Method for inhibiting neuronal hyperexcitability via AAV-mediated expression of potassium channel

Abstract

The present application relates to a method for inhibiting neuronal hyperexcitability via AAV-mediated expression of a potassium channel. The present application relates to a recombinant adeno-associated virus vector (rAAV vector) for inhibiting neuronal hyperexcitability. The rAAV vector comprises a nucleotide sequence encoding a potassium channel. Further, the present application also relates to a virus particle comprising the rAAA vector, a pharmaceutical composition comprising the rAAA vector and / or the virus particle, and a use thereof in the preparation of a drug for treating diseases related to neuronal hyperexcitability.

Description

A method for inhibiting neuronal overexcitation using AAV-expressed potassium ion channels. Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for enhancing potassium ion channel expression to inhibit neuronal overexcitation. Background Technology

[0002] Epilepsy is a common neurological disorder primarily caused by excessive discharge of neurons in the brain, manifesting as a range of clinical symptoms such as disturbances in motor, sensory, consciousness, and mental state. Focal epilepsy, also known as partial epilepsy, is caused by seizures resulting from localized lesions in the cerebral cortex. These lesions may be related to structurally abnormal brain regions, such as areas with neuronal developmental abnormalities, degeneration, or tumors.

[0003] Currently, the main treatments for focal epilepsy are medication and surgery. Medication primarily involves selecting appropriate antiepileptic drugs based on the type of epilepsy, such as carbamazepine and oxcarbazepine. However, many problems remain, such as significant side effects and short-lasting efficacy. For patients who do not respond to medication, the feasibility of surgery can be assessed, and surgery may be performed if necessary. Surgical procedures mainly include resective surgery, transection surgery, ablative surgery, and neuromodulation surgery.

[0004] Neuromodulation techniques can directly activate or inhibit specific brain regions, but they have several serious drawbacks. First, prolonged high-current conduction can cause thermal damage to the tissues surrounding the electrodes, leading to neuronal apoptosis. Second, electrical stimulation is a non-specific neuromodulation method, affecting all types of neurons near the electrodes. Finally, the use of electrical stimulation is relatively complex, requiring specialized equipment, implanted electrodes in some methods, and repeated adjustments to stimulation parameters. In addition, resection of the epileptic focus is also an effective treatment strategy; however, this surgery is invasive and not suitable for patients with epileptic foci in specific brain regions or those with unexplained epilepsy origins.

[0005] In addition, a significant number of patients are unable to achieve adequate epilepsy control with currently available drug treatments or experience unnecessary side effects.

[0006] Therefore, it is evident that the effective treatments for epilepsy are still relatively limited, and the development of an epilepsy drug with high efficacy, few side effects, and a wide range of beneficiaries is of great significance.

[0007] The pathogenesis of epilepsy is complex, involving multiple aspects such as ion channel dysfunction, neurotransmitter imbalance, and glial cell abnormalities. Among these, ion channel dysfunction, especially potassium ion channel dysfunction, plays a crucial role in the pathogenesis of epilepsy. Potassium ion channels are the most widely distributed and diverse type of ion channels in the body, participating in maintaining the resting membrane potential of cells and regulating the repolarization process of action potentials, determining the firing frequency and amplitude of action potentials. When potassium channels are open, they promote potassium ion efflux, causing neuronal repolarization and hyperpolarization, thereby inhibiting the burst firing of neurons; conversely, potassium channel blockage or downregulation increases neuronal excitability and induces epilepsy.

[0008] Therefore, enhancing the function of potassium ion channels or upregulating the number of potassium ion channels to reduce neuronal excitability holds promise for developing a specific new therapy for epilepsy and other similar diseases caused by neuronal overexcitation. Summary of the Invention

[0009] Among potassium channels, Kv (Voltage-gated potassium channels) are the largest family, divided into 12 subfamilies (Kv1–12). Kv channels control potassium efflux and are involved in the repolarization and hyperpolarization of action potentials, thereby limiting neuronal excitability. For example, mutations in the Kv1.1 channel primarily cause paroxysmal ataxia type 1 (EA-1), a disease characterized by hyperexcitability in both the central and peripheral nervous systems, leading to partial seizures and myofibrosis. Furthermore, the KCNQ2 and KCNQ3 genes encode Kv7.2 and Kv7.3 channels, respectively. The M-currents formed by these channels are major regulators of neuronal excitability, and variations in these channels can lead to benign familial neonatal seizures (BFNC) and early-onset epileptic encephalopathy (EOEE). Other potassium channels, such as the Kv2.1 channel encoded by KCNB1 and the Kv1.2 channel encoded by KCNA2, are also closely related to the pathogenesis of epilepsy.

[0010] Kir (Inwardly Rectifying Potassium Channels) are an important class of potassium ion channels, also known as inwardly rectifying potassium ion channels. They allow potassium ions to more easily flow out of the cell when the membrane potential is negative (i.e., the intracellular membrane is more negative than the extracellular membrane), while restricting potassium ion outflow when the membrane potential is positive. This property enables Kir channels to play a crucial role in maintaining the cell's resting membrane potential, regulating cell excitability, and participating in various physiological processes.

[0011] Kir channels belong to a branch of the potassium channel superfamily. Based on their structural and functional characteristics, they can be further divided into several subfamilies, including Kir1.x (also known as ROMK, mainly found in the kidneys), Kir2.x (widely found in various cell types, including neurons), Kir3.x (involved in G protein-coupled receptor signaling), Kir4.x (mainly expressed in astrocytes and some neurons), Kir5.x (possibly involved in acid-base balance regulation), Kir6.x (involved in ATP-sensitive potassium channels, involved in insulin secretion and cardioprotection), and Kir7.x (whose function is not yet fully understood).

[0012] In the nervous system, Kir channels, particularly members of the Kir2.x and Kir4.x subfamilies, play a crucial role in regulating neuronal excitability and synaptic transmission. For example, Kir2.1 channels play a key role in controlling the repolarization of neuronal action potentials, while Kir4.1 channels are expressed in astrocytes and participate in potassium ion buffering and reabsorption, thereby maintaining potassium ion homeostasis around neurons.

[0013] In a first aspect, this application provides a viral vector that can be used to inhibit neuronal overexcitation.

[0014] In some embodiments, the viral vector contains a nucleotide sequence encoding a potassium ion channel protein.

[0015] In some embodiments, the potassium ion channel in the viral vector 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 dual-pore potassium ion channels (K2P). Preferably, the potassium ion channel can be Kv and / or Kir.

[0016] In a preferred embodiment, the potassium ion channel in the viral vector 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 may be Kir2.x and / or Kir3.x.

[0017] In a more preferred embodiment, the potassium ion channel in the viral vector 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. Preferably, 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.

[0018] In some embodiments, the potassium ion channel in the viral vector comprises one or more selected from: (1) human Kir1.1 as shown in SEQ ID NO:22; (2) human Kir2.1 as shown in SEQ ID NO:23; (3) human Kir3.1 as shown in SEQ ID NO:24; (4) human Kir2.3 as shown in SEQ ID NO:25; (5) human Kir3.4 as shown in SEQ ID NO:26; (6) human Kir3.2 as shown in SEQ ID NO:27; (7) human Kir6.1 as shown in SEQ ID NO:28; (8) human Kir3.3 as shown in SEQ ID NO:29; (9) human Kir4.1 as shown in SEQ ID NO:30; and (10) human Kir6.2 as shown in SEQ ID NO:31.

[0019] 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. Preferably, Kir1.1 is encoded by the human KCNJ1 gene, Kir2.1 is encoded by the human KCNJ2 gene, Kir2.3 is encoded by the human KCNJ4 gene, Kir3.1 is encoded by the human KCNJ3 gene, Kir3.2 is encoded by the human KCNJ6 gene, Kir3.3 is encoded by the human KCNJ9 gene, Kir3.4 is encoded by the human KCNJ5 gene, Kir4.1 is encoded by the human KCNJ10 gene, Kir6.1 is encoded by the human KCNJ8 gene, and Kir6.2 is encoded by the human KCNJ11 gene.

[0020] In a specific embodiment, in the viral vector, the nucleotide sequence encoding the potassium ion channel is as shown in SEQ ID NOs:3-18 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:3-18.

[0021] In a more preferred embodiment, the potassium ion channel in the viral vector 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, more preferably selected from human Kir2.1, human Kir2.2, human Kir2.3, human Kir2.4, human Kir2.6, human Kir2.7, human Kir3.2 and human Kir4.1.

[0022] In a more preferred embodiment, the potassium ion channel in the viral vector is Kir2.1, Kir2.2, Kir2.3, Kir2.4 and / or Kir3.2, more preferably human Kir2.1, human Kir2.2, human Kir2.3, human Kir2.4 and / or human Kir3.2.

[0023] For example, the potassium ion channel is Kir2.1, preferably human Kir2.1.

[0024] For example, the potassium ion channel is Kir2.2, preferably human Kir2.2.

[0025] For example, the potassium ion channel is Kir2.3, preferably human Kir2.3.

[0026] For example, the potassium ion channel is Kir2.4, preferably human Kir2.4.

[0027] For example, the potassium ion channel is Kir3.2, preferably human Kir3.2.

[0028] In some embodiments, the virus is selected from one or more of the following: lentiviral vectors, adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, retroviral vectors, and adeno-associated virus vectors (AAV vectors), preferably AAV vectors and lentiviral vectors, and more preferably recombinant adeno-associated virus vectors (rAAV vectors).

[0029] In one specific embodiment, the viral vector is a lentiviral vector. The lentiviral vector may contain a nucleotide sequence encoding one or more of the following potassium ion channel proteins: Kir1.x, Kir2.x, Kir3.x, Kir4.x, Kir5.x, Kir6.x, and Kir7.x. Preferably, the potassium ion channel may be Kir2.x and / or Kir3.x. More preferably, 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, preferably selected from 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.

[0030] In a more specific embodiment, the lentiviral vector further comprises a promoter. The promoter is selected from one or more of the following: constitutive promoters, inducible promoters, activity-regulating promoters, and tissue-specific promoters. Specifically, the promoter is selected from one or more of the following: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) promoter, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR promoter, 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) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, heat shock protein 70kDa 5 (HSPA5) promoter, heat shock protein 90kDa β member 1 (HSP90B1) promoter, heat shock protein 70kDa ( HSP70 promoter, β-kinin (β-KIN) promoter, human ROSA26 promoter, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, β-actin promoter, transcription cofactor response promoter, tetracycline response promoter, ecdysone response promoter, cumate response promoter, glucocorticoid response promoter, estrogen response promoter, PPAR-γ promoter, RU-486 response promoter, c-fos promoter, CREB promoter, SRE promoter, Srf promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter, PRAM (Promoter Robust Activity Marker, Npas4 specific robust activity marker promoter (NRAM), fos specific robust activity marker (FRAM), Enhanced Synaptic Active Response Element (ESARE), EpiPro promoter, Human Synaptic Protein-1 (SYN-1) promoter, Calcium-Calmodulin Dependent Protein Kinase II (CaMKII) promoter, Calcium-Calmodulin Dependent Protein Kinase IIα (CaMKIIα) promoter, Tubulin α1 promoter, Neuron-Specific Enolase (NSE) promoter, Derived Growth Factor β Chain Promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.The following promoters are used: Advilin 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, Dlx-free (Dlx) promoter, astrocyte fibrillary acidic protein (GFAP) promoter, Gfabc1D promoter, myelin basal protein (MBP) promoter, oligodendrocyte myelin glycoprotein (MOG) promoter, CD11b promoter, Iba1 promoter, and Foxj1 promoter. Preferably, the c-fos promoter, CaMKII promoter, and / or CaMKIIα promoter are used.

[0031] Secondly, this application provides a recombinant adeno-associated virus vector (rAAV vector) comprising a nucleotide sequence encoding any of the potassium ion channel proteins described in the first aspect.

[0032] In some embodiments, the rAAV vector contains, between its inverted terminal repeat (ITR) sequences:

[0033] (1) Promoter;

[0034] (2) The nucleotide sequence encoding the potassium ion channel;

[0035] (3) polyA sequence.

[0036] In some embodiments, the rAAV vector further includes a marmot hepatitis virus posttranscriptional regulatory element (WPRE) sequence between its inverted terminal repeat (ITR) sequences.

[0037] In some embodiments, the promoter in the rAAV vector is selected from one or more of the following: constitutive promoters, inducible promoters, activity-regulating promoters, and tissue-specific promoters.

[0038] For example, the constitutive promoter is selected from one or more of the following: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) promoter, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR promoter, 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) promoter, ferritin H (FerH) promoter, ferritin L (FerL) promoter, glycerol Aldehyde 3-phosphate dehydrogenase (GAPDH) promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, heat shock protein 70kDa (HSPA5) promoter, heat shock protein 90kDa β member 1 (HSP90B1) promoter, heat shock protein 70kDa (HSP70) promoter, β-kinin (β-KIN) promoter, human ROSA26 promoter, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, and β-actin promoter.

[0039] For example, the inducible promoter is selected from one or more of the following: transcription cofactor response promoter, tetracycline response promoter, ecdysone response promoter, cumate response promoter, glucocorticoid response promoter, estrogen response promoter, PPAR-γ promoter, and RU-486 response promoter.

[0040] For example, the activity-regulating promoter is selected from one or more of the following: immediate early gene (IEG) promoters (such as c-fos promoter, CREB promoter, SRE promoter, Srf 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), Npas4-specific robust activity marker promoter (NRAM), fos-specific robust activity marker (FRAM), enhanced synaptic activity response element (ESARE), EpiPro promoter). In a preferred embodiment, the promoter is a c-fos promoter. The c-fos promoter is an activity-regulating promoter, specifically, a promoter that targets overexcited neurons (see Leon G. Reijmers et al., Localization of a Stable Neural Correlate of Associative Memory, Sciences).

[0041] For example, the tissue-specific promoter is selected from one or more of the following: neuron-specific promoters, glial cell-specific promoters, heart-specific promoters, muscle-specific promoters, and retinal-specific promoters.

[0042] In some embodiments, the promoter in the rAAV vector is a neuron-specific promoter. The neuron-specific promoter is selected from one or more of the following: human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase II (CaMKII) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 promoter, neuron-specific enolase (NSE) 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, and promoter without distal homeobox (Dlx), preferably CaMKII promoter and / or CaMKIIα promoter.

[0043] For example, the promoter is the CaMKII promoter and / or the CaMKIIα promoter.

[0044] In some embodiments, the promoter in the rAAV vector is a glial cell-specific promoter selected from one or more of the following: astrocyte fibrillary acidic protein (GFAP) promoter, Gfabc1D promoter, myelin basal protein (MBP) promoter, oligodendrocyte myelin glycoprotein (MOG) promoter, CD11b promoter, Iba1 promoter, and Foxj1 promoter.

[0045] In some embodiments, the polyA sequence in the rAAV vector is selected from one or more of the following: SV40polyA, human growth hormone (hGH) polyA, and bovine growth hormone (bGH) polyA.

[0046] In a further embodiment, the rAAV vector further comprises one or more regulatory sequences selected from the following: enhancer, intron, marmot hepatitis virus posttranscriptional regulatory element (WPRE), miRNA target sequence, Kozak sequence, and Rep protein regulatory sequence.

[0047] Thirdly, this application also provides a viral particle comprising any one of the viral vectors in the first aspect or any one of the rAAV vectors in the second aspect. Preferably, the viral particle comprises any one of the rAAV vectors in the second aspect. Furthermore, the rAAV vector is located within the AAV capsid.

[0048] In some embodiments, the AAV capsid is derived from an AAV serotype that is tropism-prone to nerve cells. More specifically, the AAV capsid is derived from one or more of the following serotypes: AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAVDJ, AAV-PHP.N, AAV-PHP.eB, and AAV-PHP.B, and their variants.

[0049] In some embodiments, the nerve cells include, but are not limited to, neurons and glial cells.

[0050] For example, the neuron is selected from one or more of the following: sensory neurons, motor neurons, interneurons, pyramidal cells, basket cells, double bundle cells, lampstand cells, spiny cells, and Purkinje cells.

[0051] For example, the glial cells are selected from one or more of the following: astrocytes, oligodendrocytes, Schwann cells, microglia, ependymal cells, reticulocytes, Betz cells, and satellite cells.

[0052] Fourthly, this application also provides a cell or host cell that includes any one of the viral vectors in the first aspect and / or any one of the rAAV vectors in the second aspect.

[0053] In some embodiments, the cell is a mammalian cell.

[0054] In a preferred embodiment, the cells are commercially available cells used to produce viral particles for any of the third aspects. More preferably, the cells are HEK293 cells.

[0055] Fifthly, this application also provides a pharmaceutical composition. The pharmaceutical composition comprises (a) any one of the viral vectors of the first aspect, any one of the rAAV vectors of the second aspect, any one of the viral particles of the third aspect, and / or, any one of the cells or host cells of the fourth aspect, and (b) a pharmaceutically acceptable vector.

[0056] In some embodiments, the dosage form of the pharmaceutical composition is a solution, granules, powder, tablet, capsule, nebulizer, or injection.

[0057] In some embodiments, the route of administration of the pharmaceutical composition is selected from oral, nasal, pharyngeal, duodenal, intravenous, intramuscular, peritoneal, sternal, subcutaneous, spinal, joint, intracranial, and percutaneous injection.

[0058] In some embodiments, the pharmaceutical composition further comprises other drugs, preferably other drugs that inhibit neuronal overexcitation. More preferably, the other drugs are selected from one or more of the following: sodium valproate, carbamazepine, phenytoin sodium, lamotrigine, morphine, benzodiazepines, clonidine, propranolol, gabapentin, and baclofen.

[0059] In other embodiments, the pharmaceutical composition may also be used in conjunction with surgical treatment. The pharmaceutical composition may be administered before or after surgery.

[0060] It should be understood that the pharmaceutical composition can be manufactured in any suitable manner, and this application is by no means limited to compositions produced by the methods described herein. The selection of an appropriate method may require consideration of the characteristics of the specific portions being bound.

[0061] In some embodiments, the pharmaceutical composition is manufactured or ultimately sterilized under aseptic conditions. This ensures that the resulting composition is sterile and non-infectious, thus improving safety compared to non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the pharmaceutical composition has an immunodeficiency, is suffering from an infection, and / or is susceptible to infection.

[0062] Sixthly, this application also provides the use of any viral vector of the first aspect, any rAAV vector of the second aspect, any viral particle of the third aspect, any cell or host cell of the fourth aspect, and / or, any pharmaceutical composition of the fifth aspect, in the preparation of a medicament for treating diseases related to neuronal overexcitation.

[0063] In a specific implementation, the neuronal hyperexcitability-related diseases are selected from one or more of the following: epilepsy, Parkinson's disease, pain, drug addiction, paroxysmal dyskinesia, unipolar or bipolar affective disorder, anxiety and fear, preferably epilepsy, pain and / or drug addiction.

[0064] Seventhly, this application also provides a method for treating diseases related to neuronal overexcitation. The method comprises administering to a subject any of the viral vectors of the first aspect, any of the rAAV vectors of the second aspect, any of the viral particles of the third aspect, any of the cells or host cells of the fourth aspect, and / or, any of the pharmaceutical compositions of the fifth aspect.

[0065] In a specific implementation, the neuronal overexcitation-related diseases are selected from one or more of the following: epilepsy, Parkinson's disease, pain, obsessive-compulsive disorder, drug addiction, paroxysmal dyskinesia, unipolar or bipolar affective disorder, anxiety and phobia, preferably epilepsy, pain, obsessive-compulsive disorder and / or drug addiction.

[0066] In a preferred embodiment, the application occurs during an attack phase of the neuronal overexcitation-related disease. Those skilled in the art will understand that neuronal overexcitation typically occurs during an attack phase of the disease. By upregulating the number of potassium channel proteins on the cell membrane of the subject's nerve cells, action potential repolarization or accelerated polarization can be promoted when neurons are overexcited, thereby reducing neuronal excitability and even reducing neurotransmitter release, shortening the attack phase of the disease, or even inhibiting the onset of the disease.

[0067] Adeno-associated virus (AAV), a commonly used gene therapy vector, is characterized by low immunogenicity, high safety, and a broad host range. It can efficiently deliver exogenous genes to specific tissue sites and maintain long-term expression in vivo. By injecting potassium channel genes into the epileptic foci and epileptogenic focus of epilepsy patients via AAV vectors, the opening of potassium channels promotes potassium ion efflux, inducing neuronal repolarization and hyperpolarization, thereby inhibiting burst discharges of neurons and achieving the effect of suppressing epilepsy. This method is highly targeted and effective, with few side effects, and can provide long-lasting anti-epileptic efficacy, making it particularly suitable for patients with refractory epilepsy.

[0068] Compared with existing technologies, this invention provides a method for inhibiting epilepsy by utilizing AAV to express potassium ion channels in epileptic foci and epileptogenic foci, which has the following beneficial effects:

[0069] 1. High Targeting and Effectiveness: This invention directly targets the epileptic focus and epileptogenic focus, significantly improving the targeting and effectiveness of the treatment. By injecting the rAAV vector, the potassium ion channel gene is expressed at a specific site, ensuring that the therapeutic effect is concentrated in the lesion area of ​​epilepsy, thereby maximally inhibiting the burst discharge of neurons and reducing the frequency and severity of epileptic seizures.

[0070] 2. Fewer side effects: Compared with traditional drug therapy, this invention uses an rAAV vector to express potassium ion channels, avoiding the systemic side effects commonly associated with drug therapy. The rAAV vector can stably express potassium ion channels at specific locations, reducing the impact on other neurons and tissues, thereby lowering the incidence of adverse reactions and providing a safer treatment option.

[0071] 3. Durable Efficacy: The rAAV vector has the ability to stably express genes over a long period, enabling potassium ion channels to be continuously expressed in epileptic foci and epileptogenic foci. Compared to drug therapy, which requires long-term medication and may lead to drug resistance, this invention provides a durable anti-epileptic effect, offering patients long-term and stable therapeutic benefits while reducing the frequency and cost of treatment.

[0072] 4. Reduced Drug Resistance: Because this invention does not rely on traditional drug treatments, it avoids the common problem of drug resistance. Through gene therapy, potassium ion channels are directly expressed in nerve cells, ensuring the sustainability and stability of the therapeutic effect.

[0073] 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

[0074] 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:

[0075] Figure 1 shows the spectrum of the pAAV-CaMKIIα-mCherry plasmid.

[0076] Figure 2 shows the spectrum of the pLJM1-EF1α-mCherry_P2A_Hygro plasmid.

[0077] Figure 3 shows the different action potential current changes after different potassium ion channels were introduced into HEK293 cells in Example 3.

[0078] Figures 4a-e show the current-voltage (IV) curves of the potassium ion channels initially screened in Example 3, alone and in the negative control group.

[0079] Figures 5a-f show the inhibitory effect of the selected potassium ion channel on neuronal firing in the hippocampus in Example 4.

[0080] Figures 6a-c show the in vivo therapeutic effect of the selected potassium ion channel on epilepsy in Example 5.

[0081] Figure 7 shows the in vivo therapeutic effect of the potassium ion channel selected in Example 6 on chronic epilepsy.

[0082] Figure 8 shows the effect of the selected potassium ion channel in Example 7 on the recognition and memory ability of mice. Detailed Implementation

[0083] 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.

[0084] Terminology Definition

[0085] In this application, the term "potassium ion channel" refers to a hollow, open-topped, bottomless cylindrical protein molecule that spans the cell membrane, allowing potassium ions to be transported from one side of the membrane to the other. Those skilled in the art will understand that the direction of potassium ion transport across the potassium ion channel should be from the side with higher potassium ion concentration to the side with lower potassium ion concentration. The transport of potassium ions across the potassium ion channel does not require the consumption of bioenergy stored in the subject's body (e.g., ATP, GTP, etc.). As will be known to those skilled in the art, the rate at which potassium ions are transported by the potassium ion channel can vary with changes in the environment in which the potassium ion channel is located. For example, for some potassium ion channels, the rate of potassium ion transport is higher when the membrane potential is negative (i.e., more negative inside the cell than outside), and decreases when the membrane potential is positive.

[0086] In this application, the term "overexcitation" refers to an abnormal phenomenon of action potentials occurring in cells capable of generating action potentials. An action potential is a transient, self-propagating potential reversal generated by neurons or muscle cells upon sufficient stimulation; more specifically, it refers to a change in the potential difference from the inside of the cell membrane being lower than the outside, then higher, and finally lower again. The cells capable of generating action potentials can be nerve cells (primarily neurons), skeletal muscle cells, or cardiomyocytes. An abnormal action potential can refer to an abnormal enhancement of the action potential, specifically an increase in the potential difference across the cell membrane after action potential depolarization, a longer duration of the action potential, an increased action potential frequency, a decreased action potential threshold, or a smaller absolute value of the resting potential. Those skilled in the art should understand that abnormal enhancement of action potentials should exclude enhancement under physiological conditions (e.g., during exercise). In some embodiments, during the overexcitation, the neural network may become oversynchronized, increasing the likelihood of neuronal over-discharge. In some embodiments, the overexcitation may include the loss of inhibitory neurons (such as GABAergic interneurons, which typically balance the excitability of other neurons); or changes in the intrinsic properties of excitatory neurons that make them more likely to fire abnormally. In some embodiments, the overexcitation may include reduced GABA levels and decreased sensitivity of GABAA receptors to neurotransmitters, resulting in less inhibition.

[0087] In this application, the term "Kir" refers to an inwardly rectifying potassium ion channel, a type of potassium ion channel that plays an important role in the excitation of nerve cells. Inward rectification means that the potassium ion channel facilitates the outflow of potassium ions from the cell when the membrane potential is negative (i.e., more negative inside the cell than outside), and restricts the outflow of potassium ions when the membrane potential is positive.

[0088] In this application, the term "regulatory sequence" or "regulatory sequence" refers to a sequence in a nucleic acid vector that regulates gene expression.

[0089] In this application, the term "episode" refers to a period during the course of a neuronal hyperexcitability-related disease in which the patient's neurons experience hyperexcitability or abnormal discharge. Those skilled in the art should understand that patients with such neuronal hyperexcitability-related diseases are not necessarily in a state of continuous hyperexcitability, but rather experience intermittent hyperexcitability. The duration of hyperexcitability varies, ranging from a few seconds to several minutes, tens of minutes, hours, days, or even months. Within the episodic period, the level of neuronal hyperexcitability can be the same or different. For the same patient, the intervals between different episodic periods can be very short (e.g., minutes, tens of minutes, hours) or very long (e.g., days, tens of days).

[0090] In this application, the term "medicine" or "medicinal composition" generally refers to a compound or composition that, when properly administered to a patient, can induce a desired therapeutic effect. Preferably, the pharmaceutical composition comprises the medicament of this application (e.g., a viral vector, rAAV vector, AAV viral particle) or a variant thereof, a prodrug, or other bioactive active form, and a carrier, such as a diluent, pharmaceutical excipient, buffer, preservative, and tension modifier.

[0091] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as AAV viral particles. A pharmaceutically acceptable carrier is any drug carrier that does not itself induce the production of substances harmful to a subject receiving the composition and can be administered without causing excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0092] 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 the viral vector and / or rAAV vector 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 must be able to express the viral vector and / or rAAV vector 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 *E. coli* cells. 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 a HEK293 cell.

[0093] 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 expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0094] In this application, the term "viral vector" generally refers to a nucleic acid molecule (e.g., a transfer plasmid) or a viral particle that mediates the transfer of nucleic acid. 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 acid into a cell, or the transferred nucleic acid itself.

[0095] 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. Several AAV serotypes have been characterized. General information and reviews of AAV 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 apparently 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.

[0096] In this application, the term "AAV capsid" refers to the protein coat of adeno-associated virus (AAV). The AAV capsid encapsulates the viral genome and plays a crucial role in viral function. The AAV capsid consists of 60 identical VP1, VP2, and VP3 protein subunits, forming a pseudo-octahedral structure of 12 symmetric units. In some embodiments, the AAV capsid can also determine the tissue tropism of AAV viral particles. Tissue tropism refers to the characteristic of a virus to prefer certain specific types of cells, tissues, or organs; more specifically, tissue tropism means that when a virus infects a subject, its content in certain specific types of cells, tissues, or organs is significantly higher than in other types of cells, tissues, or organs. For example, AAV viral particles with neuronal tropism or tropism for neuronal cells are more likely to infect neuronal cells, and when these AAV viral particles infect a subject, their content in neuronal cells is significantly higher than in other cell types.

[0097] In this application, the terms "recombinant adeno-associated virus vector," "recombinant AAV vector," or "rAAV vector" generally refer to a vector containing one or more nucleotide sequences of interest (or transgenes) flanked by an AAV terminal repeat (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in host cells that have been transfected with a vector encoding and expressing the rep and cap gene products. 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 rAAV vector. If the particle contains a heterologous nucleotide sequence (i.e., a nucleotide sequence other than the wild-type AAV genome, such as a transgene intended to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply "AAV vector." Therefore, the production of AAV vector particles necessarily includes the production of rAAV vectors such that the vector is contained within the AAV vector particle.

[0098] 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.

[0099] 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).

[0100] 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.

[0101] The term "operably linked" generally refers to placing the regulatory sequences necessary for the expression of a coding sequence in the 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 (such as promoters and enhancers) in an expression vector.

[0102] 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, nucleotide structure modifications 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.

[0103] 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.

[0104] In this application, the terms "effective amount" and "therapeutic effective amount" are used interchangeably, generally referring to an amount or dose sufficient to produce the desired therapeutic outcome when a pharmaceutical composition comprising one or more viral vectors, rAAV vectors, and / or AAV viral particles is administered. More specifically, a therapeutic effective amount is an amount sufficient to treat a specified condition, ailment, or disease for a period of time, specifically referring to an amount of a drug or pharmaceutical composition thereof that improves, alleviates, reduces, and / or delays one or more of its symptoms. Determining an effective amount or therapeutic effective amount of a given pharmaceutical composition is entirely within the capabilities of those skilled in the art.

[0105] In this application, the term "treatment" is interpreted broadly, including both therapeutic treatment and prophylactic treatment. Patients requiring treatment include those with a disease, those susceptible to a disease, or those seeking to prevent a disease.

[0106] In this application, the terms "patient" or "subject" generally include humans, non-human primates (e.g., monkeys), or other animals, particularly mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. Furthermore, the term "subject" as used in this application should be interpreted in a broader sense, such as including cells or single-celled organisms of any origin. In a particularly preferred embodiment, the patient or subject is a human.

[0107] In this application, the term "and / or" should be understood as any one of the multiple elements used for connection or any combination of elements.

[0108] In this application, the term "comprising" or "including" generally means including the explicitly specified features, but does not exclude other elements.

[0109] In this application, the term “selected from” generally refers to the selection of objects and all combinations thereof. For example, “selected from A, B and C” means all combinations of A, B and C, such as A, B, C, A+B, A+C, B+C, or A+B+C.

[0110] 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.

[0111] Invention Details

[0112] potassium ion channels

[0113] Based on the correlation between potassium ion channels and epilepsy, one or more potassium ion channels with inhibitory neuronal excitability, such as Kv and Kir, are selected.

[0114] The Kv gene is selected from one or more members of the following subfamily:

[0115] (1) Kv1.x, where x represents any integer between 1 and 8, members of this subfamily are widely expressed in neurons, especially Kv1.1 to Kv1.6, which play an important role in the repolarization of action potentials and the regulation of neuronal excitability. For example, Kv1.2 and Kv1.4 are particularly critical in controlling the firing frequency and pattern of neurons.

[0116] (2) Kv2.x, where x represents 1 or 2, these channels are mainly expressed on the neuronal membrane, especially Kv2.1, which plays a central role in regulating neuronal excitability and the shape of action potentials. Dysfunction of Kv2.x subfamily members may be associated with neuronal overexcitation and neurological disorders such as epilepsy.

[0117] (3) Kv3.x, where x represents any integer between 1 and 4, these channels are characterized by rapid activation and deactivation, and are mainly involved in the rapid repolarization process of action potentials. In particular, Kv3.1 and Kv3.2 play an important role in high-speed neural conduction and neuronal network synchronization, and their dysfunction may affect the processing speed and accuracy of neural information.

[0118] (4) Kv4.x, where x represents any integer between 1 and 3. Members of this subfamily are mainly expressed in the dendrites and axons of neurons, such as Kv4.2 and Kv4.3, which are crucial for regulating the propagation of neuronal membrane potentials and action potentials. Dysfunction of Kv4.x channels may lead to changes in neuronal excitability, thereby affecting the normal function of the nervous system.

[0119] (5) In addition, there are subfamilies such as Kv5.x, Kv6.x, Kv7.x (also known as KCNQ), Kv8.x, Kv9.x, Kv10.x, Kv11.x (also known as hERG), and Kv12.x. Although the specific functions of many members in these subfamilies in the nervous system have not been fully elucidated, studies have shown that they may participate in the regulation of neuronal excitability, the generation and propagation of action potentials, and the development and disease processes of the nervous system through different mechanisms.

[0120] The Kir gene is selected from one or more members of the following subfamilies: characterized in that the Kir gene is selected from one or more members of the Kir2.x, Kir3.x, Kir4.x, Kir5.x, Kir6.x or Kir7.x subfamilies.

[0121] (1) Kir2.x, where x represents any integer between 1 and 7, is widely expressed in neurons and participates in the repolarization of action potentials;

[0122] (2) Kir3.x, where x represents any integer between 1 and 4. These channels are associated with G protein-coupled receptor signaling and may indirectly affect epilepsy by regulating neuronal excitability.

[0123] (3) Kir4.x, where x represents 1 or 2, is expressed in astrocytes and is essential for maintaining potassium balance around neurons;

[0124] (4) and members of the Kir5.x, Kir6.x and Kir7.x subfamilies, although their specific functions in the nervous system may not be fully elucidated, may participate in the pathogenesis or treatment of epilepsy through different mechanisms.

[0125] Gene sequences can be optimized for selected potassium ion channels to ensure efficient expression of these channels. The vector contains regulatory elements to enhance the expression efficiency of potassium ion channel genes, thereby improving their expression specificity and efficiency in neurons.

[0126] (1) Select one or more specific potassium ion channel genes as target genes, and perform codon optimization or functional enhancement mutations on the genes;

[0127] (2) Based on the characteristics and expected expression pattern of the target potassium ion channel gene, design and select at least one promoter. The promoter may be a constitutive promoter to ensure continuous expression of the gene in all cell types, a specific promoter to limit gene expression to neurons only, an inducible promoter to initiate gene expression in response to a specific signal or condition (wherein the inducible promoter responds to a specific external stimulus, such as a neurotransmitter, drug, light stimulation or a specific change in physiological conditions, thereby achieving precise spatiotemporal control of potassium ion channel gene expression), or an activity-regulating promoter to initiate gene expression when the activity of a specific cell changes.

[0128] (3) Optionally, one or more enhancer elements are introduced near the target gene to enhance the activity of the promoter, thereby improving the efficiency of potassium channel gene transcription; the enhancer should be selected from sequences that have been proven to effectively promote gene expression in neurons;

[0129] (4) Optionally, a silencer element is added to suppress gene expression in non-target cell types, ensuring that the expression of potassium ion channel genes is highly specific to neurons;

[0130] (5) Optionally, post-transcriptional regulatory sequences can be added to optimize the expression of potassium channel genes in specific neuron types, specific brain regions or specific time points.

[0131] (6) Construct a recombinant expression vector containing the above-mentioned regulatory elements and target potassium ion channel genes, and transfer it into host cells by appropriate means (preferably AAV and lentivirus).

[0132] The potassium ion channel used in this application for the treatment of epilepsy can be of any species origin (such as rodents such as mice, rats, and rabbits, or primates such as monkeys and humans), preferably of human origin.

[0133] Viral vector / rAAV vector

[0134] Those skilled in the art should understand that the potassium channel protein can be a modified or unmodified complete protein encoded by the corresponding coding gene, or a functional truncated protein encoded by the corresponding coding gene. The truncated protein can be formed by splicing the complete protein, or it can be a truncated protein resulting from the truncation of intermediate products of gene expression. Furthermore, the sequence of the coding gene can be a sequence that can be retrieved by those skilled in the art from publicly available databases (e.g., NCBI, Uniprot, etc.), preferably a wild-type sequence, and may also contain nucleotide sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the retrieved sequence. For example, Kir2.3 is encoded by the KCNJ4 gene, the sequence of which can be the nucleotide sequence shown on the Uniprot website as number P48050, or a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown on P48050.

[0135] In some embodiments, the viral vector, particularly the rAAV vector, may contain regulatory sequences, which may include a promoter, WPRE, and polyA sequences, and optionally one or more of the following: enhancers, introns, miRNA target sequences, Kozak sequences, and Rep protein regulatory sequences. Those skilled in the art will understand that different regulatory sequences have different functions.

[0136] For example, the promoter refers to a key regulatory sequence that controls the initiation step of gene expression, determining the intensity and cell specificity of gene expression. As another example, the enhancer refers to a regulatory sequence that can enhance gene expression. As another example, the intron is part of the mRNA precursor and can affect mRNA splicing, stability, and nuclear export, thereby regulating gene expression. As another example, the polyA sequence is a signal sequence instructing the addition of a poly(A) tail to the 3' end of mRNA, crucial for mRNA stability and translation efficiency. As another example, the WPRE is a cis-acting element that can enhance mRNA stability and nuclear export. As another example, the miRNA target sequence is a regulatory sequence that enables post-transcriptional regulation of gene expression. As another example, the Kozak sequence refers to a conserved nucleotide sequence flanking the start codon AUG in mRNA, typically enhancing gene expression; the sequence is preferably 5'-GCC(A / G)CCAUGG-3', where AUG is the start codon. As another example, the Rep protein regulatory sequence refers to a regulatory sequence that regulates viral gene expression.

[0137] Those skilled in the art will understand that the selection of the promoter can be related to the target of the vector, and in this application, it is preferably tissue-specific, and more preferably nerve cell-specific. Furthermore, the promoter can also be specific to hyperexcitable nerve cells, such as the c-fos promoter (see Leon G. Reijmers et al., Localization of a Stable Neural Correlate of Associative Memory, Sciences).

[0138] Those skilled in the art should note that the enhancer is a different element from the aforementioned promoter. The enhancer refers to a regulatory sequence that enhances gene expression when the gene can be expressed. The promoter refers to a key regulatory sequence that controls whether a gene is expressed. Those skilled in the art should understand that the promoter is an essential element of the rAAV vector, while the enhancer is an optional element. Furthermore, the enhancer can be used in conjunction with the promoter to increase the expression level of a specific gene.

[0139] In some embodiments, the recombinant adeno-associated virus vector (rAAV vector) refers to an engineered adeno-associated virus (AAV) vector. This modification involves removing the gene sequences encoding Rep or Cap from the AAV genome, retaining only the inverted terminal repeat (ITR) sequences that serve as packaging signals, and replacing these sequences with therapeutic genes and expression regulatory elements. Such modified AAV vectors cannot self-replicate but can effectively deliver therapeutic genes into host cells. Recombinant AAV vectors have become a major gene delivery platform for treating various diseases due to their diversity, extremely low immunogenicity, high safety, wide host cell range, strong diffusion capacity, and long in vivo gene expression time. The viral capsid of the rAAV vector retains the sequence and structure of wild-type AAV, but the genome within the viral capsid has been completely stripped of the Rep and Cap genes, retaining only the ITR sequences responsible for guiding viral vector genome replication and packaging. This modification significantly increases the loading capacity of the AAV vector and greatly reduces in vivo immunogenicity and toxicity. Therefore, recombinant AAV vectors have been widely used in gene therapy, gene editing, and vaccine development.

[0140] In a further embodiment, the aforementioned recombinant AAV vector can be amplified and packaged into a large number of AAV virus particles using a three-plasmid packaging method. Specifically, the three plasmids may include the recombinant AAV vector, a packaging plasmid, and an auxiliary plasmid.

[0141] In some embodiments, the packaging plasmid refers to a plasmid carrying the Rep gene and Cap gene of AAV viral particles. The protein encoded by the Rep gene is a protein capable of guiding AAV replication. The Cap gene is a gene encoding the AAV capsid protein.

[0142] The helper plasmid is a plasmid that assists AAV in autonomous replication and the production of viral particles, and is typically a plasmid containing adenovirus or herpes simplex virus proteins. Furthermore, the helper plasmid may typically contain one or more of the adenovirus's E1A / B, E2A / B, E4, and VA genes. Commonly used adenoviruses include Ad5 and Ad2, with Ad5 being preferred. The genes contained in the helper plasmid may depend on the type of production cell. For example, when using HEK293 cells as the production cell, since HEK293 cells contain E1A and E1B, a helper plasmid containing only the E2A, E4, and VA genes can be used.

[0143] In a specific embodiment, the three-plasmid packaging method refers to transfecting a certain proportion of the recombinant AAV vector, the packaging plasmid, and the helper plasmid into host cells (preferably HEK293 cells) for a period of time, and then collecting and purifying the virus-containing composition to obtain a high-titer virus solution. In a specific embodiment, the virus-containing composition can be cell lysate and / or cell culture medium. In a specific embodiment, the purification method can refer to common methods for concentrating virus solutions in the art, including but not limited to density gradient ultracentrifugation, ultrafiltration, polyethylene glycol (PEG) precipitation, mass flow cytometry, virus purification and concentration kits, ion exchange chromatography, affinity chromatography, and gel filtration.

[0144] Neuronal hyperexcitability-related diseases

[0145] The aforementioned neuronal hyperexcitability-related diseases are a class of diseases characterized primarily by neuronal hyperexcitability or abnormal discharge. Those skilled in the art should understand that the hyperexcitability or abnormal discharge is usually not persistent but rather occurs intermittently. In a preferred embodiment, the drug can be administered during an episode of neuronal hyperexcitability or abnormal discharge, before an episode of neuronal hyperexcitability or abnormal discharge, or after an episode of neuronal hyperexcitability or abnormal discharge; preferably, it can be administered before or during an episode.

[0146] Furthermore, the drug can be administered before, during, or after the first occurrence of neuronal hyperexcitation.

[0147] In a preferred embodiment, when the drug is administered before the first occurrence of neuronal hyperexcitation, the subject may be assessed as potentially susceptible to neuronal hyperexcitation, and the assessment may be performed through genetic counseling, comprehensive assessment of pathogenic genes, application of Mendelian laws of inheritance, Bayes analysis, polygenic genetic risk scoring, gene testing services, molecular diagnostic techniques, or precision medicine.

[0148] The drug is preferably, but not limited to, administered during an attack of a neuronal hyperexcitability-related disease. The drug may be administered to the subject at an appropriate carrier dose, which should be determined by a person skilled in the art through appropriate testing.

[0149] epilepsy

[0150] Those skilled in the art will understand that the viral vector, rAAV vector, AAV viral particles, and pharmaceutical composition protected in this application have the advantages of high targeting and long-lasting efficacy in the treatment of epilepsy. The treatment should be for all diseases that can be clinically termed epilepsy, including but not limited to neocortical epilepsy, temporal lobe epilepsy, and refractory epilepsy. The epilepsy can be acute or chronic. Chronic epilepsy refers to recurrent seizures over a prolonged period, typically more than one year.

[0151] In some implementations, the treatment may occur during a seizure, before a seizure, or after a seizure.

[0152] In some implementations, the treatment may occur before the first seizure of the epilepsy. The first seizure refers to the subject's first appearance of symptoms of neuronal hyperexcitability.

[0153] In a preferred embodiment, the treatment occurs during a seizure. During a seizure, the corresponding brain region of the patient may experience deep depolarization and pulses of cortical pyramidal neurons firing at frequencies greater than 50 Hz.

[0154] In some embodiments, the treatment can be used to quench or block epileptic activity. The treatment can be used to reduce the frequency of seizures. The treatment can be used to temporarily (e.g., for 2, 6, 24, 48, or 72 hours) or permanently reduce the excitability of abnormal neurons.

[0155] In a preferred embodiment, the treatment does not affect the subject's spontaneous movement or memory, and spontaneous movement or memory may optionally be measured using an open field test, a target localization test, or a T-maze test.

[0156] In some embodiments, the viral vector, the rAAV vector, or the AAV viral particles are locally active only in the epileptic foci and epileptogenic foci of the subject. In some embodiments, the viral vector, the rAAV vector, or the AAV viral particles are locally active only in neurons capable of driving seizures and / or generating sustained discharges. In some embodiments, the viral vector, the rAAV vector, or the AAV viral particles are locally active only in neurons that are excessively depolarized.

[0157] In some implementations, the therapeutic effect can be assessed by evaluating the number or frequency of action potentials, resting membrane potentials, or action potential thresholds in slices of the corresponding brain region (e.g., the hippocampus) of the subject.

[0158] In some implementations, treatment effectiveness can be assessed by evaluating the subject's corresponding behavioral patterns. These behavioral patterns include, but are not limited to, the number of seizures in the subject within the same time period, the latency of seizures, and the severity of seizures (e.g., Racine classification).

[0159] In some embodiments, the subjects used to evaluate the efficacy of the potassium ion channel therapy for epilepsy may be of the same species as the potassium ion channel or of a different species; the subjects may or may not be immunodeficient.

[0160] In some embodiments, the viral vector, the rAAV vector, or the AAV viral particles can produce a greater antiepileptic effect in neurons driving a subject's next seizure than in neurons driving a subject's previous seizure. In some embodiments, the antiepileptic effect is measured using any suitable method described herein.

[0161] In some embodiments, the viral vector, the rAAV vector, or the AAV viral particles can prevent a subject from having a next seizure, wherein the next seizure occurs after the subject's previous seizure.

[0162] In some implementations, the subject may be selectively treated with targeted therapy to one or more of the following brain regions: the anterior thalamic nucleus, the hippocampal-amygdala complex, the central thalamic nucleus, the subthalamic nucleus, the temporal lobe, the hippocampus, the amygdala, and the prefrontal lobe.

[0163] pain

[0164] Those skilled in the art should understand that pain refers to a class of diseases characterized by a painful state. The pain typically occurs concurrently with other diseases or is a residual symptom after the cure or incurability of other diseases. The causes of pain may generally originate from organic organ diseases (such as cancer, neurological dysfunction, etc.) or emotional and psychological factors.

[0165] In some implementations, the treatment may occur during the onset of pain, before the onset of pain, or after the onset of pain.

[0166] In some implementations, the treatment may occur before the first onset of pain. The first onset refers to the subject's first appearance of symptoms of neuronal hyperexcitability. The period preceding the first onset of pain may be after treatment for the other disease, or it may be any time period.

[0167] In some implementations, the pain state may include, but is not limited to, sharp pain, dull pain, burning pain, stabbing pain, and electric shock-like pain.

[0168] In some implementations, the pain state may include, but is not limited to, mild pain, moderate pain, and severe pain.

[0169] In some implementations, the pain state may include, but is not limited to, acute pain and chronic pain.

[0170] In some implementations, the pain state may include, but is not limited to, somatic pain, visceral pain, and neuralgia.

[0171] In some implementations, the pain state may include, but is not limited to, nociceptive pain, inflammatory pain, and neuropathic pain.

[0172] In some implementations, the pain state may include, but is not limited to, localized pain, radiating pain, and widespread pain.

[0173] In some implementations, the pain state may include, but is not limited to, spontaneous pain and induced pain (such as pain induced by touch, pressure, or temperature changes).

[0174] Those skilled in the art should understand that current treatments for pain primarily rely on medication (e.g., nonsteroidal anti-inflammatory drugs or opioids). These treatments typically carry a high risk of drug tolerance and addiction, and have significant side effects. As the disease progresses, patients may be forced to increase the dosage, further exacerbating the risk of addiction.

[0175] The treatment described in this application can be administered to subjects suffering from pain, including but not limited to treatment before drug treatment, before drug addiction occurs, or after drug addiction occurs.

[0176] In some implementations, the subject may be selectively treated with targeted therapy to one or more of the following brain regions: locus coeruleus, rostral ventromedial nucleus of medulla oblongata, caudal ventrolateral nucleus of medulla oblongata, orbitofrontal cortex, periaqueductal gray matter, thalamus, anterior cingulate cortex, somatosensory cortex, insula, and paraventricular nucleus of hypothalamus.

[0177] Administration method

[0178] The viral particles and vectors described herein can be delivered to the subject in a variety of ways, such as by direct injection of the viral particles into neural tissue. For example, the treatment may involve direct injection of viral particles into the cerebral cortex, particularly the neocortex or hippocampus. Another injection site is the area of ​​cortical malformation or hamartoma suspected of causing seizures, as occurs in focal cortical dysplasia or tuberous sclerosis. The treatment may also involve direct injection of viral particles into locations in the brain believed to be functionally related to the impairment. For example, in the case of the treatment for myoclonic epilepsy, this may involve direct injection of viral particles into the motor cortex; in the case of the treatment for pain (e.g., chronic or paroxysmal pain), this may involve direct injection of viral particles into the dorsal root ganglion, trigeminal ganglion, or sphenopalatine ganglion; in the case of the treatment for drug addiction, this may involve direct injection of viral particles into the substantia nigra, amygdala, or hippocampus. The specific method and site of administration will be determined by the physician, who will also use his / her common sense and techniques known to skilled practitioners to select the administration technique.

[0179] This application can also be used to treat multiple epileptic foci simultaneously by direct injection into multiple identified loci.

[0180] The patient may be someone who has been diagnosed with drug-resistant or drug-demand-resistant epilepsy, which means that seizures continue despite adequate administration of antiepileptic drugs.

[0181] Subjects can be individuals already diagnosed with a defined focal epilepsy affecting a single area of ​​the neocortex. For example, focal epilepsy can be caused by developmental abnormalities or following a stroke, tumor, penetrating brain injury, or infection.

[0182] After administration of viral particles, recipients may experience a reduction in symptoms of the disease or disorder being treated. For example, for individuals undergoing treatment for a seizure disorder (such as epilepsy), recipients may show a decrease in the frequency or severity of seizures. This may have a beneficial effect on the individual's disease condition.

[0183] 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 even 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.

[0184] In specific implementations, 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 2 genome particles / mL, or at least about 8 × 10^12 genome particles / mL, or at least about 9 × 10^12 genome particles / mL, or at least about 10 × 10^12 genome particles / mL, or at least about 15 × 10^12 genome particles / mL, or at least about 20 × 10^12 genome particles / mL, or at least about 25 × 10^12 genome particles / mL, or at least about 50 × 10^12 genome particles / mL, or at least about 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 genome particles in a particular vector preparation can be measured, for example, by the method described in the examples herein.

[0185] The carrier of the present invention can be administered in a volume of fluid (such as water or other solvents). 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).

[0186] In specific implementations, the vectors considered herein are administered to subjects at titers of at least about 5 × 10^9 infectious units / mL, at least about 6 × 10^9 infectious units / mL, at least about 7 × 10^9 infectious units / mL, at least about 8 × 10^9 infectious units / mL, at least about 9 × 10^9 infectious units / mL, at least about 10 × 10^9 infectious units / mL, at least about 15 × 10^9 infectious units / mL, at least about 20 × 10^9 infectious units / mL, at least about 25 × 10^9 infectious units / mL, at least about 50 × 10^9 infectious units / mL, or at least about 100 × 10^9 infectious units / mL. When referring to viral titers, the terms “infectious unit (iU),” “infectious particle,” or “replication unit” refer to a assay of the number of infectious and replicable recombinant AAV vector particles measured at the center of infection, also known as the center of replication assay.

[0187] In specific implementations, the vectors 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. As used with respect to viral titers, the term “transduction unit (tu)” refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgenic product, as measured in the functional assays described in the examples herein.

[0188] The carrier dosage is generally determined by the route of administration. In one specific embodiment, intraganglionic injection may include about 1×10^9 to about 1×10^13 carrier genomes in a volume of about 0.01 mL to about 10.0 mL. In another specific embodiment, intrathecal injection may include about 1×10^9 to about 1×10^13 carrier genomes in a volume of about 0.01 mL to about 10.0 mL. In another specific embodiment, intracranial injection may include about 1×10^9 to about 1×10^13 carrier genomes in a volume of about 0.01 mL to about 10.0 mL. In another specific embodiment, intraneural injection may include about 1×10^9 to about 1×10^13 carrier genomes in a volume of about 0.01 mL to about 10.0 mL. In another specific embodiment, intraspinal injection may include about 1×10^9 to about 1×10^13 carrier genomes in a volume of about 0.1 mL to about 0.01 mL to about 10.0 mL. In yet another specific embodiment, 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 another specific embodiment, 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.

[0189] In some implementations, the ligand is administered to the subject at the same time as the carrier is administered to the subject.

[0190] In some implementations, the ligand is administered to the subject after the carrier is administered to the subject.

[0191] In some implementations, the ligand is administered to the subject before the carrier is administered to the subject.

[0192] In some implementations, 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 carrier administration. In some cases, a therapeutically effective amount of 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 ligand is administered to the subject at least one week after carrier delivery.

[0193] 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.

[0194] In some embodiments, the dose of the ligand administered to the subject is at least about 0.001 micrograms per kilogram (μg / kg), at least about 0.005 μg / kg, at least about 0.01 μg / kg, at least about 0.05 μg / kg, at least about 0.1 μg / kg, at least about 0.5 μg / kg, 0.001 milligrams per kilogram (mg / kg), at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 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.

[0195] 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.

[0196] In some embodiments, the therapeutically effective amount of ligand may be expressed as a molar concentration (i.e., M or mol / L). In some cases, the therapeutically effective amount of ligand may be about 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, 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.

[0197] 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, 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, a 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 ligand dose may be administered to the subject continuously, or 1, 2, 3, 4, or 5 times daily; 1, 2, 3, 4, 5, 6, or 7 times weekly; 1, 2, 3, or 4 times monthly; once every two, three, four, five, or six months; or once a year; or once, twice, three, four, five, or six times monthly, or even at longer intervals. 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.

[0198] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the viral vector, rAAV vector, viral particles, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0199] Example

[0200] Example 1: Selection of Potassium Ion Channels

[0201] In the Kv and Kir potassium ion channels, the inventors selected 17 channels for selective investigation and used the EKC gene (the sequence of the EKC gene is from Albert Snowball et al., PMID:30755487), which is known to suppress epileptic seizures through overexpression, as a positive control. The nucleotide sequence of the EKC gene is shown in SEQ ID NO:2. The inventors selected 17 channels: Kv1.1 (SEQ ID NO:1), Kir1.1 (SEQ ID NO:3), Kir2.1 (SEQ ID NO:4), Kir3.1 (SEQ ID NO:5), Kir2.3 (SEQ ID NO:6), Kir3.4 (SEQ ID NO:7), Kir3.2 (SEQ ID NO:8), Kir6.1 (SEQ ID NO:9), Kir3.3 (SEQ ID NO:10), Kir4.1 (SEQ ID NO:11), Kir6.2 (SEQ ID NO:12), Kir2.2 (SEQ ID NO:13), Kir7.1 (SEQ ID NO:14), Kir2.4 (SEQ ID NO:15), Kir4.2 (SEQ ID NO:16), Kir5.1 (SEQ ID NO:17), and Kir2.6 (SEQ ID NO:18). The coding gene sequences for these potassium ion channels are shown in parentheses.

[0202] Example 2: Vector Construction and Virus Purification

[0203] Vectors were constructed targeting the aforementioned potassium ion channel genes. These vectors contained regulatory elements to enhance the expression efficiency of potassium ion channel genes, thereby improving the expression specificity and efficiency of potassium ion channel genes in neurons.

[0204] The methods for constructing the rAAV vector and obtaining the rAAV virus are as follows:

[0205] (1) Use pAAV-CaMKIIα-mCherry plasmid (addgene#Plasmid#114469) containing the nucleotide sequence shown in Figure 1 and SEQ ID NO:19 as template plasmid. The position of mCherry is the insertion position of the potassium ion channel coding gene mentioned above. mCherry will be completely replaced by the potassium ion channel coding gene mentioned above in subsequent steps.

[0206] (2) Potassium ion channel genes (such as kir2.3) were amplified by PCR, and the sequences of BamHI and EcoRI restriction endonuclease sites were added to the PCR primers.

[0207] (3) The target gene fragment obtained in step (2) and the pAAV-CaMKIIα-mCherry vector were digested with restriction endonucleases BamHI and EcoRI.

[0208] (4) Use DNA ligase to ligate the target gene fragment treated in step (3) to the vector backbone to construct a recombinant plasmid.

[0209] (5) Transform the recombinant plasmid into a host cell (such as Escherichia coli) and identify plasmids with correct sequences (i.e., 100% identity with the corresponding sequence shown in Example 1) by enzyme digestion and Sanger sequencing.

[0210] (6) Select the Stabl3 strain to prepare a large number of plasmids selected in step (5).

[0211] (7) Select HEK293T cells as production cells. The target plasmid or control plasmid with the correct sequence, pHelper (addgene#Plasmid#112867) plasmid and pAAV2 / 9 pRC plasmid (addgene#Plasmid#112865, AAV packaging plasmid expressing Rep2 and Cap9) were co-transfected into HEK-293T cells at a molar ratio of 1:1:1 using PEI transfection reagent (approximately 1 μg of plasmid per million cells). After culturing in an incubator at 37°C with 5% carbon dioxide for 3 days, the cells were digested and centrifuged at 1000g. The cell pellet was transferred to a 50ml centrifuge tube, washed once with PBS buffer, and the cells were collected in PBS buffer. The cells were then subjected to five freeze-thaw cycles. Solid NaCl was added to make the final NaCl concentration 500mM, and the cells were centrifuged at 10000g for half an hour. After filtering the supernatant through a 0.45 μm filter membrane, prepare gradient iodixanol solutions and add them to centrifuge tubes (from bottom to top: 5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 mL 25% iodixanol, 8 mL 15% iodixanol). Add the sample to the top layer and centrifuge at 350,000 g for 1 hour. Aspirate the virus layer at the 40% and 60% interfaces. Change the medium using a 50 kDa ultrafiltration tube (centrifuge at 4000 g for 10-15 minutes), changing the medium 5-6 times. Each time, use 0.01% poloxamer PBS buffer to ensure the virus is finally dissolved in the 0.01% poloxamer PBS buffer. After determining the viral titer using qPCR, the titer was adjusted (if concentration is required, the aforementioned method of centrifuging at 4000g for 10-15 minutes and changing the medium can be used), finally obtaining a viral titer of 1×10^13 vg / ml.

[0212] The method for constructing an instantaneous reload object is as follows:

[0213] (1) The base plasmid pLJM1-EF1α-mCherry_P2A_Hygro, which contains the nucleotide sequence shown in Figure 2 and SEQ ID NO:20, is used (addgene#Plasmid#135003). The position of mCherry is the insertion site of the potassium ion channel encoding gene mentioned above. mCherry_ will be completely replaced by the potassium ion channel encoding gene mentioned above in subsequent steps.

[0214] (2) Potassium ion channel genes (such as kir2.3) were amplified by PCR, and the sequences of BamHI and NheI restriction endonuclease sites were added to the PCR primers.

[0215] (3) The target gene fragment obtained in step (2) and the pLJM1-EF1α-mCherry_P2A_Hygro vector were digested with restriction endonucleases BamHI and NheI.

[0216] (4) Use DNA ligase to ligate the target gene fragment treated in step (3) to the vector backbone to construct a recombinant plasmid.

[0217] (5) Transform the recombinant plasmid into a host cell (such as Escherichia coli) and identify plasmids with correct sequences (i.e., 100% identity with the corresponding sequence shown in Example 1) by enzyme digestion and Sanger sequencing.

[0218] (6) Select DH5α strain to prepare a large number of plasmids screened in step (5).

[0219] Example 3: Validation of the function of selected ion channels in HEK cells

[0220] (1) HEK293 cells, as a commonly used heterologous expression system, have advantages such as ease of culture, high transfection efficiency, and low background current, making them an ideal model for studying ion channel function. A transient transfection vector containing genes encoding the 17 selected ion channels shown in Example 1 (such as the Kir2.3 gene) and the EKC gene was introduced into HEK293 cells to study the cellular electrophysiological changes after overexpression of the 17 selected ion channels, thereby conducting preliminary screening for selected potassium ion channels.

[0221] (2) Since the opening or closing of potassium ion channels on the cell membrane can generate measurable current changes, the inventors chose to use whole-cell patch clamp recording. After 24 to 72 hours of ion channel introduction, the current changes of HEK293 cells under different voltage conditions were detected in voltage clamp mode. At the same time, HEK293 cells expressing fluorescent protein mCherry were used as negative control group, and HEK293 cells overexpressing EKC gene were used as positive control group to plot current-voltage (IV) curves.

[0222] (3) By recording and analyzing the current changes measured in step (2), the inventors can assess the functional status of selected ion channels in HEK293 cells and analyze the open state, potassium current magnitude, voltage gating characteristics and inward rectification characteristics of potassium ion channels after different gene introductions.

[0223] Figure 3 shows the different action potential current changes after overexpression of some of the 17 potassium ion channels in Example 1. Each overexpressed potassium ion channel in Figure 3 was analyzed individually and in a negative control group. Screening was conducted based on a decrease in resting potential and / or the presence of significant inward rectification, resulting in four potassium ion channels: Kir2.1, Kir2.3, Kir3.2, and Kir4.1, which were selected for further research.

[0224] The IV curves of the four selected potassium ion channels, the positive control EKC gene alone, and the negative control group are shown in Figures 4a-e. EKC opens at approximately -30 mV, exhibiting typical electrophysiological characteristics of a voltage-gated potassium channel. When Kir2.1 and Kir2.3 channels are open, their potassium currents are significantly higher than those of the control group, while their resting potentials are lower than those of the negative control group. Furthermore, with increasing clamping voltage, Kir2.1 and Kir2.3 exhibit significant inward rectification characteristics. Figure 4d shows that the expression of Kir3.2 alone in HEK293 cells did not lead to a significant difference in its current-voltage curve compared to the control group. Figure 4e indicates that although Kir4.1 expression in HEK293 cells produces a certain potassium current, no significant inward rectification phenomenon was observed.

[0225] Example 4: Inhibitory effect of ion channels on neuronal firing in the CA1 region of the hippocampus

[0226] To further investigate the inhibitory effects of EKC and Kir channels on neuronal over-discharge, this study used stereotactic localization to inject AAVs (such as AAV9::CamkII-EKC, AAV9::CamkII-Kir2.1, AAV9::CamkII-Kir2.3, AAV9::CamkII-Kir3.2, and AAV9::CamkII-Kir4.1) carrying four potassium ion channels selected in Example 3 and the positive control EKC into the CA1 region of the mouse hippocampus.

[0227] In this application, the experimental group underwent unilateral injection of AAV carrying the four potassium ion channels and the EKC gene selected in Example 3 into the CA1 brain region of the hippocampus; the control group underwent injection of AAV9::CamkII-mcherry into the same brain region.

[0228] The specific experimental steps are as follows.

[0229] 1. After anesthetizing the mouse, remove the hair from the surgical area using a razor. Secure the shaved mouse to the stereotaxic apparatus. To secure it, first clamp the mouse's incisors onto the adapter's incisor clamp, gently press down on the clamp's crossbar, cut open the head skin, and apply erythromycin ointment to the eyes. Adjust the adapter's height and position to allow the ear rod to easily enter the mouse's external auditory canal.

[0230] 2. After securing the mouse, use a skull drill to drill two small holes in the mouse's cerebellum. Stop drilling when the holes are almost completely punctured, then use a screwdriver to tighten the skull screws. Tighten the screws halfway, leaving space to wrap around the screws.

[0231] 3. Adjust the skull level using a positioning pin. The standard for level is that Bregma and Lambda have the same ML(X) and DV(Z), and X is arbitrarily chosen. When X = -X', Z = Z' (the deviation is less than 0.1mm).

[0232] 4. Carefully use a skull drill to gently abrade the skull at the injection site, gradually thinning it. When a crack appears in the skull, carefully puncture it with the needle of a medical syringe to prevent damage. If bleeding occurs during this process, use a small medical cotton ball stretched into a long strip to absorb the blood. Care must be taken when drilling; otherwise, the drill bit may accidentally enter the brain tissue after penetrating the skull, causing damage. Because electrodes need to be implanted in this experiment, the following settings were used: hip: AP: -2mm, ML: -1.5mm, DV: -1.5mm, with openings on both sides.

[0233] 5. Fill the glass electrode with mineral oil, insert a nanoliter, and aspirate a volume larger than the injection volume. Place the needle above the drilled hole, with the needle tip parallel to the skull (Z=0). Fine-tune the syringe position to match the drilling position, and slowly lower the needle to the predetermined depth. Inject 400 nL of 4×10^12 vg / ml AAV into the right side at a rate of 40 nL / min, retain for 5 minutes, and then remove the needle.

[0234] Two weeks after viral injection, mice were anesthetized with sodium pentobarbital, and their brains were removed and sliced ​​into 200 μm thick sections using a Leica vibratory slicer for electrophysiological recording. In the electrophysiological experiments, target neurons were located using fluorescent labeling. Membrane capacitance and membrane resistance were recorded in the "membrane test" mode, and then the current clamp mode was switched to record the resting potential of neurons and the number of action potentials generated under different intensities of current stimulation.

[0235] The experimental results are shown in Figures 5a-f. Figure 5a shows that the firing frequency of action potentials in neurons of the CA1 region of the mouse hippocampus gradually increased with the increase of stimulation current. Compared with neurons in the control group that were not infected with AAV, Kir2.3 showed the most significant inhibitory effect on action potential firing in AAV-infected neurons; Kir2.1, Kir3.2, and EKC also effectively inhibited action potential firing in the stimulation range of 100-300 pA, while the inhibitory effect of Kir4.1 was relatively weak.

[0236] Figure 5b compares the number of maximum action potentials generated under different current stimulation conditions. The results show that the number of maximum action potentials in the CA1 neurons of the hippocampus of mice infected with Kir2.3, Kir2.1 and Kir3.2 is significantly reduced, while there is no significant difference between neurons infected with EKC and Kir4.1 and the control group.

[0237] Figure 5c shows the minimum current stimulation (i.e., threshold current) required to induce action potentials. The results showed that the threshold current of neurons infected with Kir2.3, Kir2.1, and Kir3.2 was significantly increased, while the threshold current of neurons infected with EKC and Kir4.1 was not statistically different from that of the control group.

[0238] Figure 5d compares the changes in resting potential of neurons infected with different ion channels. It was found that infection with Kir2.3, Kir2.1 and Kir3.2 significantly reduced the resting potential of neurons, while infection with EKC and Kir4.1 had no significant effect on the resting potential.

[0239] Figure 5e analyzes the effect of infection with different potassium ion channels on neuronal membrane resistance. Membrane resistance affects neuronal excitability. Higher membrane resistance means that a smaller current can cause a larger change in membrane potential, making neurons more easily excitable; lower membrane resistance has the opposite effect. The results show that the membrane resistance of neurons infected with Kir2.3 is significantly reduced, while other ion channels have no significant effect on membrane resistance. Therefore, Kir2.3 can inhibit neuronal excitability by reducing neuronal membrane resistance.

[0240] Figure 5f shows that the membrane capacitance of neurons did not change significantly after infection with different ion channels, indicating that AAV infection had no significant effect on the membrane surface area of ​​neurons in the CA1 region of the hippocampus.

[0241] In summary, overexpression of potassium channels Kir2.3, Kir2.1, and Kir3.2 significantly affected neuronal firing in the mouse hippocampus in vitro, while the inhibitory effects of other potassium channels were not significant compared to the control group. Kir2.3 exhibited strong inhibitory effects on all observed indicators. Kir2.3 effectively inhibited neuronal firing in multiple dimensions by suppressing action potential firing, reducing the number of action potentials, increasing the action potential threshold current, lowering the resting potential of neurons, and reducing neuronal membrane resistance. Therefore, future research will primarily focus on in vivo epilepsy treatment using Kir2.3.

[0242] Example 5: In vivo verification of the therapeutic effect of rAAV vector on epilepsy.

[0243] Using stereotactic localization technology, the constructed rAAV carrier is precisely injected into the epileptic focus and / or epileptogenic focus of the subject; the recombinant AAV carrier expresses the potassium ion channel in the neurons of the epileptic focus and / or epileptogenic focus, thereby promoting neuronal repolarization and / or hyperpolarization by increasing potassium ion outflow and / or enhancing inward rectification, inhibiting abnormal neuronal discharge, and achieving the purpose of treating epilepsy.

[0244] This application utilizes kainic acid (KA) to construct a mouse model of focal epilepsy in the hippocampus. KA is an effective agonist of excitatory amino acid receptor subtypes in the central nervous system. In this application, the experimental group underwent unilateral injection of AAVs carrying Kir2.3 and EKC (such as AAV9::CamkII-Kir2.3 or AAV9::CamkII-EKC) into the CA1 region of the hippocampus; the control group received AAV9::CamkII-mcherry at the same brain region.

[0245] The specific experimental steps are as follows.

[0246] 1. Take two silver wires and two tungsten wires, and burn off the insulation layer at one end. Solder them to the nutplate. Take a plastic board of appropriate size, drill holes, pass the nutplate through the plastic board, and fix it with hot melt glue to seal the exposed wires and spaces, ensuring there is no gap between the electrode and the plastic board, and minimizing the electrode size as much as possible. Then, solder the drug delivery tube to the right side of the electrode with hot melt glue, ensuring the tip of the drug delivery tube is close to but does not touch the right electrode. Use a multimeter to check if the electrode circuit is continuous and short-circuited. If short-circuited, remove the short-circuited tungsten or silver wire and resolder it. Continue to test the circuit condition after soldering. If the contact is good, it is ready for use.

[0247] 2. After anesthetizing the mouse, remove the hair from the surgical area using a razor. Secure the shaved mouse to the stereotaxic apparatus. To secure it, first clamp the mouse's incisors onto the adapter's incisor clamps, gently press down on the clamp's crossbar, cut open the head skin, and apply erythromycin ointment to the eyes. Adjust the adapter's height and position to allow the ear rod to easily enter the mouse's external auditory canal.

[0248] 3. After securing the mouse, use a skull drill to drill two small holes in the mouse's cerebellum. Stop drilling when the holes are almost completely punctured, then use a screwdriver to screw on the skull screws. Tighten the screws halfway, leaving space to wrap around them.

[0249] 4. Adjust the skull level using a positioning pin. The standard for level is that Bregma and Lambda have the same ML(X) and DV(Z), and X is arbitrarily chosen. When X = -X', Z = Z' (the deviation is less than 0.1mm).

[0250] 5. Carefully use a skull drill to gently abrade the skull at the injection site, gradually thinning it. When a crack appears in the skull, carefully puncture it with the needle of a medical syringe to prevent damage. If bleeding occurs during this process, use a small medical cotton ball stretched into a long strip to absorb the blood. It is crucial to control the drill carefully during drilling; otherwise, the drill bit may accidentally enter the brain tissue after penetrating the skull, causing damage. Because this experiment requires electrode implantation, the following settings were used: hip: AP: -2mm, ML: -1.5mm, DV: -1.5mm, with openings on both sides.

[0251] 6. Fill the glass electrode with mineral oil, insert a nanoliter, and aspirate a volume larger than the injection volume of virus. Place the needle above the drilled hole, with the needle tip parallel to the skull (Z=0). Fine-tune the syringe position to match the drilling position, and slowly lower the needle to the predetermined depth. Inject AAV doses of AAV9::CamkII-mcherry 1.8E9vg (mcherry), AAV9::CamkII-EKC 1.8E9vg (EKC), and AAV9::CamkII-Kir2.3 6E8vg and 1.8E9vg (kir2.3) at a rate of 40 nL / min, and retain for 5 minutes.

[0252] 7. After removing the nanoliter, use a cranial drill to grind the skull surface to create an uneven surface, making it easier for the dental cement to adhere better. At this point, implant the tested electrode 1.5mm in diameter.

[0253] 8. After wrapping the silver wire around the skull nail several times, tighten the remaining half-turn with a screwdriver. Prepare the dental cement in the exact amount needed to connect the electrode to the mouse skull. After applying, wait approximately 20-30 minutes for it to solidify before removing it. Removing it too early will result in the electrode becoming unstable.

[0254] 9. The electrodes and cannulas of mice are prone to colliding, causing the cannulas to fall off and block the drug delivery port. Therefore, mice should be kept in cages of 1-2 at a time to prevent damage to the cannulas.

[0255] 10. Seven days after AAV injection, draw an appropriate amount of 1 mg / mL KA, remove the mouse cannula cap, insert the administration tube, and inject 300 nL at a rate of 0.5 μL / min. Leave the needle in place for 3 minutes. Remove the administration tube, replace the cannula cap, and begin recording.

[0256] 11. During recording, immediately after connecting the EEG markers to the mouse electrodes, the mouse's EEG activity was recorded for 3 hours using the "Sirenia Acquisition" software. The collected data were statistically analyzed using the "Sirenia Seizure" software.

[0257] 12. According to the Racine classification: Grade 1: Mouth and facial tics. Grade 2: Head nodding. Grade 3: Forelimb clonic seizures. Grade 4: Forelimb clonic seizures with orthostatic movement. Grade 5: Orthostatic movement with falling and forelimb clonic seizures. Grade 6: Jumping, complete tonic-clonic seizures. Racine grades 4–6 are defined as generalized seizures (GS).

[0258] The experimental results are shown in Figures 6a-c. Figure 6a shows that 6E8vg, 1.8E9vg Kir2.3, and 1.8E9vg EKC significantly reduced the number of seizures in mice with gastrointestinal tract (GS). Comparison of different Kir2.3 concentrations shows that the inhibitory effect of Kir2.3 overexpression on epilepsy is dose-dependent, with higher concentrations of AAV showing stronger inhibitory effects. Figure 6b shows that 6E8vg, 1.8E9vg Kir2.3, and EKC reduced the racine grade of seizures compared to the control group. Figure 6c shows that the experimental groups of 6E8vg, 1.8E9vg Kir2.3, and 1.8E9vg EKC increased the latency of seizures in mice compared to the control group.

[0259] Therefore, it can be seen that overexpression of Kir2.3 has a therapeutic effect on epilepsy in mice, and the therapeutic effect is better than that of the positive control EKC at the same dose.

[0260] Example 6: Pharmacodynamic Validation in a Mouse Model of Chronic Epilepsy

[0261] A mouse model of chronic epilepsy was established using stereotactic injection of kainic acid (KA). The specific steps are as follows:

[0262] (1) Mice were anesthetized with isoflurane by inhalation and fixed in a stereotaxic apparatus. The skull was exposed and adjusted to a horizontal position (Bregma and Lambda were in the same plane on the ML and DV axes). The hippocampus was used as the target point, and the positioning coordinates were: AP-2.0mm, ML-1.5mm, DV-1.5mm (relative to Bregma).

[0263] (2) After drilling at the target location, slowly inject 300 nL KA (1 mg / mL) into the right hippocampus using a nano-level injection system at a rate of 50 nL / min. After injection, retain the needle for 10 minutes to prevent reflux, then slowly withdraw the needle and suture the scalp.

[0264] (3) Mice were placed in individual cages for observation after surgery. One hour after the onset of a continuous epileptic seizure, 0.3 mL of avertin was injected intraperitoneally to terminate the seizure and reduce the risk of death caused by status epilepticus. Mice were fed normally after recovery.

[0265] (4) One month after KA injection, mice were screened for chronic epilepsy models, and their free-movement behavior was recorded continuously for 48 hours. Mice that exhibited spontaneous epileptic seizures during the monitoring period were considered to have successfully established a chronic epilepsy model; mice that did not show epileptic seizures were considered to have failed to establish a model.

[0266] (5) Mice with successful modeling were further subjected to bilateral stereotactic injections of AAV9::CamKIIα-Kir2.3 or an equal volume of solvent as a control in the hippocampus. The bilateral hippocampal injection sites are as follows (relative to Bregma):

[0267] ①AP-1.5mm, ML±1.5mm, DV-2.0mm;

[0268] ②AP-2.5mm, ML±2.0mm, DV-2.0mm;

[0269] ③AP-3.1mm, ML±3.0mm, DV-3.0mm.

[0270] (6) Inject 300 nL of viral solution (4 × 10¹² vg / mL) or solvent into each site at a rate of 50 nL / min. After injection, leave the needle in place for 10 min, then slowly withdraw the needle and suture.

[0271] (7) Behavioral monitoring began 14 days after AAV injection. Free movement behavior was recorded for 7 consecutive days. The number of spontaneous epileptic seizures in mice during the monitoring period was counted to assess the effect of Kir2.3-mediated neuronal inhibition on chronic epileptic seizures.

[0272] The experimental results are shown in Figure 7. Compared with the solvent group, the Kir2.3 group mice had a significantly lower number of seizures during the monitoring period, indicating that Kir2.3 overexpression can significantly reduce the frequency of spontaneous seizures in KA-induced chronic epilepsy mice. In Figure 7, the bar chart represents the mean ± SEM, and the scatter plot represents data from a single mouse.

[0273] Example 7: Effects of rAAV vector injection on recognition and memory abilities in mice

[0274] The experimental animals used in this study were the aforementioned KA-induced chronic epilepsy model mice. All mice were first used to establish a chronic epilepsy model via stereotactic injection of KA, and animals that successfully established the model were screened through continuous behavioral monitoring one month after injection. Only mice that experienced spontaneous seizures during the monitoring period were included for subsequent experiments. Mice that successfully established the model then received bilateral hippocampal stereotactic injections of AAV9::CamKIIα-Kir2.3 or an equal volume of solvent as a control. After AAV injection was completed and expression stabilized (14 days after injection), mice underwent seizure monitoring and behavioral assessment. The Novel Object Recognition (NOR) experiment was conducted in the aforementioned chronic epilepsy model mice to evaluate the effect of Kir2.3 expression on learning and memory-related behaviors. All behavioral experiments were performed on the same batch of animals to minimize the interference of individual differences on the results.

[0275] (1) Before the experiment, the experimental animals were given acclimatization treatment to reduce stress response. Specifically, this included: the experimental personnel handling and touching the animals for short periods of time every day for one week before the experiment; and the animals were transferred to the experimental environment for several consecutive days before the experiment to acclimatize to the environment.

[0276] (2) The experiment was conducted in a closed open field device, such as a cubic box with a side length of about 45cm. The experimental process included three stages: adaptation period, familiarization period, and recognition period.

[0277] (3) During the habituation phase, each experimental animal is placed in an experimental box without any objects and allowed to move freely for about 10 minutes to adapt to the experimental environment.

[0278] (4) During the familiarization phase, two identical objects are placed in the experimental chamber, positioned diagonally opposite each other and approximately 7 cm away from the side wall of the chamber. The experimental animal is then allowed to move freely in the chamber for about 10 minutes to allow it to fully come into contact with and become familiar with the two identical objects.

[0279] (5) During the identification phase (test phase), after a predetermined time interval (e.g., 4 hours) following the familiarization phase, one of the familiar objects is replaced with a new object that is significantly different in shape or color, while the other experimental conditions remain unchanged. The experimental animal is then placed back into the experimental chamber to move freely for about 10 minutes, and its exploratory behavior is recorded.

[0280] (6) The entire experiment was recorded using a video recording system. Exploratory behavior was defined as the behavior of experimental animals approaching an object with their heads and engaging in sniffing or touching actions. Based on the video analysis system, the exploratory behaviors of experimental animals with new and familiar objects during the recognition period were quantitatively analyzed. The main indicators included the exploration time of experimental animals with new and familiar objects.

[0281] (7) Based on the above data, calculate behavioral parameters to characterize the experimental animals' preference for new objects, such as the proportion of exploration time for new objects to total exploration time (novel object preference index), or the difference in exploration time between new objects and familiar objects. Statistical analysis of the behavioral parameters obtained from different experimental groups is used to compare the differences in recognition and memory abilities of experimental animals under different treatment conditions.

[0282] The experimental results are shown in Figure 8. Compared with the solvent group and the control C57 group, the Kir2.3 group mice showed no significant difference in their exploration preference for new objects, indicating that Kir2.3 expression did not cause significant impairment of learning and memory function under the experimental conditions. This demonstrates that overexpression of Kir2.3 in the hippocampus of mice does not affect their recognition and memory abilities. Data are presented as mean ± SEM, with scatter points representing individual animals.

[0283] The sequence of the pAAV-CaMKIIα-mCherry plasmid is as follows (SEQ ID NO:19):

[0284] The first and sixth bolded underlined sections are ITR.

[0285] The second bolded underlined part is the CamkII promoter.

[0286] The third bolded and underlined part is mCherry

[0287] The fourth bolded underlined part is WPRE.

[0288] The fifth bolded underlined part is poly A.

[0289] The sequence of the plasmid Pljm1-EF1α_mCherry_P2A_Hygro is as follows (SEQ ID NO:20):

[0290] The first and sixth bolded underlined parts represent LTR.

[0291] The second bolded underlined part is the EF1α promoter.

[0292] The third bolded and underlined part is mcherry

[0293] The fourth underlined part is P2A.

[0294] The fifth bolded and underlined part is the hygromycin resistance gene.

[0295] The sequence of the pAAV-CamKII-kir2.3 plasmid is as follows (SEQ ID NO:21):

Claims

1. A recombinant adeno-associated virus vector (rAAV vector) for inhibiting neuronal overexcitation, comprising a nucleotide sequence encoding a potassium ion channel protein.

2. The rAAV carrier according to claim 1, 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); preferably, the potassium ion channel is selected from one or more of Kv and / or Kir.

3. The rAAV carrier according to claim 1 or 2, wherein the potassium ion channel is selected from one or more Kirs.

4. The rAAV carrier according to claim 3, 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 selected from one or more of Kir2.x and / or Kir3.x.

5. The rAAV carrier according to any one of claims 1-4, 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.

6. The rAAV carrier according to any one of claims 1-5, 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.

7. The rAAV carrier according to any one of claims 1-6, wherein the potassium ion channel is selected from one or more of the following: (1) Kir1.1, as shown in SEQ ID NO:22; (2) Kir2.1, as shown in SEQ ID NO:23; (3) Kir3.1, as shown in SEQ ID NO:24; (4) Kir2.3, as shown in SEQ ID NO:25; (5) Kir3.4, as shown in SEQ ID NO:26; (6) Kir3.2, as shown in SEQ ID NO:27; (7) Kir6.1, as shown in SEQ ID NO:28; (8) Kir3.3 as shown in SEQ ID NO:29; (9) Kir4.1 as shown in SEQ ID NO:30; and (10) Kir6.2, as shown in SEQ ID NO:

31.

8. The rAAV vector according to any one of claims 1-7, wherein the nucleotide sequences encoding the potassium ion channel are as shown in SEQ ID NOs:3-12, or have at least 80% sequence identity with the nucleotide sequences shown in SEQ ID NOs:3-12.

9. The rAAV carrier according to any one of claims 1-8, wherein the potassium ion channel is selected from one or more of the following: Kir2.1, Kir2.3, Kir3.2 and Kir4.1, preferably selected from human Kir2.1, human Kir2.3, human Kir3.2 and human Kir4.

1.

10. The rAAV carrier according to any one of claims 1-9, wherein the potassium ion channel is Kir2.1, Kir2.3 and / or Kir3.2, preferably human Kir2.1, human Kir2.3 and / or human Kir3.

2.

11. The rAAV carrier according to any one of claims 1-10, wherein the potassium ion channel is Kir2.3, preferably human Kir2.3, and more preferably human Kir2.3 as shown in SEQ ID NO:

25.

12. The rAAV vector according to claim 11, wherein the nucleotide sequence encoding Kir2.3 is as shown in SEQ ID NO:6, or has at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:

6.

13. The rAAV vector according to any one of claims 1-12, comprising between inverted terminal repeats (ITRs): (1) Promoter; (2) The nucleotide sequence encoding the potassium ion channel; (3) polyA sequence in, The promoter and the nucleotide sequence are operatively linked.

14. The rAAV vector according to claim 13, further comprising a marmot hepatitis virus posttranscriptional regulatory element (WPRE) sequence between the inverted terminal repeat (ITR) sequences.

15. The rAAV vector according to claim 13 or 14, wherein the promoter is selected from one or more of the following: constitutive promoters, inducible promoters, activity-regulating promoters, and tissue-specific promoters, preferably activity-regulating promoters and / or tissue-specific promoters.

16. The rAAV vector according to any one of claims 13-15, wherein the promoter is an activity-regulating promoter, said activity-regulating promoter being selected from one or more of the following: immediate early gene (IEG) promoters or synthetic activity-dependent promoters; The IEG bootstrap is selected from one or more of the following: c-fos bootstrap, CREB bootstrap, SRE bootstrap, Srf bootstrap, Egr1 bootstrap, Arc bootstrap, mArc bootstrap, Homer1a bootstrap, Bdnf bootstrap, Mef2 bootstrap, Fosb bootstrap, Npas4 bootstrap, AP1 bootstrap; The activity-dependent promoter is selected from one or more of the following: PRAM (Promoter Robust Activity Marker), Npas4-specific robust active marker promoter (NRAM), fos-specific robust active marker (FRAM), enhanced synaptic active response element (ESARE), EpiPro promoter; The preferred activity regulator is the c-fos promoter.

17. The rAAV vector according to any one of claims 13-16, wherein the promoter is a tissue-specific promoter, said tissue-specific promoter being selected from one or more of the following: neuron-specific promoter, glial cell-specific promoter, heart-specific promoter, muscle-specific promoter, retina-specific promoter, preferably a neuron-specific promoter.

18. The rAAV vector according to claim 15 or 17, wherein the tissue-specific promoter is a neuron-specific promoter, said neuron-specific promoter being selected from one or more of the following: human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase II (CaMKII) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, microtubule α1 promoter, neuron-specific enolase (NSE) promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, etc. The promoters include Nav1.7, Nav1.8, Nav1.9, Advilin, 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, and promoters without a distal homeobox (Dlx), preferably CaMKII promoter and / or CaMKIIα promoter.

19. A viral particle comprising the rAAV vector according to any one of claims 1-18, wherein the rAAV vector is located within an AAV capsid.

20. The viral particle according to claim 19, wherein the AAV capsid is derived from an AAV serotype that is tropism-dependent on nerve cells.

21. The viral particle according to claim 19 or 20, wherein the AAV capsid is derived from one or more of the following serotypes: AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAVDJ, AAV-PHP.N, AAV-PHP.eB, and AAV-PHP.B and their variants.

22. A pharmaceutical composition comprising: (a) the rAAV vector according to any one of claims 1-18 or the viral particle according to any one of claims 19-21; and, (b) Pharmaceutically acceptable carriers.

23. Use of the rAAV vector according to any one of claims 1-18, the viral particle according to any one of claims 19-21, or the pharmaceutical composition according to claim 22 in the preparation of a medicament for treating diseases related to neuronal hyperexcitability.

24. The use according to claim 23, wherein the neuronal hyperexcitability-related disease is selected from one or more of the following: epilepsy, Parkinson's disease, pain, obsessive-compulsive disorder, drug addiction, paroxysmal dyskinesia, unipolar or bipolar affective disorder, anxiety and phobia, preferably epilepsy, pain, obsessive-compulsive disorder and / or drug addiction.

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