Gene therapy vector for treating drug-refractory epilepsy and application thereof

Abstract

The invention relates to a gene therapy vector for treating drug-refractory epilepsy and application of the gene therapy vector. Specifically, based on the new discovery that the expression level of A1R is obviously reduced in ANT brain region excitatory neurons of a chronic spontaneous epilepsy mouse model, the invention constructs a recombinant adeno-associated virus vector AAV2 / 9-CamKIIA-A1R-P2A-GFP on the basis of a pAV-CamKIIA-P2A-GFP vector for the first time. Wherein the CamKIIA promoter is only activated in excitatory neurons, so that the specific expression of a target gene is ensured; the P2A self-cleaving peptide sequence ensures the independence of respective functions of the target gene and the reporter gene. The vector has the advantages of high specificity, visual expression efficiency, safety, reliability, optimized packaging capacity and the like. A gene therapy scheme based on the vector has the technical advantages of strong targeting, low invasiveness, lasting treatment effect, clear mechanism, high safety and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular, relates to a gene therapy vector for treating drug-resistant epilepsy and application thereof. BACKGROUND

[0002] Epilepsy is a chronic neurological disease characterized by repeated and transient central nervous system dysfunction caused by abnormal discharges of neurons in the brain. Patients who do not respond to existing antiepileptic drugs are defined as drug-resistant epilepsy (DRE). DRE patients not only have poor disease control, but also long-term use of multiple antiepileptic drugs may cause cognitive dysfunction, liver damage and other serious adverse reactions, which seriously affect the quality of life of patients.

[0003] The anterior nucleus of thalamus (ANT) is an important part of the limbic system, which forms a complex neural circuit with the hippocampus and cingulate gyrus, and is involved in emotion regulation and memory consolidation. Existing studies have confirmed that the ANT brain region is a key node of the Papez loop, which plays a "relay station" role in the initiation and propagation of seizures, and promotes the propagation of abnormal discharges between different brain regions. Clinical research data shows that deep brain stimulation targeting the ANT brain region (ANT-DBS) for DRE treatment can significantly reduce seizure frequency and improve quality of life. However, DBS treatment as an invasive surgery has the risk of intracranial hemorrhage, infection and other surgical complications, and the cost of device implantation and long-term maintenance is high, which limits its widespread clinical application.

[0004] Adenosine (Ado) is an endogenous purine nucleoside that is widely distributed in the central nervous system and plays an important role in neural regulation. Adenosine regulates neuronal excitability, synaptic transmission efficiency and regional cerebral blood flow by binding to specific receptors on the cell membrane, thereby producing multiple biological effects such as sedation, anticonvulsant and neuroprotection. Among them, adenosine A1 receptor (A1R) as a member of the G protein-coupled receptor family is mainly distributed in the excitatory neurons of the central nervous system, and its activation can open potassium channels and inhibit calcium influx, thereby reducing neuronal excitability and effectively inhibiting abnormal discharge activity.

[0005] In recent years, adeno-associated virus (AAV) vectors have shown great application prospects in the field of gene therapy for neurological diseases due to their low immunogenicity, long-term expression, and multiple serum type options. Combined with neuron-specific promoters (such as CaMKIIA, Syn1, etc.), precise expression of exogenous genes in specific neuronal subgroups can minimize the risk of adverse reactions caused by non-specific expression.

[0006] Currently, the main intervention strategies for treating drug-refractory epilepsy include the combination of traditional antiepileptic drugs, neural modulation techniques (such as deep brain stimulation, vagus nerve stimulation, etc.), and surgical resection. However, these methods have the following obvious defects: (1) The effect of multi-drug combination therapy is limited, and may cause serious adverse reactions and drug interactions; (2) Although ANT-DBS is effective, it is highly invasive, has surgical risks, is costly, and requires long-term maintenance; (3) Surgical resection has limited indications and may cause permanent damage to patient's cognitive and language functions.

[0007] It is particularly important to note that there is no specific intervention strategy in the prior art that can successfully target neuron cells A1R. Although preclinical studies have shown that A1R agonists have good antiepileptic effects, systemic administration of A1R agonists can cause bradycardia, hypotension, and other serious cardiovascular system adverse reactions due to the wide expression of A1R in multiple tissues and organs throughout the body, limiting its clinical application. In addition, existing A1R modulating drugs cannot achieve targeted effects in specific regions of the brain or specific neuronal subgroups, lacking the necessary targeting and specificity.

[0008] Therefore, there is an urgent need in the art to develop a new gene therapy strategy that can precisely target excitatory neurons in the ANT brain region and specifically upregulate the expression level of A1R, thereby effectively treating drug-refractory epilepsy, in order to achieve efficient, low-invasive, long-acting, and safe control of epilepsy and meet the urgent needs of clinical treatment.

[0009] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present application, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0010] The present application aims to provide an adeno-associated virus vector (AAV)-based gene therapy system for treating drug-refractory epilepsy (DRE). This system specifically targets excitatory neurons to upregulate the expression of adenosine A1 receptor (A1R), thereby inhibiting the abnormal firing of epilepsy-related neurons and effectively controlling seizures.

[0011] In a first aspect of the present invention, there is provided a use of an A1R coding sequence or an A1R gene expression upregulator for preparing a drug for reducing neuronal excitability or for preparing a drug for treating epilepsy.

[0012] In another preferred embodiment, the amino acid sequence of A1R is shown in Seq ID No.1.

[0013] In another preferred embodiment, the A1R coding sequence is shown as Seq ID No.2.

[0014] In a second aspect of the present invention, an expression cassette is provided, wherein the expression cassette contains an A1R coding sequence.

[0015] In another preferred embodiment, the expression cassette has a structure of formula I from the 5'-3' end:

[0016] Z0-Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0017] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0018] Z0 is none or 5′ITR sequence;

[0019] Z1 is the promoter;

[0020] Z2 is the A1R coding sequence (e.g., the full-length coding sequence of the ADORA1 gene);

[0021] Z3 is a self-cleaving peptide;

[0022] Z4 is a reporter gene;

[0023] Z5 is absent or a transcription terminator;

[0024] Z6 is absent or has a 3' ITR sequence.

[0025] In another preferred embodiment, the promoter is an excitatory neuron-specific promoter.

[0026] In another preferred embodiment, the promoter is selected from the following group: CamkIIA promoter, Syn1 promoter, Vglut2 promoter.

[0027] In another preferred embodiment, the A1R coding sequence is shown as Seq ID No.2.

[0028] In another preferred embodiment, the full-length coding sequence of the ADORA1 gene is shown as Seq ID No.2.

[0029] In another preferred embodiment, the self-cleaving peptide is selected from the following group: P2A self-cleaving peptide, T2A self-cleaving peptide, E2A self-cleaving peptide, F2A self-cleaving peptide.

[0030] In another preferred embodiment, the reporter gene is selected from the group consisting of GFP reporter gene, mCherry reporter gene, EYFP reporter gene.

[0031] In another preferred embodiment, the transcription terminator is selected from the group consisting of SV40 PolyA.

[0032] In another preferred embodiment, the expression cassette has the structure of Formula II from 5'-3' end:

[0033] CamkIIA-ADORA1-P2A-GFP (II).

[0034] In a third aspect of the present application, there is provided a vector, which carries or contains an A1R coding sequence or a coding sequence that facilitates A1R expression or the expression cassette of the second aspect of the present application.

[0035] In another preferred embodiment, the vector is an expression vector.

[0036] In another preferred embodiment, the vector is a gene therapy vector that can target upregulate A1R expression.

[0037] In another preferred embodiment, the vector comprises a viral vector or a non-viral vector.

[0038] In another preferred embodiment, the viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0039] In another preferred embodiment, the A1R coding sequence is as set forth in Seq ID No. 1.

[0040] In another preferred embodiment, the upregulation of A1R expression is in a neural cell (neuron).

[0041] In another preferred embodiment, the vector contains the expression cassette of the second aspect of the present application.

[0042] In another preferred embodiment, the vector contains the expression cassette CamkIIA-ADORA1-P2A-GFP (II).

[0043] In another preferred embodiment, the vector is an AAV2 / 9-CamKIIA-A1R-P2A-GFP vector.

[0044] In a fourth aspect of the present application, there is provided a host cell, which contains the vector of the third aspect of the present application or has integrated in its chromosome the expression cassette of the second aspect of the present application.

[0045] In another preferred embodiment, the host cell is selected from the group consisting of a neural cell, a human embryonic kidney cell.

[0046] In a fifth aspect of the present application, there is provided use of the expression cassette of the second aspect of the present application or the vector of the third aspect of the present application, for the manufacture of a composition or medicament for: (a) preventing and / or treating epilepsy; and / or (b) reducing neuronal excitability.

[0047] In another preferred embodiment, the epilepsy is drug-resistant epilepsy.

[0048] In a sixth aspect of the present application, there is provided a pharmaceutical preparation comprising (a) the expression cassette of the second aspect of the present application or the vector of the third aspect of the present application, and (b) a pharmaceutically acceptable carrier or excipient.

[0049] In another preferred embodiment, the vector is selected from the group consisting of:

[0050] (i) a lentiviral vector targeting up-regulation of A1R expression;

[0051] (ii) an adeno-associated viral vector targeting up-regulation of A1R expression;

[0052] (iii) a retroviral vector targeting up-regulation of A1R expression;

[0053] (iv) any combination of (i) to (iii) above.

[0054] In a seventh aspect of the present application, there is provided a pharmaceutical composition comprising:

[0055] (a) the pharmaceutical preparation of the sixth aspect of the present application; and (b) another biologically active agent.

[0056] In another preferred embodiment, the other biologically active agent is an agent for treating epilepsy.

[0057] In another preferred embodiment, the agent for treating epilepsy is selected from the group consisting of sodium valproate, levetiracetam, topiramate, and the like.

[0058] In an eighth aspect of the present application, there is provided use of A1R or its gene for the manufacture of a reagent or kit for detecting epilepsy.

[0059] In another preferred embodiment, the kit comprises a reagent for quantitatively detecting A1R protein or mRNA, and a corresponding label or instruction.

[0060] In a ninth aspect of the present application, a kit for detecting epilepsy is provided, which comprises a container containing a detection reagent for detecting A1R protein or mRNA; and a label or instruction indicating that the kit is used for detecting epilepsy.

[0061] In a tenth aspect of the present application, a method for reducing neuronal excitability in vitro is provided, which comprises the step of culturing neuronal cells in the presence of the vector or A1R gene expression up-regulator of the third aspect of the present application, thereby reducing neuronal excitability.

[0062] In another preferred embodiment, the neurons are central nervous system excitatory neurons.

[0063] It should be understood that the general description above and the detailed description below are merely exemplary and explanatory, and are not restrictive of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0064] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the application. It is to be understood that the drawings are only schematic and that they do not necessarily represent a limiting embodiment of the application. For the purpose of clarity, not every component can be shown in each drawing.

[0065] Figure 1 Vector plasmid for pAAV-CamKIIa-P2A-GFP in the recombinant adenovirus of the present application.

[0066] Figure 2 Virus titer detection in the recombinant adenovirus of the present application.

[0067] Figure 3 Fluorescent brain section images and overexpression efficiency after stereotactic microinjection of the recombinant adenovirus into the brain of mice.

[0068] Figure 4 EEG / EMG monitoring of seizure after stereotactic microinjection of the recombinant adenovirus into the brain of KA-induced epilepsy model mice.

[0069] Figure 5 Overexpression of A1R in chronic epilepsy mouse models and its effect on seizures.

[0070] Figure 6 Detection of A1R expression in the brain tissue of epilepsy mice and cell localization analysis. DETAILED DESCRIPTION

[0071] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be embodied in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the example embodiments to those skilled in the art.

[0072] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have in the art, unless explicitly stated to the contrary or clearly apparent from the context in which the terms and phrases are used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described.

[0073] The inventors have found through extensive and in-depth research that in the chronic spontaneous epilepsy mouse model ( Figure 6 A) In the excitatory neurons of the ANT brain region, there was a significant downregulation of A1R expression ( Figure 6 B, 6C). Further studies confirmed that the AAV-shRNA strategy, driven by the CaMKIIα promoter, specifically knocked down A1R expression in excitatory neurons in the hippocampal CA1 region or the anterior thalamic nucleus (ANT) ( Figure 5 A). Immunofluorescence staining results showed that A1R expression in Vglut2-positive neurons in the A1R knockout group was significantly reduced compared with the control group ( Figure 5 B), Quantitative analysis confirmed that the number of Adora1-positive neurons in each Vglut2-positive neuron was significantly decreased (P=0.021, Figure 5 C). In vivo EEG recording results showed that A1R knockdown in both the hippocampal CA1 region and the anterior thalamic nucleus could induce spontaneous epileptiform discharges ( Figure 5 D). Further analysis of epilepsy parameters showed that A1R knockdown in the two brain regions had no significant differences in multiple indicators such as epileptic seizure frequency and duration ( Figure 5 E), suggesting that A1R downregulation can effectively induce epileptic seizures in different brain regions. At the same time, the inventors confirmed through systematic research that activation of the adenosine-A1R signaling pathway in the ANT brain region is the core molecular mechanism for ANT-DBS to exert its anti-epileptic effect ( Figure 6 D. Figure 6 E. Figure 6 F) These findings suggest that gene therapy, by specifically upregulating A1R expression in excitatory neurons in the ANT brain region, may achieve similar or even superior anti-epileptic effects as ANT-DBS while avoiding the risks of complications associated with invasive surgery.

[0074] The application belongs to the technical field of biological medicine, and particularly relates to a recombinant adeno-associated virus with neuron cell-specific overexpression of adenosine A1 receptor (ADORA1, referred to as A1R) and application thereof in treatment of drug-resistant epilepsy (DRE).

[0075] In the application, a recombinant adeno-associated virus (AAV2 / 9-CamKIIA-A1R-P2A-GFP) vector is constructed based on a pAV-CamKIIA-P2A-GFP vector for the first time, and the vector comprises the following core components: ① target gene: human adenosine A1 receptor (A1R) gene sequence (reference sequence number: NC_000067.7); ② specific promoter: excitatory neuron-specific promoter CamKIIA, which ensures that the target gene is expressed only in excitatory neurons; ③ P2A self-cleavage peptide: located between the target gene and the marker gene, which realizes the synergistic expression and functional independence of the two proteins; ④ marker gene: green fluorescent protein (GFP) gene sequence, which is used for in vivo tracing and transfection efficiency evaluation; and ⑤ vector skeleton: adeno-associated virus type 2 / 9 (AAV2 / 9) skeleton, which has high neuron affinity and good transduction efficiency.

[0076] Specifically, the recombinant adeno-associated virus vector (AAV2 / 9-CamKIIA-A1R-P2A-GFP) has the following technical advantages: (1) high specificity: the CamkIIA promoter ensures that ADORA1 is expressed only in excitatory neurons, reducing non-specific effects; (2) expression efficiency visualization: the P2A-GFP system realizes the co-expression of ADORA1 and GFP, which facilitates intuitive evaluation of transduction efficiency; (3) safe and reliable: AAV does not integrate the host genome, has long-term expression and high safety, and is suitable for gene therapy of the nervous system; (4) optimized packaging capacity: the vector design takes into account the AAV packaging limit, ensuring efficient virus production. Based on this, the application is completed.

[0077] The gene operation tool system combines the precise targeting ability of the CamkIIA promoter with the high-efficiency transduction ability of the AAV vector, providing an ideal research platform for the application of ADORA1 in the treatment of drug-resistant epilepsy. The verification results show that the system has excellent neuron targeting and significant antiepileptic effect, and shows broad clinical conversion prospects.

[0078] Terminology

[0079] As used in the present application, the terms "containing", "including" or "having" include "comprising", "consisting essentially of", "consisting substantially of" and "consisting of"; "consisting essentially of", "consisting substantially of" and "consisting of" are sub-concepts of "containing", "having" or "including".

[0080] As used herein, "about," "approximately," "around," or "substantially" shall generally mean an acceptable range of variability from plus to minus ten percent (20%), preferably from plus to minus ten percent (10%), and more preferably from plus to minus five percent (5%) of a stated value or range. Numerical values used herein are approximations that represent a reasonable amount of experimental error for the measurement techniques used.

[0081] Gene (A1R) sequence

[0082] In the present application, A1R is adenosine A1 receptor ADORA1.

[0083] The target gene used in the present application is human adenosine A1 receptor gene (ADORA1) with reference sequence number NC_000067.7. The gene product is adenosine A1 receptor protein, which is a member of G protein-coupled receptor superfamily, and can play a role in inhibiting neuronal excitability by inhibiting adenylyl cyclase, reducing intracellular cyclic adenosine monophosphate (cAMP) concentration, and reducing the release of excitatory neurotransmitter glutamate. In the present application, the A1R gene is designed to be inserted between the AsiSI and MluI enzyme cutting sites of the vector, downstream of the CamKIIA promoter sequence, to ensure efficient expression in excitatory neurons.

[0084] A1R coding sequence

[0085] In the present application, "A1R coding sequence" refers to a nucleotide sequence encoding A1R polypeptide / protein (such as the amino acid sequence shown in Seq ID No. 1), which includes the nucleotide sequence shown in Seq ID No. 2 and its degenerate variants. As used herein, "degenerate variants" in the present application refer to nucleotide sequences that encode polypeptides / proteins (such as the amino acid sequence shown in Seq ID No. 1) with the same amino acid sequence as the polypeptides / proteins of the present application, but differ from the nucleotide sequence shown in Seq ID No. 2 (differences conform to codon degeneracy).

[0086] In the present application, "ADORA1 gene full-length coding sequence" and "A1R coding sequence", "complete coding sequence of A1R gene" have the same meaning and can be used interchangeably.

[0087] In the present application, a typical A1R coding sequence is shown in Seq ID No. 2.

[0088] Cell-specific promoter

[0089] Under the control of this promoter, the expression of the exogenous gene is specific to certain cells. The CamkIIA promoter used in the present application can specifically target excitatory neurons.

[0090] Specifically, the application uses CamKII alpha promoter to drive A1R gene expression, ensuring that the gene is only expressed in excitatory neurons without affecting the normal function of inhibitory neurons. This design has the following advantages: ① precise targeting: avoids expression in non-target cells, reducing non-specific effects; ② efficient utilization: overcomes the waste of resources caused by non-specific expression mediated by constitutive promoters; ③ safe and controllable: reduces the risk of systemic adverse reactions and improves treatment safety.

[0091] In addition to the CamkIIA promoter, the application can also use Syn1 promoter and Vglut2 promoter as excitatory neuron-specific promoters.

[0092] Recombinant adeno-associated virus (rAAV)

[0093] The AAV vector modified by genetic engineering technology deletes the Rep and Cap gene coding sequences, and only retains the ITR sequence at both ends as a packaging signal. rAAV does not integrate with the host genome after infecting cells, effectively avoiding the risk of cancer.

[0094] Expression vector

[0095] The recombinant plasmid pAV-CamKIIA-A1R-P2A-GFP constructed by the application has a circular structure with a total length of about 5350 bp, and contains the following functional elements: ① plasmid backbone: pUC backbone, containing a bacterial replication origin and an ampicillin resistance gene (Amp), for amplification and selection of the plasmid in bacteria; ② excitatory neuron-specific promoter: CamKIIA promoter sequence is selected, which is only activated in excitatory neurons, ensuring specific expression of the target gene; ③ target gene sequence: ADORA1 gene sequence is inserted into the AsiSI / MluI enzyme cutting site of the plasmid, which is located between the CamKIIA promoter and the P2A sequence; ④ P2A self-cleavage peptide sequence: located between the target gene and the reporter gene, allowing the two proteins to automatically separate after translation, ensuring the independence of their respective functions; ⑤ reporter gene: GFP gene sequence, for real-time monitoring of transfection efficiency and localization of expression site; ⑥ SV40 PolyA tail: viral-derived polyadenylate signal sequence, for improving mRNA stability and enhancing gene expression efficiency; ⑦ ITR sequence (inverted terminal repeat sequence): located on both sides of the expression cassette, ensuring the integrity of the target gene during virus packaging and infection, and is a necessary element for AAV virus genome replication and packaging.

[0096] The application selects an adeno-associated virus 2 / 9 type (AAV2 / 9) as a gene delivery carrier, and has the following significant advantages: ① high safety: AAV does not have replication ability and pathogenicity, and is not integrated into the host genome, and the safety is significantly higher than that of retrovirus and adenovirus; ② high-efficiency transduction: AAV2 / 9 serotype has high affinity for neurons and can efficiently transduce central nervous system cells; ③ long-term expression: AAV-mediated expression of exogenous genes can last for several months to several years, realizing long-acting treatment; ④ low immunogenicity: The immune response caused by AAV is significantly lower than that of other viral vectors, and is suitable for central nervous system application.

[0097] In the application, "pAV-CamkIIA-ADORA1-P2A-GFP recombinant plasmid", "recombinant plasmid pAV-CamKIIA-A1R-P2A-GFP", "AAV2 / 9-CamKIIA-A1R-P2A-GFP vector", "pAV-CamKIIA-A1R-P2A-GFP recombinant plasmid", "AAV-CamKIIa-A1R-P2A-GFP vector" have the same meaning and can be used interchangeably.

[0098] Live monitoring system

[0099] In the virus vector of the application, the GFP fluorescent protein is co-expressed with the target gene through the P2A peptide sequence, and the transfected positive cells can be directly observed by fluorescence microscopy, which is beneficial to: ① evaluating the transfection efficiency: judging the expression level of the target gene by the fluorescence intensity; ② locating the expression area: accurately determining the range of brain regions infected by the virus; ③ studying the transfection persistence: long-term monitoring of the stability of transgene expression; ④ the use of P2A self-cleavage peptide ensures the synergistic expression and functional independence of A1R and GFP, compared with the fusion protein strategy, which does not affect the membrane localization and functional activity of A1R.

[0100] The main advantages of the application include:

[0101] Based on the above findings, the application provides a new gene therapy strategy, which realizes effective treatment of drug-refractory epilepsy by specifically up-regulating the expression level of A1R in excitatory neurons in the ANT brain area. The application has the following advantages compared with the prior art:

[0102] (1) Strong targeting: through the combination of AAV vector and neuron-specific promoter, precise expression of A1R gene in excitatory neurons in the ANT brain area is realized, and systemic adverse reactions caused by systemic administration are avoided;

[0103] (2) Low invasiveness: compared with ANT-DBS surgery, the application only needs one stereotactic injection to achieve long-term therapeutic effect, greatly reducing the risk of surgery and the pain of patients;

[0104] (3) Treatment effect is long-lasting: AAV vector-mediated expression of exogenous genes can last for several months to several years, avoiding the problem of frequent administration of conventional drug therapy;

[0105] (4) Mechanism is clear: The present application interferes with the key molecular target A1R of epilepsy onset, the mechanism is clear, and it shares the core molecular pathway with ANT-DBS, and the efficacy is predictable;

[0106] (5) High safety: The endogenous inhibitory signal pathway is used to regulate neuronal activity, avoiding the toxic side effects of exogenous drugs.

[0107] The present application will be further described below in conjunction with specific examples.

[0108] It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified in detail, which are usually carried out according to the conventional conditions, such as the conditions described in Sambrook. J et al. Molecular Cloning: A Laboratory Manual (Huang Peitang et al. Transl, Beijing: Science Press, 2002) or the conditions recommended by the manufacturer (for example, product instructions). Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples are commercially available or can be prepared according to the literature method unless otherwise specified.

[0109] Materials and methods

[0110] 1. Vector construction and verification

[0111] a. Preparation of starting vector:

[0112] The present application uses pAV-CamKIIA-P2A-GFP vector (Addgene, #118276) as a backbone vector, which has a total length of about 5350 bp, containing AAV inverted terminal repeat (ITR), calmodulin kinase II alpha (CamKII alpha) promoter, P2A self-cleavage peptide sequence, green fluorescent protein (GFP) reporter gene, SV40 polyadenylation signal sequence, and ampicillin resistance gene and pUC replication origin required for bacterial replication.

[0113] b. Preparation of target gene:

[0114] Human adenosine A1 receptor (A1R) gene acquisition: The complete coding sequence of A1R gene (Seq ID No. 2) is obtained from human genomic DNA or cDNA library. The A1R gene fragment is obtained by PCR amplification technology. The upstream primer is designed to contain the AsiSI enzyme cutting site, and the downstream primer is designed to contain the MluI enzyme cutting site.

[0115] PCR reaction condition: initial denaturation 95°C for 5 min, cycling reaction (95°C for 30 s, 58-62°C for 30 s, 72°C for 1-2 min) x 30 cycles, final extension 72°C for 10 min. The PCR product was purified by agarose gel electrophoresis.

[0116] c. Vector enzyme treatment:

[0117] Vector linearization: pAV-CamKIIA-P2A-GFP vector was double digested with AsiSI and MluI; enzyme reaction system: vector DNA 2-5 μg, AsiSI 10-20 U, MluI 10-20 U, NEB CutSmart Buffer (10x) 2 μL, BSA (100x) 0.2 μL, ddH2O to a total volume of 20 μL. Reaction at 37°C for 2-4 hours; linearized vector was separated and purified by agarose gel electrophoresis, and the cut fragments were removed.

[0118] d. Target gene enzyme treatment

[0119] The PCR amplified A1R gene fragment was double digested with AsiSI / MluI, and the enzyme digestion conditions were the same as the vector enzyme treatment: PCR product 1-3 μg, AsiSI 5-10 U, MluI 5-10 U, NEB CutSmart Buffer (10x) 2 μL, BSA (100x) 0.2 μL, ddH2O to 20 μL, 37°C for 2-4 hours, 65°C heat inactivation for 20 minutes, agarose gel electrophoresis purification of target fragment.

[0120] e. Ligation reaction

[0121] The digested A1R gene fragment was ligated with the linearized vector using T4 DNA ligase. Ligation reaction system: vector: insert molar ratio 1:3-1:5, T4 DNA ligase 5-10 U, ligation buffer, T4 DNA ligase buffer (10x) 2 μL, PEG-4000 (50%) 1 μL (optional, to improve ligation efficiency), ddH2O to 20 μL, 16°C overnight or room temperature for 2-4 hours. The ligation product was transformed into competent E. coli DH5a cells (Thermo, 18265017).

[0122] f. Clone screening and verification

[0123] Positive clone screening: positive transformants were screened on LB agar plates containing ampicillin (100 μg / mL), and single colonies were picked for preliminary verification by colony PCR using primers spanning the insertion site (Seq ID No. 3, Seq ID No. 4).

[0124] Restriction endonuclease analysis and verification: Extract plasmid DNA from positive clones and perform single and double enzyme digestion analysis. Use AsiSI and MluI to verify the insertion site. Use other enzyme digestion sites for map analysis to confirm the integrity of the vector structure. Analyze the digestion products by agarose gel electrophoresis and compare them with the theoretical expected fragment size.

[0125] DNA sequencing verification: Full-length sequencing was performed on positive clones verified by enzyme digestion, focusing on verifying the integrity and correctness of the A1R gene sequence, confirming that the promoter, P2A sequence, GFP gene, and other functional elements had not mutated, and verifying the correctness of the insertion direction and reading frame.

[0126] g. Final product confirmation

[0127] The successfully constructed pAV-CamKIIA-A1R-P2A-GFP recombinant plasmid is expected to have a total length of approximately 6-7 kb. This recombinant plasmid can simultaneously express A1R and GFP under the drive of the CamKIIα promoter, and achieve equimolar expression of the two proteins through the P2A self-cleaving peptide.

[0128] 2. Virus Preparation

[0129] High-titer AAV2 / 9-CamKIIA-A1R-P2A-GFP virus was prepared in HEK293T cells using a three-plasmid co-transfection system: ① packaging plasmid (pUCmini-iCAP-PHP.eB, Addgene plasmid, #103005, PMID: 28671695): provides AAV2 / 9 type viral capsid protein; ② helper plasmid (pAdDeltaF6, Addgene plasmid, #112867): provides auxiliary factors required for viral replication; ③ transfer plasmid: pAV-CamKIIA-A1R-P2A-GFP constructed in the present invention, containing the target gene expression cassette protected by the ITR sequence.

[0130] The virus was purified by polyethylene glycol precipitation and density gradient centrifugation, and finally a high titer (5.4×10 12 vg / mL) of recombinant AAV virus preparation.

[0131] 3. Clinical application process

[0132] The specific clinical application steps of the present invention are as follows: ① Targeted delivery: Through stereotactic surgery, 300nL of viral solution is precisely injected into the anterior nucleus of the thalamus of the patient; ② Expression window: 2-3 weeks after injection is the gene expression window period, during which the target gene begins to express and function; ③ Effect evaluation: The therapeutic effect is evaluated through methods such as electroencephalogram / electromyogram (EEG / EMG) monitoring, epileptic seizure frequency recording, and behavioral observation.

[0133] 4. Therapeutic effect

[0134] The technical solution of the present application can realize precise treatment of drug-refractory epilepsy. The mechanism is to up-regulate the expression of A1R in the excitatory neurons of the anterior nucleus of the thalamus region, regulate neurotransmitter release and ion channel function, and then inhibit abnormal discharge activity, thereby fundamentally relieving seizures. The results are shown in Figure 3 and Figure 4 To verify the effectiveness of AAV vector-mediated A1R overexpression and its anti-epileptic effect, the inventors conducted multi-level experimental verification. Histological analysis showed that fluorescence microscope detection of GFP signal confirmed the transduction region and efficiency of the vector Figure 3 A), and the results showed that the AAV-CamKIIa-A1R-P2A-GFP vector was successfully transduced into the target brain region ANT, and the GFP positive cells were mainly distributed in the ANT subregion (AD and AV region) at the injection site, with good transduction efficiency and good regional specificity of vector expression. Molecular biology analysis verified the overexpression level of A1R by qPCR and Western blot Figure 3 B), and the results showed that compared with the control group, the ADORA1 expression level of the A1R overexpression group was significantly increased (P<0.001), and the relative expression amount was about 30 times that of the control group, confirming the high-efficiency overexpression of A1R gene in the target brain region mediated by AAV vector. Further electrophysiological evaluation monitored various indicators of seizures by in vivo electrophysiological recording Figure 4 B), and the results showed that compared with the control group, the A1R overexpression group was significantly improved in multiple epilepsy-related indicators: the seizure frequency was significantly reduced (P=0.033), the total duration of epilepsy per day was significantly shortened (P=0.004), the duration of single seizure was significantly reduced (P=0.007), and the frequency of epileptiform discharge was significantly decreased (P<0.001). These results collectively confirmed that A1R overexpression in the ANT region has a significant anti-epileptic effect and can effectively inhibit spontaneous seizures in the KA-induced chronic epilepsy model.

[0135] Compared with traditional treatment methods, the present application has the following significant advantages: ①Based on clear mechanism: targeting key neural regulation pathways with clear mechanism of action; ②Precise targeting: specifically acting on excitatory neurons in the epileptogenic brain region; ③Low invasiveness: one injection can achieve long-term therapeutic effect, avoiding repeated surgery; ④Visual monitoring: real-time monitoring of gene expression is achieved through the P2A-GFP system; ⑤High safety: using endogenous signaling pathway regulation with fewer adverse reactions.

[0136] Example 1

[0137] 1. ADORA1 overexpression vector design

[0138] ADORA1 gene overexpression system was designed based on pAV-CamkIIA-P2A-GFP vector. pAV-CamkIIA-P2A-GFP vector (5350 bp) is the basic vector of this study, which contains the following key elements: ① ITR sequences at both ends: as AAV packaging signal; ② CamkIIA promoter: specifically targeting excitatory neurons; ③ P2A self-cleavage peptide: achieving multi-protein co-expression; ④ GFP reporter gene: for monitoring transduction efficiency; ⑤ SV40 PolyA: providing transcription termination signal; ⑥ pUC and Amp: for bacterial amplification and selection; ⑦ multiple restriction enzyme sites: AsiSI, AscI, RsrII and MluI, etc.

[0139] This vector has the following advantages: ① CamkIIA promoter ensures specific and efficient expression in excitatory neurons; ② P2A sequence can achieve equimolar co-expression of ADORA1 and GFP; ③ GFP label facilitates real-time monitoring of transduction efficiency; ④ the composition of vector elements is simple, meeting the AAV packaging capacity limit.

[0140] 2. Precise construction of recombinant AAV vector

[0141] Vector preparation: Use high-fidelity restriction enzymes (such as AsiSI / AscI combination) to accurately cut pAV-CamkIIA-P2A-GFP vector at specific sites.

[0142] Gene insertion: The full-length coding sequence of ADORA1 gene (specific sequence Seq ID No. 2) amplified and purified by PCR is connected with the vector through the same enzyme cutting site to form pAV-CamkIIA-ADORA1-P2A-GFP recombinant plasmid.

[0143] Cloning verification: Through colony PCR preliminary screening, restriction enzyme analysis and sequencing, it is confirmed that ADORA1 gene has been correctly inserted and the sequence is complete and accurate.

[0144] 3. Adeno-associated virus production and purification

[0145] Co-transfection: For the packaging of AAV2 / 9-CamKIIA-A1R-P2A-GFP vector, a three-plasmid co-transfection system is required. The verified pAV-CamkIIA-ADORA1-P2A-GFP vector is co-transfected with the AAV packaging plasmid (pUCmini-iCAP-PHP.eB, Addgene plasmid, #103005, PMID: 28671695) and the auxiliary plasmid (pAdDeltaF6, Addgene plasmid, #112867). The co-transfection ratio uses a 1:1:1 molar ratio (normalized according to plasmid size) or a 2:1.6:1 weight ratio (auxiliary plasmid: packaging plasmid: transfer plasmid), and the total plasmid DNA dosage is 0.5 μg / 10 6 For live cells, the ratio of PEI transfection reagent to DNA was 1:2. HEK293 cells were selected from the ATCC CRL-1573 standard strain (less than 30 passages) (ATCC, CRL-1573), cultured at 37°C, 5% CO2, and transfected when the cells reached 80-90% confluence.

[0146] Virus collection: 48-72 hours after transfection, cells and culture medium were collected and the virus particles were released by cell lysis.

[0147] Purification process: AAV vector purification adopts a comprehensive process of differential centrifugation, iodide hexadecane density gradient ultracentrifugation and affinity chromatography. First, differential centrifugation is performed to separate cells and culture medium at 3000×g for 15 minutes, and the virus is released by three freeze-thaw cycles using a lysis buffer containing 150mM NaCl, 50mM Tris-HCl pH 8.5, and 2mM MgCl2. The supernatant is then collected by centrifugation at 3000×g for 20 minutes and treated with DNase (Thermo, EN0525) at 37°C for 30 minutes. Next, fine purification is performed by iodide hexadecane density gradient ultracentrifugation, and OptiPrep TMSigma-Aldrich D1556) were prepared with gradient from bottom to top: 60% Ioxaglate layer 3.5 mL, 40% Ioxaglate layer 6 mL, 25% Ioxaglate layer 5 mL, 15% Ioxaglate layer 5.5 mL (with 1 M NaCl), buffer used 10x PBS-MK. Centrifugation was performed using Beckman Coulter Optima L-90K ultracentrifuge equipped with JS-5.3 rotor at 58,400 x g, 18°C for 2-3 hours in QuickSeal centrifuge tubes, followed by puncturing the interface at 40-60% with 18 ga needle to collect the fractions. As an alternative, further purification can be performed using Thermo Fisher Scientific POROS CaptureSelect AAVX affinity resin (A36740) with a binding capacity > 1 x 10 13 copies / mL, operating flow rate 1 mL / min, eluted with 0.1 M glycine pH 3.0. Finally, ultrafiltration concentration was performed by Amicon Ultra-15 centrifugal filters (100 kDa), removing Ioxaglate repeated 3 times, adding glycerol to 10%, sterile filtered and stored at -80°C, obtaining a final laboratory grade AAV particles > 5 x 10 12 vg of high purity.

[0148] 4. Quality control and functional validation

[0149] Titer determination: Viral genome copies were accurately determined using real-time quantitative PCR to confirm viral titer.

[0150] In vitro validation: Viral transduction efficiency and ADORA1 expression levels were validated in primary neuronal cultures or neuronal cell lines.

[0151] Direct observation of GFP fluorescence confirmed successful transduction Figure 3 A).

[0152] Western blot and qPCR to detect ADORA1 expression levels Figure 3 B).

[0153] Functional test to assess A1 receptor activity.

[0154] 5. Animal model application

[0155] Virus preparation: AAV-ADORA1-P2A-GFP was taken out from -80°C freezer and thawed on ice.

[0156] Stereotactic injection: Using brain stereotactic technology, the virus (300 uL) was accurately injected into the right anterior thalamic nucleus region (ML-1.0 mm, AP-0.85 mm, DV-3.0 mm) of the DRE mouse model. Figure 4 A). 2 weeks ago, WT mice hippocampus (C57BL / 6J, Strain NO. N000013, Jiangsu Jiqie Yekang Biotechnology Co., Ltd.) CA1 region (ML-2.2 mm, AP-1.6 mm, DV-1.4 mm) was first injected with 0.20 ug / kg KA (kainic acid, sodium alginate) to form a DRE mouse model.

[0157] 6. Multidimensional evaluation system

[0158] Histological analysis: Fluorescence microscopy detected GFP signal to confirm the transduction region and efficiency, and the results are shown in Figure 3 A. The results showed that AAV-CamKIIa-A1R-GFP vector was successfully transduced into the target brain region ANT, and GFP positive cells were mainly distributed in the ANT subregion (AD and AV region) at the injection site, with good transduction efficiency and good regional specificity of vector expression.

[0159] Molecular biology analysis: qPCR and Western blot verified the ADORA1 overexpression level, and the results are shown in Figure 3 B. The results showed that compared with the control group, the ADORA1 expression level of the A1R overexpression group was significantly increased (P<0.001), and the relative expression amount was more than 30 times that of the control group, confirming the high-efficiency overexpression of ADORA1 gene in the target brain region mediated by AAV vector.

[0160] Electrophysiological evaluation: In vivo electrophysiological recording monitored the frequency, intensity and duration of seizures, and the results are shown in Figure 4 B. The results showed that compared with the control group, the A1R overexpression group significantly improved in multiple epilepsy-related indicators: (1) the frequency of seizures was significantly reduced (P=0.033); (2) the total duration of seizures per day was significantly shortened (P=0.004); (3) the duration of single seizure was significantly reduced (P=0.007); (4) the frequency of epileptiform discharges was significantly decreased (P<0.001). These results confirmed that ADORA1 overexpression in the ANT region had a significant antiepileptic effect, which could effectively inhibit spontaneous seizures in the KA-induced chronic epilepsy model.

[0161] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the application being indicated by the following claims.

[0162] Sequence information related to the present application:

[0163] Amino acid sequence of A1R Seq ID No. 1

[0164] MPPYISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCFIVSLAVADVAVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILALLAIAVDRYLRVKIPLRYKTVVTQRRAAVAIAGCWILSLVVGLTPMFGWNNLSEVEQAWIANGSVGEPVIKCEFEKVISMEYMVYFNFFVWVLPPLLLMVLIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALILFLFALSWLPLHILNCITLFCPTCQKPSILIYIAIFLTHGNSAMNPIVYAFRIHKFRVTFLKIWNDHFRCQPKPPIEEDIPEEKADD

[0165] Nucleotide sequence encoding A1R Seq ID No. 2

[0166] ATGCCGCCGTACATCTCGGCCTTCCAGGCTGCCTACATTGGCATCGAGGTGCTCATTGCCTTGGTCTCTGTGCCCGGAAATGTACTGGTGATTTGGGCTGTGAAGGTGAACCAGGCACTTCGCGATGCTACCTTCTGCTTCATCGTATCCCTGGCGGTAGCTGATGTGGCTGTTGGCGCCCTGGTCATCCCACTGGCCATCCTTATCAACATTGGGCCACAGACCTACTTCCACACCTGCCTCATGGTGGCCTGTCCTGTCCTCATCCTCACCCAGAGCTCCATCCTGGCTCTGCTTGCTATTGCTGTGGATCGGTACCTCCGAGTCAAGATCCCTCTCCGGTACAAGACAGTGGTGACTCAGCGGCGGGCAGCAGTGGCCATAGCCGGCTGCTGGATTCTCTCCCTTGTGGTAGGCCTGACACCCATGTTTGGCTGGAACAACCTGAGTGAGGTGGAACAAGCCTGGATAGCCAACGGCAGTGTTGGGGAGCCCGTGATCAAGTGTGAGTTCGAGAAGGTTATCAGCATGGAGTACATGGTCTACTTCAACTTCTTCGTCTGGGTGCTGCCGCCATTGCTCCTCATGGTTCTTATCTACCTGGAGGTCTTCTACCTGATCCGCAAGCAGCTCAACAAAAAGGTGTCAGCCTCCTCTGGTGACCCCCAGAAGTACTACGGGAAGGAGCTCAAGATCGCCAAGTCGCTGGCCCTCATCCTCTTCCTCTTTGCCCTCAGCTGGCTACCGCTACACATCTTGAACTGCATCACCCTCTTCTGCCCCACCTGCCAGAAACCCAGCATCCTCATCTACATTGCCATCTTCCTCACACACGGCAACTCGGCCATGAACCCCATCGTCTATGCCTTCCGAATCCACAAGTTCCGGGTCACCTTTCTGAAGATTTGGAACGACCACTTCCGATGCCAGCCCAAGCCCCCCATTGAGGAAGATATCCCAGAGGAGAAAGCTGATGACTAG

[0167] A1 R forward primer Seq ID No. 3: ATCCTGGCTCTGCTTGCTATTGC

[0168] A1 R reverse primer Seq ID No. 4: CGCTGAGTCACCACTGTCTTGTAC.

Claims

1. A use of an A1R coding sequence or an A1R gene expression upregulator, characterized in that: It is used to prepare drugs that reduce neuronal excitability or to prepare drugs for treating epilepsy.

2. An expression cassette, characterized in that The expression cassette contains the A1R coding sequence.

3. The expression cassette according to claim 2, wherein The expression cassette has a structure of formula I from the 5' to 3' end: Z0-Z1-Z2-Z3-Z4-Z5-Z6(I) In the formula, each "-" is independently a bond or a nucleotide linking sequence; Z0 is none or 5′ITR sequence; Z1 is the promoter; Z2 is the A1R coding sequence; Z3 is a self-cleaving peptide; Z4 is a reporter gene; Z5 is absent or a transcription terminator; Z6 is absent or has a 3' ITR sequence.

4. The expression cassette according to claim 2, wherein The expression cassette has a structure of Formula II from the 5' to 3' end: CamkIIA-ADORA1-P2A-GFP(II).

5. A carrier, characterized in that The vector carries or contains an A1R coding sequence or a coding sequence that promotes A1R expression or the expression cassette according to claim 2.

6. A host cell, characterized in that The host cell contains the vector according to claim 5, or the expression cassette according to claim 2 is integrated into its chromosome.

7. Use of the expression cassette according to claim 2 or the vector according to claim 5, characterized in that, Used for preparing a composition or preparation for: (a) preventing and / or treating epilepsy; and / or (b) reducing neuronal excitability.

8. A pharmaceutical preparation, characterized in that The formulation contains (a) the expression cassette of claim 2 or the vector of claim 5, and (b) a pharmaceutically acceptable carrier or excipient.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the pharmaceutical preparation according to claim 8; and (b) other biologically active drugs.

10. A use of A1R or its gene, characterized in that: Used for preparing a reagent or kit for detecting epilepsy.

11. A kit for detecting epilepsy, characterized in that: The kit comprises a container containing a detection reagent for detecting A1R protein or mRNA; and a label or instructions indicating that the kit is used for detecting epilepsy.

12. An in vitro non-therapeutic method for reducing neuronal excitability, characterized in that: The method comprises the steps of culturing neuronal cells in the presence of the vector or the A1R gene expression upregulator according to claim 5, thereby reducing neuronal excitability.

Images