24、EDG8、GPR80、GPR2、SLT、TACR 1、GPR82、CASR、CCRL1、MC1R、TACR2、GPR84、GABBR1、CCRL2、MC2R、TACR3、GP R88、GPR51、CCBP2、MC3R、TRHR、GPR91、GPRC5B、CMKBR1L1、MC4R、TRHR2、GPR9 2, GPRC5C, CMKBR1L2, MC5R, GPR57, GPR101, GPRC5D, CCXCR1, MTNR1A, GPR58, H963, RAI3, CX3CR1, MTNR1B, PNR, HGPCR2, GRM1, IL8RA, GPR50, TAR1, HGPCR 19、GRM2、IL8RB、GPR66、TAR2、HUMNPIIY20、GRM3、GPR9、NMU2R、TAR3、MRG、G RM4、CXCR4、NPFF1R、TAR4、MRGE、GRM5、BLR1、GPR74、GPR102、MRGF、GRM6、CXC R6, GPR7, TA7, MRGG, GRM7, CCKAR, GPR8, TA8, OPN3, GRM8, CCKBR, NPY1R, TA10, OPN4, GPRC6A, CYSLT1, NPY2R, TA11, PGR4, PGR28, CYSLT2, PPYR1, TA12, PG R5、DRD1、NPY5R、TA14、PGR6、DRD2、NPY6R、TA15、PGR7、DRD3、NTSR1、GPR14、 PGR8、DRD4、NTSR2、AVPR1A、PGR10、FZD1、DRD5、OPRD1、AVPR1B、PGR11、FZD2、FY, OPRK1, AVPR2, PGR12, FZD3, TG1019, OPRM1, OXTR, PGR13, FZD4, HM74, OPRL1, GPR48, PGR14, FZD5, GPR81, OPN1LW, GPR49, RDC1, FZD6, EDNRA, OPN1MW, LG R6, RE2, FZD7, EDNRB, OPN1SW, GPR27, RRH, FZD8, FPR1, RHO, GPR85, FZD9, FPRL1, HCRTR1, SREB3, FZD10, FPRL2, HCRTR2, GPR3, SMOH, FPR-RS1, PTAFR, GPR6, CALCR, FPR-RS2, PTGDR, GPR12, CALCRL, FPR-RS3, PTGER1, GPR21, CRHR1, FPR-RS4, PTGER2, GPR52, CRHR2, GALR1, PTGER3, GPR26, GIPR, TM7SF1, GALR2, PTG ER4, GPR78, GCGR, TM7SF1L1, GALR3, PTGFR, GPR37, GLP1R, TM7SF1L2, GHSR, PTGIR, GPR37L1, GLP2R, TM7SF3, GPR38, TBXA2R, GPR41, GHRHR, TPRA40, and GPR43. ,

[0185] In some embodiments, the G protein-coupled receptor is a designer receptor DREADD or a light-sensitive protein that is specifically activated by the designer drug.

[0186] In some embodiments, the reactivity of DREADD to endogenous ligands may be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 86%, less than 87%, less than 88%, less than 89%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100%.

[0187] In some embodiments, the DREADD includes hM4Di, hM3Dq, AlstR, or KORD. When the switch receptor is hM4Di or hM3Dq, the exogenous ligand includes clozapine, olanzapine, desclozapine, perlapine, JHU 37152, and JHU37160. The photosensitive GPCR includes Lamplight (Lamprey Parapinopsin), rod opsin, cone opsin, Mu opioid receptor-rod opsin chimera, and Mu opioid receptor.

[0188] In some implementations, the anion channel protein is a ligand-gated ion channel (LGIC) protein or a light-gated anion channel protein.

[0189] Among them, the anion channel proteins LGIC ligand-gated ion channels include GlyR-M, GluCl, PSAM-5HT3HC, PSAM-GlyR, PSAM-nAChR, TRPV1, or GABAA.

[0190] When the switch acceptor is an LGIC ligand-gated ion channel, the exogenous ligand includes ivermectin, selamectin, doramectin, emamectin, epramectin, abamedin, moxicillin, PSEM22S, PSEM89S, PSEM9S, capsaicin, or zolpidem.

[0191] Light-gated anion channel proteins include NpHR, eNpHR3.0, Arch, eArch 3.0, ArchT, eArchT 3.0, eBR, iC1C2, iChloC, GtACR1, GtACR2, SwiChRca, PsChR1, Phobos, Aurora, Jaws, Mac, and eMac 3.0.

[0192] In some implementations, the cation channel protein is the LGIC protein.

[0193] The LGIC cation channel protein is selected from the group consisting of: 5-HT3 receptor, acid-sensitive ion channel (ASIC) protein, epithelial sodium channel (ENaC), ionotropic glutamate receptor, IP3 receptor, nicotinic acetylcholine receptor, P2X receptor, reniform base receptor, and zinc activated channel protein (ZAC). In some embodiments, the LGIC cation channel protein is expressed in ACC neurons, and a ligand or agonist of the LGIC cation channel protein is administered to the subject.

[0194] In some embodiments, the cation channel protein is a light-gated cation channel protein. In some embodiments, the light-gated cation channel protein is selected from the group consisting of ChR2, CheRiff, Chronos, and variants thereof. Examples of ChR2 variants include, but are not limited to, ChR2(H134R), ChETA, ReaChR, bReaChES, Chrimson, ChrimsonR, C1C2, C1V1, C1V1(t), C1V1(t / t), oChIEF, ChRmine, ChRmine2.0, ChRger1, ChRger2, and ChRger3. Examples of Chronos include, but are not limited to, CsChR, CoChR, VChR1, and CheRiff.

[0195] In some embodiments, the ion channel protein is a potassium ion channel. In some embodiments, in the viral vector, the potassium ion channel is selected from one or more of the following: voltage-gated potassium ion channels (Kv), inwardly rectified potassium ion channels (Kir), calcium-activated potassium ion channels (KCa), and two-pore potassium ion channels (K2P). Preferably, the potassium ion channel can be Kv and / or Kir.

[0196] In a preferred embodiment, the potassium ion channel is selected from one or more of the following: Kir1.x, Kir2.x, Kir3.x, Kir4.x, Kir5.x, Kir6.x, and Kir7.x. Preferably, the potassium ion channel can be Kir2.x and / or Kir3.x.

[0197] In a more preferred embodiment, the potassium ion channel is selected from one or more of the following: Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, Kir3.3, Kir3.4, Kir4.1, Kir6.1 and Kir6.2.

[0198] In a specific implementation, Kir1.1 is encoded by the KCNJ1 gene, Kir2.1 is encoded by the KCNJ2 gene, Kir2.3 is encoded by the KCNJ4 gene, Kir3.1 is encoded by the KCNJ3 gene, Kir3.2 is encoded by the KCNJ6 gene, Kir3.3 is encoded by the KCNJ9 gene, Kir3.4 is encoded by the KCNJ5 gene, Kir4.1 is encoded by the KCNJ10 gene, Kir6.1 is encoded by the KCNJ8 gene, and Kir6.2 is encoded by the KCNJ11 gene.

[0199] In a more preferred embodiment, the potassium ion channel is selected from one or more of the following: Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir2.6, Kir2.7, Kir3.2 and Kir4.1.

[0200] In a more preferred embodiment, the potassium ion channel is Kir2.1, Kir2.2, Kir2.3, Kir2.4, and / or Kir3.2. For example, the potassium ion channel is Kir2.1. Another example, the potassium ion channel is Kir2.2. Another example, the potassium ion channel is Kir2.3. Another example, the potassium ion channel is Kir2.4. Another example, the potassium ion channel is Kir3.2. In some embodiments, any vector that introduces a receptor or protein (e.g., a switch receptor) into neuronal cells can be used to introduce the receptor or protein (e.g., a switch receptor). Illustrative examples of suitable vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, viscera, bacterial artificial chromosomes, and viral vectors. In some cases, the vector is a circular nucleic acid, such as a plasmid, BAC, PAC, YAC, viscera, fosmid, etc. In some cases, circular nucleic acid molecules can be used to deliver switch receptor nucleic acid molecules to a subject. For example, a plasmid DNA molecule encoding a switch receptor can be introduced into a subject's cells, thereby transcribing the DNA sequence encoding the switch receptor into mRNA and translating the mRNA "information" into a protein product. Circular nucleic acid vectors typically include regulatory elements that control the expression of target proteins. For example, circular nucleic acid vectors can include any number of promoters, enhancers, terminators, splicing signals, origins of replication, initiation signals, etc.

[0201] In a specific embodiment, in the viral vector, the nucleotide sequence encoding the potassium ion channel is as shown in SEQ ID NOs:27-42 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs:27-42.

[0202] In some embodiments, the vector is a viral vector. For example, the viral vector may include a replication-defective virus. Non-limiting examples of viral vectors suitable for delivering the nucleic acid molecules of the present invention to a subject include vectors derived from adenoviruses, retroviruses (e.g., lentiviruses), adeno-associated viruses (AAVs), and herpes simplex virus-1 (HSV-1). For example, viral vectors include, but are not limited to, retroviral vectors (e.g., lentiviral vectors), herpesvirus-based vectors, and parvovirus-based vectors (e.g., adenovirus-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors).

[0203] As used herein, the term "parvovirus" encompasses all parvoviruses, including autonomously replicating parvoviruses and virus-dependent viruses. Autonomous parvoviruses include members of the genera parvovirus, erythrovirus, lysinic virus, iteravirus, and contravirus. Exemplary autonomous parvoviruses include, but are not limited to, mouse parvovirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline leukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, for example, Fields et al., Virology, 1996, Vol. 2, Chapter 69 (3rd edition, Lippincott-Raven Publishers).

[0204] In some embodiments, the dependent virus includes adeno-associated viruses, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV Retro, AAV DJ, AAVrh10, and Anc80.

[0205] In some embodiments, AAV includes poultry AAV, cattle AAV, dog AAV, horse AAV, and sheep AAV.

[0206] The adeno-associated virus vector (AAV vector) may also include a recombinant adeno-associated virus vector (rAAV vector).

[0207] In some cases, the vector may further include a restriction enzyme site downstream of the promoter to allow insertion of the polynucleotide encoding the switch receptor, wherein the promoter and restriction enzyme site may be located downstream of the 5'AAV ITR and upstream of the 3'AAV ITR. In some cases, the vector may further include a post-transcriptional regulatory element located downstream of the restriction enzyme site and upstream of the 3'AAV ITR. In some cases, the vector may further include a polynucleotide inserted at the restriction enzyme site and operatively linked to the promoter, wherein the polynucleotide may contain the coding region of the switch receptor. As those skilled in the art will appreciate, any of the AAV vectors disclosed in this application can be used as viral constructs in the methods to generate recombinant AAV.

[0208] In some embodiments, the vector includes a promoter, and a switch receptor nucleic acid is operatively linked to the promoter.

[0209] The promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0210] The constitutive promoters mentioned include the immediate early promoter of cytomegalovirus (CMV), viral simian virus 40 (SV40), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus thymidine kinase (HSV-tk) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and eukaryotic translation initiation factor 4A1. EIF4A1, heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa β member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), human ROSA26 promoter, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter or β-actin promoter.

[0211] The inducible promoters mentioned therein include, but are not limited to, antibiotic response promoters, ultraviolet-induced promoters, metallothionein promoters, tetracycline response promoters, ecdysone response promoters, cumate response promoters, glucocorticoid response promoters and estrogen response promoters, PPAR-γ promoters or RU-486 response promoters.

[0212] In some embodiments, tissue specificity may include, but is not limited to: neuron-specific promoters, glial cell-specific promoters, heart-specific promoters (e.g., cTNT, POSTN), muscle-specific promoters (e.g., MHCK7, SM22a, ACTA1), lung-specific promoters (e.g., SP-C), liver-specific promoters (e.g., TBG), kidney-specific promoters (e.g., nphs1, nphs2), pancreas-specific promoters (e.g., padx1, insulin2), adipose-specific promoters (e.g., FABP4), endothelial-specific promoters (e.g., TIE), retinal-specific promoters (e.g., rpe65), prostate-specific promoters (e.g., DD3), skin-specific promoters (e.g., keratin14), and macrophage-specific promoters (e.g., F4 / 80). In some embodiments, the tissue-specific promoter is a neuron-specific promoter or a glial cell-specific promoter. Neuron-specific promoters include human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, Advillin promoter, Drosophila single homologue 1 (SIM1) promoter, oxytocin (OXT) promoter, spiky mouse-associated protein (AgRP) promoter, and protein kinase C-δ (PKC). -δ) promoters, auxin-releasing peptide promoters, glutamate decarboxylase (GAD1 / 2) promoters, choline acetyltransferase (ChAT) promoters, vesicle GABA transporter (VGAT) promoters, glutamate decarboxylase (GAD65) promoters, tyrosine hydroxylase (TH) promoters and promoters without a distal homeobox (Dlx), cell activity-dependent promoters (c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter or synthetic activity-dependent promoters, such as PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), FRAM (Fos Robust Activity Marker)).Glial cell-specific promoters include, but are not limited to, the astrocyte fibrillary acidic protein (GFAP) promoter, the Gfabc1D promoter, the ALDH1L1 promoter, the Pirt promoter, the Cst3 promoter, the Cx30 promoter, the myelin basal protein (MBP) promoter, the oligodendrocyte myelin glycoprotein (MOG) promoter, the CNP (NPPC) promoter, the PLP promoter, the Pdgfra promoter, the olig2 promoter, the NG2 promoter, the CD11b promoter, the Iba1 promoter, the CD68 promoter, the TMEM119 promoter, the CX3CR1 promoter, and the Foxj1 promoter.

[0213] In some embodiments, the promoter is CaMKIIα.

[0214] In some implementations, the subject is a human. In other implementations, the subject is a non-human mammal, bird, fish, reptile, or amphibian.

[0215] The methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cerebellomedullary cistern administration, and intraneural administration.

[0216] In some embodiments, application may involve a liquid formulation that can be injected into a carrier.

[0217] In some embodiments, administration may involve oral delivery of a solid formulation of an exogenous ligand. In some cases, the oral formulation may be administered with food. In some embodiments, the carrier is administered to a subject via parenteral, intravenous, intramuscular, intraperitoneal, intrathecal, intraneural, intraganglionic, intraspinal, or intracardiac administration to introduce the carrier into one or more neuronal cells (e.g., target cells). In various embodiments, the carrier is an AAV.

[0218] In some embodiments, the carrier can be administered to the subject via intracranial delivery (i.e., direct entry into the brain). In non-limiting examples of intracranial delivery, the carrier of the present invention can be delivered to brain regions such as the mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi for treatment. In another specific case, the carrier can be administered to the subject via intraneural injection (i.e., direct injection into a nerve). The nerve can be selected based on the indications for treatment, for example, injected into brain regions such as the mPFC, OFC, ACC, NAc, VS / VC, Caudate nucleus, BNST, STN, and GPi to treat OCD.

[0219] Vector dosage can be expressed as the number of vector genome units delivered to a subject. As used herein, "vector genome unit" refers to the number of individual vector genomes administered in a dose. The size of a single vector genome typically depends on the type of viral vector used. The vector genomes of this invention can be approximately 1.0 kbps, 1.5 kbps, 2.0 kbps, 2.5 kbps, 3.0 kbps, 3.5 kbps, 4.0 kbps, 4.5 kbps, 5.0 kbps, 5.5 kbps, 6.0 kbps, 6.5 kbps, 7.0 kbps, 7.5 kbps, 8.0 kbps, 8.5 kbps, 9.0 kbps, 9.5 kbps, 10.0 kbps, or more than 10.0 kbps. Therefore, a single vector genome can contain up to or more than 10,000 base pairs of nucleotides. In some cases, the carrier dose can be approximately 1×10^6, 2×10^6, 3×10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^7, 3×10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^8, 3×10^8, 4×10^8, 5×10^8, 6× 10^8, 7×10^8, 8×10^8, 9×10^8, 1×10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^9, 1×10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^10, 1×10^11, 2×10^11, 3× 10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8×10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^12, 8×10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×10^ 13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6×10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^15, 6×10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^163×10^16, 4×10^16, 5×10^16, 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10^17, 5×10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18, 4×10^18, 5×10^18, 6×10^18, 7 Vector genome units of ×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10^20, 3×10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20, 9×10^20, or higher.

[0220] In specific embodiments, the vectors considered herein have a density of at least approximately 1×10^9 genome particles / mL, at least approximately 1×10^10 genome particles / mL, at least approximately 5×10^10 genome particles / mL, at least approximately 1×10^11 genome particles / mL, at least approximately 5×10^11 genome particles / mL, at least approximately 1×10^12 genome particles / mL, at least approximately 5×10^12 genome particles / mL, at least approximately 6×10^12 genome particles / mL, and at least approximately 7×10^12 genome particles / mL. A titer of at least approximately 8 × 10^12 genome particles / mL, at least approximately 9 × 10^12 genome particles / mL, at least approximately 10 × 10^12 genome particles / mL, at least approximately 15 × 10^12 genome particles / mL, at least approximately 20 × 10^12 genome particles / mL, at least approximately 25 × 10^12 genome particles / mL, at least approximately 50 × 10^12 genome particles / mL, or at least approximately 100 × 10^12 genome particles / mL is administered to the subject. When referring to viral titers, the terms “genome particles (gp),” “genome equivalent,” or “genome copy (gc)” refer to the number of viral particles containing the recombinant AAV DNA genome, regardless of infectivity or function. The number of genomic particles in a particular vector preparation can be measured, for example, in the embodiments described herein, or, for example, in Clark et al. (1999) Human Gene Therapy, 10:1031-1039; and Veldwijk et al. (2002) Molecular Therapy, 6:272-278.

[0221] The carrier of the present invention can be administered in a volume of fluid. In some cases, the carrier can be administered in volumes of about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, 10.0 mL, 11.0 mL, 12.0 mL, 13.0 mL, 14.0 mL, 15.0 mL, 16.0 mL, 17.0 mL, 18.0 mL, 19.0 mL, 20.0 mL, or greater than 20.0 mL. In some cases, the carrier dose can be expressed as the concentration or titer of the carrier administered to the subject. In this case, the carrier dose can be expressed as the number of carrier genomic units per volume (i.e., genomic units / volume).

[0222] In specific embodiments, the carriers considered herein are administered to subjects at titers of at least about 5 × 10^9 infection units / mL, at least about 6 × 10^9 infection units / mL, at least about 7 × 10^9 infection units / mL, at least about 8 × 10^9 infection units / mL, at least about 9 × 10^9 infection units / mL, at least about 10 × 10^9 infection units / mL, at least about 15 × 10^9 infection units / mL, at least about 20 × 10^9 infection units / mL, at least about 25 × 10^9 infection units / mL, at least about 50 × 10^9 infection units / mL, or at least about 100 × 10^9 infection units / mL. The terms “infectious unit (iU),” “infectious particle” or “replication unit” used in relation to viral titers refer to the number of infectious and reproducible recombinant AAV vector particles measured by the center of infection, also known as the center of replication assay, as described, for example, in the Journal of Virology (J. Virol.), 62:1963-1973, by McLaughlin et al. (1988).

[0223] In specific embodiments, the carriers considered herein are administered to subjects at titers of at least about 5 × 10^10 transduction units / mL, at least about 6 × 10^10 transduction units / mL, at least about 7 × 10^10 transduction units / mL, at least about 8 × 10^10 transduction units / mL, at least about 9 × 10^10 transduction units / mL, at least about 10 × 10^10 transduction units / mL, at least about 15 × 10^10 transduction units / mL, at least about 20 × 10^10 transduction units / mL, at least about 25 × 10^10 transduction units / mL, at least about 50 × 10^10 transduction units / mL, or at least about 100 × 10^10 transduction units / mL. The term “transduction unit (tu)” as used in relation to viral titers refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgenic product, as measured in the examples herein or, for example, in the functional assays described in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) Journal of Virology, 70:520-532 (LFU analysis).

[0224] The carrier dosage is generally determined by the route of administration. In one specific example, intraganglionic injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intrathecal injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intracranial injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intraneural injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In yet another example, intraspinal injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.1 mL to approximately 0.01 mL to approximately 10.0 mL. In yet another specific case, cerebellomedullary cistern infusion may include approximately 0.01 mL to approximately 10.0 mL of vector genomes of approximately 1 × 10^9 to approximately 1 × 10^13. In yet another specific case, subcutaneous injection may include approximately 0.01 mL to approximately 10.0 mL of vector genomes of approximately 1 × 10^9 to approximately 1 × 10^13.

[0225] In one embodiment, the ligand is administered to the subject at the same time as the vector is administered to the subject.

[0226] In one embodiment, the ligand is administered to the subject after the vector is administered to the subject.

[0227] In one embodiment, the ligand is administered to the subject before the vector is administered to the subject.

[0228] In one embodiment, the ligand is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 hours, days, weeks, months, or years after administration of the carrier. In some cases, a therapeutically effective amount of the ligand may be administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, or more than 30 days after carrier delivery. In one specific instance, a therapeutically effective amount of the ligand is administered to the subject at least one week after carrier delivery.

[0229] The therapeutically effective amount or dose of the ligand of the present invention can be expressed as mg or μg of the ligand per kilogram of subject body weight. In some cases, the therapeutically effective amount of the ligand may be about 0.001 μg / kg, about 0.005 μg / kg, about 0.01 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 5 μg / kg, or about 5 μg / kg. 0 μg / kg, approximately 60 μg / kg, approximately 70 μg / kg, approximately 80 μg / kg, approximately 90 μg / kg, approximately 100 μg / kg, approximately 120 μg / kg, approximately 140 μg / kg, approximately 160 μg / kg, approximately 180 μg / kg, approximately 200 μg / kg, approximately 220 μg / kg, approximately 240 μg / kg, approximately 260 μg / kg, approximately 280 μg / kg, approximately 300 μg / kg, approximately 320 μg / kg, approximately 340 μg / kg, approximately 360 μg / kg, approximately 380 μg / kg, approximately 4 00μg / kg, approximately 420μg / kg, approximately 440μg / kg, approximately 460μg / kg, approximately 480μg / kg, approximately 500μg / kg, approximately 520μg / kg, approximately 540μg / kg, approximately 560μg / kg, approximately 580μg / kg, approximately 600μg / kg, approximately 620μg / kg, approximately 640μg / kg, approximately 660μg / kg, approximately 680μg / kg, approximately 700μg / kg, approximately 720μg / kg, approximately 740μg / kg, approximately 760μg / kg, approximately 780μg / kg, approximately 800 μg / kg, approximately 820 μg / kg, approximately 840 μg / kg, approximately 860 μg / kg, approximately 880 μg / kg, approximately 900 μg / kg, approximately 920 μg / kg, approximately 940 μg / kg, approximately 960 μg / kg, approximately 980 μg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, or greater than 10 mg / kg.

[0230] In some embodiments, the dose of the ligand administered to the subject is at least about 0.001 μg / kg, at least about 0.005 μg / kg, at least about 0.01 μg / kg, at least about 0.05 μg / kg, at least about 0.1 μg / kg, at least about 0.5 μg / kg, 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, or at least about 10 or higher mg / kg.

[0231] In some embodiments, the dose of the ligand administered to the subject is at least about 0.001 μg / kg to at least about 20 mg / kg, at least about 0.01 μg / kg to at least about 20 mg / kg, at least about 0.1 μg / kg to at least about 20 mg / kg, at least about 1 μg / kg to at least about 20 mg / kg, at least about 0.01 mg / kg to at least about 20 mg / kg, at least about 0.1 mg / kg to at least about 20 mg / kg, or at least about 1 mg / kg to at least about 20 mg / kg, or any range thereof.

[0232] In some respects, the therapeutically effective amount of ligand can be expressed as a molar concentration (i.e., M or mol / L). In some cases, the therapeutically effective amount of ligand can be approximately 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM. nM, 800nM, 900nM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM , 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or greater.

[0233] A therapeutically effective dose of the ligand may be administered once or more daily. In some cases, a therapeutically effective dose of the ligand is administered as needed (e.g., when relief of obsessive-compulsive disorder is required). The ligand may be administered continuously (e.g., daily without interruption for the duration of the treatment regimen). In some cases, the treatment regimen may be less than one week, one week, two weeks, three weeks, one month, or more than one month. In some cases, a therapeutically effective dose of the ligand may be administered for one day, for at least two consecutive days, for at least three consecutive days, for at least four consecutive days, for at least five consecutive days, for at least six consecutive days, for at least seven consecutive days, for at least eight consecutive days, for at least nine consecutive days, for at least ten consecutive days, or for more than ten consecutive days. In certain cases, a therapeutically effective dose of the ligand may be administered for three consecutive days. In some cases, therapeutically effective doses of ligand may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, thirty times, thirty times, forty times, or more than forty times per week. In other cases, therapeutically effective doses of ligand may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times per day. In some cases, the therapeutically effective dose of ligand is administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine hours, at least every ten hours, at least every eleven hours, at least every twelve hours, at least every thirteen hours, at least every fourteen hours, at least every fifteen hours, at least every sixteen hours, at least every seventeen hours, at least every eighteen hours, at least every nineteen hours, at least every twenty hours, at least every twenty-one hours, at least every twenty-two hours, at least every twenty-three hours, or at least daily. The dose of ligand may be administered to the subject continuously, or once, twice, three times, four times, or five times daily; once, twice, three times, four times, five times, or six times weekly; once, twice, three times, four times, five times, or six times monthly; once, twice, three times, four times, five times, or six times monthly; or at intervals of even longer. Treatment duration can last from one 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.

[0234] This application further provides the use of M4Di (hM4Di, chicken M4Di, macaque M4Di, guinea pig M4Di, or rabbit M4Di) and its encoding nucleic acid, as well as its mutants (M4Di-399, M4Di-PD, M4Di-ENDO, M4Di-PD-ENDO, hM4Dnrxn) and their encoding nucleic acids for the treatment of obsessive-compulsive disorder and / or substance abuse. This application further provides the use of M4Di (hM4Di, chicken M4Di, macaque M4Di, guinea pig M4Di, rabbit M4Di) and its encoding nucleic acid, as well as its mutants (M4Di-399, M4Di-PD, M4Di-ENDO, M4Di-PD-ENDO, hM4Dnrxn) and their encoding nucleic acids for the preparation of a treatment for obsessive-compulsive disorder and / or substance abuse. This application further provides a method for preparing a treatment for obsessive-compulsive disorder and / or substance abuse, comprising administering to a subject in need an effective dose of an M4Di (hM4Di, chicken M4Di, macaque M4Di, guinea pig M4Di, rabbit) encoded nucleic acid or a mutant thereof (M4Di-399, M4Di-PD, M4Di-ENDO, M4Di-PD-ENDO, hM4Dnrxn) encoded nucleic acid.

[0235] This application further provides the use of potassium ion channels and their encoded nucleic acids for the treatment of obsessive-compulsive disorder and / or substance abuse.

[0236] This application further provides the use of potassium ion channels and their encoding nucleic acids in the preparation of drugs for treating obsessive-compulsive disorder and / or substance abuse. In some embodiments, the potassium ion channel is Kv and / or Kir. In some embodiments, the potassium ion channel is human Kv and / or human Kir. In some embodiments, the potassium ion channel is selected from one or more of the following: Kir1.x, Kir2.x, Kir3.x, Kir4.x, Kir5.x, Kir6.x, and Kir7.x; preferably, the potassium ion channel is Kir2.x and / or Kir3.x. In some embodiments, the potassium ion channel is selected from one or more of the following: human Kir1.x, human Kir2.x, human Kir3.x, human Kir4.x, human Kir5.x, human Kir6.x, and human Kir7.x; preferably, the potassium ion channel is human Kir2.x and / or human Kir3.x. In some embodiments, the potassium ion channel is selected from one or more of the following: Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, Kir3.3, Kir3.4, Kir4.1, Kir6.1, and Kir6.2. In some embodiments, the potassium ion channel is selected from one or more of the following: human Kir1.1, human Kir2.1, human Kir2.3, human Kir3.1, human Kir3.2, human Kir3.3, human Kir3.4, human Kir4.1, human Kir6.1, and human Kir6.2. In some embodiments, Kir1.1 is encoded by the KCNJ1 gene, Kir2.1 by the KCNJ2 gene, Kir2.3 by the KCNJ4 gene, Kir3.1 by the KCNJ3 gene, Kir3.2 by the KCNJ6 gene, Kir3.3 by the KCNJ9 gene, Kir3.4 by the KCNJ5 gene, Kir4.1 by the KCNJ10 gene, Kir6.1 by the KCNJ8 gene, and Kir6.2 by the KCNJ11 gene. In some embodiments, the human Kir1.1 is encoded by the human KCNJ1 gene, the human Kir2.1 is encoded by the human KCNJ2 gene, the human Kir2.3 is encoded by the human KCNJ4 gene, the human Kir3.1 is encoded by the human KCNJ3 gene, the human Kir3.2 is encoded by the human KCNJ6 gene, the human Kir3.3 is encoded by the human KCNJ9 gene, the human Kir3.4 is encoded by the human KCNJ5 gene, the human Kir4.1 is encoded by the human KCNJ10 gene, the human Kir6.1 is encoded by the human KCNJ8 gene, and the human Kir6.2 is encoded by the human KCNJ11 gene.

[0237] Example

[0238] Example 1 Sapap3 - / - Treatment of OCD through chemogenetic inhibition of ACC neurons in transgenic mouse models

[0239] 1. Administration:

[0240] (1) OCD model animal: Sapap3 - / - Mice;

[0241] (2) Virus: AAV-CaMKIIα-hM4Di-T2A-mCherry;

[0242] (3) Injection brain regions: bilateral anterior cingulate cortex (ACC);

[0243] (4) Dosage: 9 × 10⁹ injections per unilateral ACC 8 vg.

[0244] (6) Operation procedure:

[0245] AAV preparation: pAAV-CaMKIIα-hM4Di-T2A-mCherry plasmid (encoding hM4Di, SEQ ID NO 43) or pAAV-CaMKIIα-mcherry (SEQ ID NO 44) plasmid, along with pHelper plasmid and AAV9 pRC plasmid at a molar ratio of 1:1:1, were co-transfected into HEK-293F cells using PEI transfection reagent (approximately 1 μg plasmid per million cells). After culturing at 37°C in an incubator containing 5% CO2 for 3 days, the cells were washed once with PBS buffer. After collecting the cells, the cells were subjected to five freeze-thaw cycles. Solid NaCl was added to bring the final concentration to 500 mM. The cells were centrifuged at 10000g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane. A gradient of iodixanol solutions was then added to centrifuge tubes (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 mL 25% iodixanol, 8 mL iodixanol). 15% iodixanol was used to add the sample to the top layer, and the mixture was centrifuged at 350,000g for 1 hour. The virus layer at the 40% and 60% interface was then aspirated. The mixture was centrifuged at 4000g using a 50 kDa ultrafiltration tube, and the medium was changed 5 times with 0.01% poloxamer PBS buffer. The viral titer was determined by qPCR and adjusted accordingly. Finally, a viral titer of 1 × 10^13 vg / ml was obtained.

[0246] Virus injection: Mice were anesthetized with tribromoethanol, their scalp hair was shaved, and they were fixed in a stereotaxic apparatus. The scalp was cut open to expose the skull. The ACC coordinates were: AP: +0.38mm; ML: ±0.25mm; DV: -1.12mm. After locating the ACC, the skull was shaved to create an injection site. An appropriate amount of virus was drawn up and injected into the ACC at the predetermined location and depth, yielding 400 nL of virus. 400 nL of virus was injected into the ACC of the contralateral brain using the same method. The wound was cleaned, the scalp was sutured, and the mice were returned to their cages.

[0247] CNO injection: One week after viral injection, CNO was injected intraperitoneally to verify the efficacy of chemogenetic treatment for OCD. CNO dosage: 2 mg / kg.

[0248] 2. Efficacy evaluation:

[0249] (1) Before administration, record the free movement of OCD model mice in a quiet environment for 1 hour;

[0250] (2) Intraperitoneal injection of CNO, 2 mg / kg;

[0251] (3) After CNO injection, the free movement of OCD mice was continuously recorded on video for 4 hours;

[0252] (4) Observation indicators: Compare the changes in the grooming phenotype of mice in the same duration while they are fully awake before and after drug administration, including the number of attacks and the total duration. Phenotypic recording criteria: The grooming phenotype includes mice grooming their fur with their mouths and scratching their face and behind their ears with their forepaws; the interval between different attacks is more than 3 seconds.

[0253] II. Experimental Results

[0254] As shown in Figure 1A, two Sapap3 cells expressing hM4Di in the ACC brain region - / - In mice, intraperitoneal injection of CNO reduced the frequency of grooming attacks. The phenotype was more pronounced in mouse #10, with a more significant reduction in the number of grooming attacks and a significantly shorter duration of attacks (Figure 1B).

[0255] Example 2: Treatment of OCD by chemogenetic inhibition of ACC neurons in 8-OH-DPAT model mice

[0256] Yadin et al. first reported an animal model of obsessive-compulsive disorder (OCD) established by inducing a reduction in alternating back-and-forth activity using drugs. The experimental method involved placing starved rats in a T-maze. The T-arms were divided into black and white sections, approximately 50cm × 10cm in size, with a small amount of food placed inside each arm as bait. The rats would then move back and forth between the black and white sections in the T-maze to obtain the small amount of food. This process was repeated every other day until the rats exhibited alternating back-and-forth behavior. Next, the rats were subcutaneously injected with the 5-HT receptor agonist 8-OH-DPAT to deplete 5-HT in the tissues, resulting in a reduction in alternating back-and-forth behavior, thus establishing a serotonin-induced OCD animal model. This reduction in alternating back-and-forth behavior could be suppressed by repeated injections (for 3 weeks) of the serotonin reuptake inhibitor fluoxetine.

[0257] Experimental steps:

[0258] (1) Experimental Groups:

[0259] (2) Laboratory animals:

[0260] ICR mice, 8 weeks old, male

[0261] (3) AAV injection:

[0262] AAV was prepared as in Example 1. After successful preparation, stereotactic injection was used to inject 400 nL of AAV9::CaMKIIα-hM4Di-T2A-mCherry or control virus AAV9::CaMKIIα-mCherry into both ACC of mice.

[0263] (4) T-maze experiment

[0264] After the virus injection, T-maze training began. The experimental procedure for the T-maze Spontaneous Alternation Behavior (SAB) was as follows:

[0265] Preparation of the T-maze: Use a tabletop T-maze, with all arms measuring 20cm x 10cm x 7cm, including a starting box and two target boxes. The starting box and target boxes are separated from the main body of the maze by manually operated wooden doors.

[0266] Mouse adaptation: Mice were placed in a maze to explore freely for 20 minutes, and then each mouse was confined to a target box for 5 minutes to allow them to adapt to the environment. Chocolate cake was used as a reward.

[0267] Behavioral training: On the second day after acclimatization training, mice that had been deprived of food for 24 hours were placed in a starting box. The wooden door was opened, allowing the mice to choose one of two target arms, each containing a chocolate cake as bait. After the mice ate the cake, they were removed from the maze and placed in a holding cage for 15 seconds. Food was then replenished, and this process was repeated 7 times per mouse.

[0268] Behavioral recording: Record the mice's behavior in the maze, including their choice of the target arm (left or right) and the latency to reach the target box (in seconds, up to 90 seconds). Record the spontaneous alternation behavior score, which is the number of repeated choices before spontaneous alternation occurs. A score of 1 is given for spontaneous alternation on the first choice, and a score of 7 is given for no spontaneous alternation after 7 tests.

[0269] 8-OH-DPAT Modeling and Efficacy Evaluation: After adaptation and training (more than 14 days after AAV injection, with stable viral expression), baseline levels of spontaneous alternation behavior in mice within the T-maze were tested. Once stable baseline scores were obtained, efficacy testing began. We tested the efficacy of three small-molecule ligands activating artificially modified chemogenetic receptors: DCZ (0.3 mg / kg, intraperitoneal injection), CNO (3 mg / kg, intraperitoneal injection), and clozapine (2 mg / kg, gavage). Based on the pharmacokinetic characteristics of DCZ, CNO, and clozapine, we induced fixation behavior by intraperitoneal injection of 8-OH-DPAT (1.4 mg / kg) 45 minutes after CNO administration, 3 hours and 45 minutes after clozapine administration, and immediately after DCZ administration. The normal control group received an equal volume of saline intraperitoneally. Fifteen minutes after injection of 8-OH-DPAT or saline, the mice's behavior in the T-maze was tested and scored.

[0270] (2) Experimental Results

[0271] As shown in Figure 2, acute administration of 8-OH-DPAT significantly increased the spontaneous alternation behavior score in mice, i.e., induced fixation behavior. All three small molecule ligands, upon activation of the chemogenetic receptor, significantly inhibited 8-OH-DPAT-induced fixation behavior in OCD model mice.

[0272] Example 3 Sapap3 - / - In transgenic mouse models, ion channel expression driven by constitutive promoters was inhibited, and ACC neurons were used to treat OCD.

[0273] I. Experimental Procedure:

[0274] The main experimental steps are the same as in Example 1, using Sapap3. - / -Transgenic mouse models were bilaterally injected with the ACC of AAV9::CaMKIIα--kir2.3 (SEQ ID NO 45) virus. In this embodiment, CNO administration to the mice was not required, allowing direct measurement of drug efficacy. Facial lesions in the model mice were photographed before virus injection. Facial lesions were photographed weekly after virus injection. The effects of Sapap3 after virus injection were compared. - / - Changes in facial damage caused by excessive grooming in transgenic mouse models.

[0275] II. Experimental Results:

[0276] As shown in Figure 3, Sapap3 - / - Excessive grooming in transgenic model mice resulted in significant facial lesions, with bilateral ACC expression of kir2.3 and Sapap3. - / - The facial lesions in the transgenic model mice were significantly improved.

[0277] Example 4: Treatment of OCD by inhibiting ACC neurons through constitutive promoter-driven ion channel expression in 8-OH-DPAT model mice.

[0278] I. Experimental Procedure:

[0279] The main experimental steps were the same as in Example 2. The experimental group was injected with four different batches of AAV expressing Kir2.3, while the model control group was injected with the solvent. After T-maze adaptation, training, and baseline testing, the virus had been expressed for four weeks. Fifteen minutes after injection of 8-OH-DPAT or saline, the mice's behavior in the T-maze was tested and scored.

[0280] II. Experimental Results:

[0281] As shown in Figure 4, acute administration of 8-OH-DPAT significantly increased the spontaneous alternation behavior score in mice, i.e., induced fixed behavior. Meanwhile, four different batches of AAV expressing Kir2.3 significantly inhibited 8-OH-DPAT-induced fixed behavior.

[0282] Example 5 Sapap3 - / - In transgenic mouse models, ion channel expression driven by an inducible promoter was used to inhibit ACC neurons in the treatment of OCD.

[0283] I. Experimental Procedure:

[0284] The main experimental steps are the same as in Example 1, using Sapap3. - / -Transgenic mouse models were bilaterally injected with AAV9::cfos-kir2.3-T2A-mcherry (SEQ ID NO 46) virus via ACC. Two weeks after injection, tissue samples were collected to verify activity-dependent expression. Facial lesions in the model mice were photographed before injection. After injection, grooming behavior was video-recorded weekly, and facial lesions were photographed. The effects of Sapap3 on the expression of the virus were compared after injection. - / - Changes in excessive grooming behavior and facial damage caused by excessive grooming in transgenic model mice.

[0285] II. Experimental Results:

[0286] As shown in Figure 5A, the AAV9::cfos-kir2.3-T2A-mcherry virus in Sapap3 - / - ACC expression was observed in transgenic model mice, but almost absent in littermate Wildtype mice. As shown in Figure 5B, bilateral ACC-induced Kir2.3 expression inhibited Sapap3. - / - The transgenic model mouse exhibited excessive grooming behavior and improved facial damage caused by excessive grooming behavior, as shown in Figure 5C.

[0287] Example 6: Treatment of OCD in 8-OH-DPAT model mice by inhibiting ACC neurons through inducible promoter-driven ion channel expression.

[0288] I. Experimental Procedure:

[0289] The main experimental steps were the same as in Example 2. The experimental group was injected with AAV9::cfos-kir2.3-T2A-mcherry virus, and the model control group was injected with AAV9::cfos-mcherry (SEQ ID NO 47) control virus. After the virus injection, T-maze adaptation and spontaneous alternating behavior training were performed sequentially. After the training, 8-OH-DPAT was injected intraperitoneally to induce virus expression (once each in the morning and afternoon for 3 consecutive days). Baseline testing was performed before expression induction on the second day. After the baseline stabilized, the efficacy test was started, i.e., 15 minutes after the injection of 8-OH-DPAT or physiological saline, the behavior of mice in the T-maze was tested and scored.

[0290] II. Experimental Results

[0291] As shown in Figure 6A, 8-OH-DPAT modeling successfully induced the expression of AAV9::cfos-kir2.3-T2A-mcherry virus at the injection site. Furthermore, bilateral ACC-induced expression of kir2.3 significantly inhibited 8-OH-DPAT-induced immobilization behavior, as shown in Figure 6B.

Claims

1. A method for treating obsessive-compulsive disorder, comprising: a) Identify one or more specific brain regions that exhibit abnormal activation in the subject; b) To inhibit or disrupt neuronal activity in one or more specific brain regions, or to interrupt or block neuronal signals in the subject’s brain region.

2. The method according to claim 1, wherein the subject is a patient with obsessive-compulsive disorder.

3. The method according to any one of claims 1-2, wherein identifying one or more specific brain regions exhibiting abnormal activation in the subject further comprises: Neuroimaging was used to identify one or more specific brain regions that were abnormally activated in the subjects.

4. The method of claim 3, wherein the neuroimaging comprises: Neuronal firing signal (Spikes) measurement, cortical potential (ECoG) measurement, PET scan imaging, electroencephalography (EEG) with electromagnetic signal detection, magnetoencephalography (MEG), functional magnetic resonance imaging (fMRI), and near-infrared spectroscopy (NIRS).

5. The method of claim 4, wherein the PET scan imaging comprises: Aβ-PET scanning imaging and / or tau-PET.

6. The method according to any one of claims 3-5, wherein the method further comprises: Neuroimaging is used to obtain brain region detection results of the subject. Based on the comparison of the subject's brain region detection results with reference values, one or more specific brain regions with abnormal activation in the subject are identified.

7. The method of claim 6, wherein the reference value is the brain region detection result of the healthy control group obtained by the neuroimaging.

8. The method of claim 6, wherein identifying one or more specific brain regions of abnormal activation in the subject further comprises: The following results were used to identify one or more specific brain regions in the subjects that exhibited the abnormal activation. 1) Under specific task stimulation, determine that the BOLD signal intensity in a specific brain region of the subject is significantly higher than that of the healthy control group or that the activation pattern of a specific brain region is significantly different from that of the healthy control group, including the range, intensity or timing of the activated brain region, and that such difference is statistically significant by statistical test. 2) Under resting conditions, the spontaneous neural activity in a specific brain region of the subject was significantly higher than that of the healthy control group, or the activation pattern of a specific brain region was significantly different from that of the healthy control group, including the range, intensity or timing of the activated brain region, and the difference was determined to be statistically significant by statistical testing.

9. The method according to any one of claims 7-8, wherein the brain region detection results of the subject and / or the brain region detection results and / or reference values ​​of the control group are generated based on the brain oxygen metabolism rate.

10. The method according to any one of claims 1-9, wherein the one or more specific brain regions comprise: The medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), nucleus accumbens (NAc), ventral striatum (VS), caudate nucleus (Cd), bed nucleus of the stria terminalis (BNST), subthalamus nucleus (STN), and globus pallidus internus (GPi).

11. The method according to any one of claims 1-10, wherein inhibiting or disrupting neuronal activity in a specific brain region comprises severing fiber bundles of the inferior thalamic peduncles (ITP), the medial forebrain bundle (MFB), and the anterior limb of the internal capsule (ALIC).

12. A method for treating obsessive-compulsive disorder, comprising inhibiting or disrupting neuronal activity in a specific brain region, or interrupting or blocking neuronal signals in a specific brain region of a subject.

13. The method of claim 12, wherein the specific brain region includes mPFC, OFC, ACC, NAc, VS, Cd, BNST, STN, and GPi.

14. The method of any one of claim 1 or 12, wherein blocking neuronal signaling in a specific brain region of the subject comprises expressing an exogenous receptor to the subject.

15. The method of any one of claim 1 or 12, wherein blocking neuronal signals in a specific brain region of the subject comprises expressing an exogenous receptor in the specific brain region of the subject.

16. The method according to any one of claims 14-15, wherein blocking neuronal signals in a specific brain region of the subject further comprises administering an exogenous ligand to the subject.

17. The method according to any one of claims 14-16, wherein the exogenous receptor comprises: G protein-coupled receptors (GPCRs) or ion channel proteins.

18. The method of claim 17, wherein the G protein-coupled receptor is a designer receptor DREADD specifically activated by the designer drug.

19. The method of claim 18, wherein the DREADD comprises Rq (R165L), hM1Dq, hM5Dq, rM3D, hM2Di, M4Di and variants thereof, hM3Dq, AlstR or KORD.

20. The method of claim 19, wherein when the exogenous receptor is hM4Di or its variants or hM3Dq, the exogenous ligand comprises clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160, or compound 21 (C21).

21. The method according to any one of claims 15-17, wherein the exogenous receptor comprises: Photosensitive GPCR.

22. The method of claim 21, wherein the photosensitive GPCR comprises Lamplight (Lamprey Parapinopsin), rod opsin, cone opsin, Mu opioid receptor-rod opsin chimera, and Mu opioid receptor.

23. The method of claim 17, wherein the ion channel protein is a ligand-gated ion channel (LGIC) protein or a light-gated ion channel protein.

24. The method of claim 23, wherein the LGIC ligand-gated ion channel comprises GlyR-M, GluCl, PSAM-5HT3HC, PSAM-GlyR, PSAM-nAChR, PSAM4-5HT3, PSAM4-GlyR, TRPV1, or GABAA.

25. The method according to any one of claims 23-24, wherein when the exogenous receptor is an LGIC ligand-gated ion channel, the exogenous ligand comprises ivermectin, selamectin, doramectin, emamectin, epramectin, abamectin, moxicillin, or PSEM. 22S PSEM 89S PSEM 9S Varenic acid, capsaicin, or zolpidem.

26. The method of claim 23, wherein the light-gated ion channel proteins include NpHR, eNpHR3.0, Arch, eArch 3.0, ArchT, eArchT 3.0, eBR, iC1C2, iChloC, GtACR1, GtACR2, SwiChRca, PsChR1, Phobos, Aurora, Jaws, Mac, eMac 3.0, ChR2, ChIEF, C1V1, ReaChR, ChrimsonR, and Chronos.

27. The method according to any one of claims 14-26, further comprising delivering a nucleic acid molecule encoding an exogenous receptor to the subject prior to administration of the exogenous ligand.

28. The method of claim 27, wherein the nucleic acid molecule is delivered to the subject in a viral vector.

29. The method according to claim 28, wherein the viral vector is an adeno-associated virus (AAV), a herpesvirus vector, a retroviral vector, a vaccinia virus vector, an adenovirus vector, or a lentiviral vector.

30. The method of claim 29, wherein the AAV carrier comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV Retro, AAV DJ, AAVrh10, Anc80, or a variant of the above AAV carriers.

31. The method according to any one of claims 27-30, wherein the nucleic acid encoding the modified exogenous receptor is operatively linked to the promoter.

32. The method according to claim 31, wherein the promoter is one of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

33. The method of claim 32, wherein the constitutive promoter comprises the immediate early promoter of cytomegalovirus (CMV), simmon virus 40 (SV40), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus thymidine kinase (HSV-tk) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and eukaryotic translation initiation factor 4A1. One or more of the following: EIF4A1, heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa β member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), human ROSA26 promoter, ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, or β-actin promoter.

34. The method according to claim 32, wherein the inducible promoter comprises one of a tetracycline-responsive promoter, a ecdysone-responsive promoter, a cumate-responsive promoter, a glucocorticoid-responsive promoter, an estrogen-responsive promoter, a PPAR-γ promoter, and a RU-486-responsive promoter.

35. The method of claim 32, wherein the tissue-specific promoters include neuron-specific promoters, glial cell-specific promoters, heart-specific promoters, muscle-specific promoters, lung-specific promoters, liver-specific promoters, kidney-specific promoters, pancreas-specific promoters, adipose-specific promoters, endothelial-specific promoters, retinal-specific promoters, prostate-specific promoters, skin-specific promoters, and macrophage-specific promoters.

36. The method of claim 35, wherein the promoter is a neuron-specific promoter, the neuron-specific promoter comprising: Human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, Advillin promoter, Drosophila single homologue 1 (SIM1) promoter, oxytocin (OXT) promoter, spiky mouse-associated protein (AgRP) promoter, protein kinase C-δ (PKC-δ) promoter. , auxin-releasing peptide promoter, glutamate decarboxylase (GAD1 / 2) promoter, choline acetyltransferase (ChAT) promoter, vesicle GABA transporter (VGAT) promoter, glutamate decarboxylase (GAD65) promoter, tyrosine hydroxylase (TH) promoter and promoter without a distant homeobox (Dlx), cell activity-dependent promoters (c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter or synthetic activity-dependent promoters, such as PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), FRAM (Fos Robust Activity Marker)).

37. The method of claim 36, wherein the promoter is CaMKIIα.

38. The method according to claim 35, wherein the glial cell-specific promoters include the astrocyte fibrillary acidic protein (GFAP) promoter, Gfabc1D promoter, ALDH1L1 promoter, Pirt promoter, Cst3 promoter, Cx30 promoter, myelin basal protein (MBP) promoter, oligodendrocyte myelin glycoprotein (MOG) promoter, CNP (NPPC) promoter, PLP promoter, Pdgfra promoter, olig2 promoter, NG2 promoter, CD11b promoter, Iba1 promoter, CD68 promoter, TMEM119 promoter, CX3CR1 promoter, and Foxj1 promoter.

39. The method of claim 27, wherein the nucleic acid molecule is delivered to the subject using a non-viral method.

40. The method of claim 39, wherein the non-viral method is liposome transfection, nanoparticle delivery, particle bombardment, electroporation, sonication, or microinjection.

41. The method of claim 17, wherein the G protein-coupled receptor is Gi-coupled or Gq-coupled.

42. The method according to any one of claims 1 or 12, wherein the method of destroying neurons in a specific brain region comprises surgically removing neurons in the specific brain region or severing fiber bundles of the inferior thalamic peduncles (ITP), the medial forebrain bundle (MFB), and the anterior limb of the internal capsule (ALIC).

43. The method of any one of claims 1 or 12, wherein the method of destroying neurons in a specific brain region comprises inducing apoptosis of neurons in the specific brain region.

44. The method of claim 43, wherein the method of destroying neurons in a specific brain region comprises administering an apoptosis protein to the subject or administering a nucleic acid encoding an apoptosis protein.

45. The method of claim 44, wherein the apoptosis protein is caspase 3 / 7.

46. ​​The method of claim 1 or 12, wherein the method of neurons in a specific brain region comprises administering a toxin receptor protein and its ligand to a subject.

47. The method of claim 45, wherein the method of destroying neurons in a specific brain region comprises administering to a subject a coding sequence of a toxin receptor protein and its ligand.

48. The method according to any one of claims 46-47, wherein the toxin receptor protein is DTR (diphtheria toxin receptor).

49. The method according to any one of 1 or 12, wherein the method of interrupting or blocking neuronal signals in a specific brain region comprises administering to a subject an inhibitor of neurotransmitters synthesized or secreted by neurons in the aforementioned brain region.

50. The method of claim 49, wherein the method of interrupting or blocking neuronal signals in a specific brain region comprises administering to the subject an antagonist of a neurotransmitter synthesized or secreted by neurons in the aforementioned brain region.

51. The method of claim 49, wherein the method of interrupting or blocking neuronal signals in a specific brain region comprises administering to the subject a neutralizer of neurotransmitters synthesized or secreted by neurons in the aforementioned brain region.

52. The method according to any one of claims 50-51, wherein interrupting or blocking neuronal signals in a specific brain region comprises administering TeNT (tetanus toxin) to the subject.

53. The method according to any one of claims 50-51, wherein interrupting or blocking neuronal signals in a specific brain region comprises inhibiting the release of neurotransmitters.

54. The method according to any one of claims 50-51, wherein interrupting or blocking neuronal signals in a specific brain region comprises inhibiting the synthesis of neurotransmitters.

55. The method according to any one of claims 1-54, wherein the administration comprises oral, intrathecal, intraganglionic, intracranial, subcutaneous, intraspinal, intracisional, or local administration.

56. The use of any one of the exogenous receptors, exogenous ligands, nucleic acid molecules encoding exogenous receptors, apoptosis proteins, nucleic acids encoding apoptosis proteins, toxin receptor proteins and their ligands, coding sequences of toxin receptor proteins, inhibitors of neurotransmitters synthesized or secreted by neurons in a specific brain region, antagonists of neurotransmitters synthesized or secreted by neurons in a specific brain region, and neutralizers of neurotransmitters synthesized or secreted by neurons in a specific brain region in the preparation of a medicament for treating obsessive-compulsive disorder.

57. The use of any one of the exogenous receptors, exogenous ligands, nucleic acid molecules encoding exogenous receptors, apoptosis proteins, nucleic acids encoding apoptosis proteins, toxin receptor proteins and their ligands, coding sequences of toxin receptor proteins, and inhibitors of neurotransmitters synthesized or secreted by neurons in a specific brain region, antagonists of neurotransmitters synthesized or secreted by neurons in a specific brain region, and neutralizers of neurotransmitters synthesized or secreted by neurons in a specific brain region for the treatment of obsessive-compulsive disorder.

58. The method of claim 17, wherein the receptor is a potassium ion channel protein.

59. The method of claim 58, wherein the receptor is an ion channel protein, specifically a human potassium ion channel.

60. The method according to any one of claims 58-59, wherein the potassium ion channel is selected from one or more of the following: voltage-gated potassium ion channel (Kv), inward rectified potassium ion channel (Kir), calcium-activated potassium ion channel (KCa), and dual-pore potassium ion channel (K2P).

61. The method according to any one of claims 58-60, wherein the potassium ion channel is Kv and / or Kir.

62. The method according to any one of claims 58-61, wherein the potassium ion channel is human Kv and / or human Kir.

63. The method according to any one of claims 58-62, wherein the potassium ion channel is selected from one or more of the following: Kir1.x, Kir2.x, Kir3.x, Kir4.x, Kir5.x, Kir6.x and Kir7.x; preferably, the potassium ion channel is Kir2.x and / or Kir3.x.

64. The method according to any one of claims 58-63, wherein the potassium ion channel is selected from one or more of the following: human Kir1.x, human Kir2.x, human Kir3.x, human Kir4.x, human Kir5.x, human Kir6.x and human Kir7.x; preferably, the potassium ion channel is human Kir2.x and / or human Kir3.x.

65. The method according to any one of claims 58-64, wherein the potassium ion channel is selected from one or more of the following: Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, Kir3.3, Kir3.4, Kir4.1, Kir6.1 and Kir6.

2.

66. The method according to any one of claims 58-65, wherein the potassium ion channel is selected from one or more of the following: human Kir1.1, human Kir2.1, human Kir2.3, human Kir3.1, human Kir3.2, human Kir3.3, human Kir3.4, human Kir4.1, human Kir6.1 and human Kir6.

2.

67. The method according to claim 65, wherein Kir1.1 is encoded by the KCNJ1 gene, Kir2.1 is encoded by the KCNJ2 gene, Kir2.3 is encoded by the KCNJ4 gene, Kir3.1 is encoded by the KCNJ3 gene, Kir3.2 is encoded by the KCNJ6 gene, Kir3.3 is encoded by the KCNJ9 gene, Kir3.4 is encoded by the KCNJ5 gene, Kir4.1 is encoded by the KCNJ10 gene, Kir6.1 is encoded by the KCNJ8 gene, and Kir6.2 is encoded by the KCNJ11 gene.

68. The method according to claim 66, wherein the human Kir1.1 is encoded by the human KCNJ1 gene, the human Kir2.1 is encoded by the human KCNJ2 gene, the human Kir2.3 is encoded by the human KCNJ4 gene, the human Kir3.1 is encoded by the human KCNJ3 gene, the human Kir3.2 is encoded by the human KCNJ6 gene, the human Kir3.3 is encoded by the human KCNJ9 gene, the human Kir3.4 is encoded by the human KCNJ5 gene, the human Kir4.1 is encoded by the human KCNJ10 gene, the human Kir6.1 is encoded by the human KCNJ8 gene, and the human Kir6.2 is encoded by the human KCNJ11 gene.

69. The method according to any one of claims 58-68, wherein the nucleotide sequences encoding potassium ion channels are as shown in SEQ ID NOs:27-42 or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NOs:27-42.

70. The method according to any one of claims 58-69, wherein the sequence of the potassium ion channel is consistent with the polypeptide encoded by the nucleotides shown in SEQ ID NOs:27-42.

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