Gene construct and use thereof

Abstract

The present invention relates to a gene construct and use thereof. The gene construct is used for inhibiting the excessive excitation of neurons caused by excessive use or abuse of addictive substances. The gene construct comprises a neuronal inhibitory protein-coding gene and a promoter operably linked to the neuronal inhibitory protein-coding gene. The gene construct exhibits significant advantages in terms of precise targeting, closed-loop regulation, retention of natural reward responses, reduction of side effects, and improvement of safety, as well as potential long-acting properties and sustainability. This strategy provides a novel idea and method for the treatment of drug use disorders, and is expected to become an important breakthrough in the field of drug use disorders in the future.

Description

Gene constructs and their uses

[0001] Cross-disciplinary applications

[0002] This application claims priority to international application PCT / CN2025 / 071659, filed on January 10, 2025, the contents of which are incorporated herein by reference in their entirety.

[0003] Related sequence list

[0004] The sequence list submitted with this application is incorporated herein by reference. The sequence list is an XML file entitled "TFJ01379PCT Sequence Listing.XML", created on January 8, 2026, and is 66,167 bytes in size. Technical Field

[0005] This application relates to the field of gene technology, and in particular to a gene construct and its uses. Background Technology

[0006] Substance Use Disorders (SUDs) are defined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) as a group of disorders resulting from the excessive use or abuse of a legal or illegal substance. These substances may include alcohol, prescription drugs (such as opioids), illicit drugs (such as marijuana and cocaine), and synthetic drugs. Key characteristics of substance use disorders include compulsive seeking and use of the substance, loss of control over the intake of the substance, and negative emotional and behavioral responses when the substance is unavailable.

[0007] The United Nations' 2023 World Drug Report vividly reveals the rampant spread of SUD: In 2021, more than 296 million people worldwide engaged in drug use, and the number of people with SUD surged to 39.5 million, a 45% increase compared to 10 years ago. Behind these alarming figures lies the predicament of treatment.

[0008] The substances that trigger SUD are numerous and highly complex, including opioids such as heroin, fentanyl, and morphine. These substances bind tightly to opioid receptors, powerfully interfering with the pain transmission and neurotransmitter release in the central nervous system, and are highly addictive while providing pain relief. Central nervous system stimulants, such as methamphetamine and cocaine, can strongly activate the brain's reward circuits, causing a large and disordered release of neurotransmitters such as dopamine, making users feel excited in a short time, but causing irreversible damage to brain nerve cells. There are also substances that are common in daily life, such as nicotine and alcohol, but which have hidden addiction risks. Long-term and excessive use gradually erodes the nervous system and changes the normal function and metabolic patterns of the brain.

[0009] Substance use disorders not only impair an individual's physical health but also lead to serious psychological and social problems. Long-term substance abuse can cause biological consequences such as immunosuppression, dysregulation of the self-reward and punishment mechanisms, and neurohormonal deficiencies, increasing the risk of mental illnesses such as major depressive disorder and anxiety / stress-related disorders. Furthermore, substance abuse can lead to the spread of infectious diseases (such as HIV / AIDS and hepatitis), damage family relationships, cause public safety problems, and impose a significant burden on the socioeconomic system.

[0010] Faced with the severe situation of drug abuse, drug rehabilitation strategies and drug treatment methods are constantly being innovated and developed. Existing treatment options include drug replacement therapy, psychotherapy, and drug intervention. (1) Drug replacement therapy, as the mainstay of the opioid addiction treatment system, plays an indispensable key role. Among them, buprenorphine and methadone have gained a foothold in the treatment of opioid addiction due to their specific chemical structures and pharmacological properties. Both belong to the category of opioid receptor agonists, with buprenorphine being a partial agonist and methadone being a complete agonist. From the perspective of mechanism of action, they can bind specifically to μ-opioid receptors in the central nervous system with high affinity, mimicking the biological effects of endogenous opioid peptides, and accurately and effectively activating downstream related signaling pathways. In this way, the imbalance of the neurotransmitter system in the brain can be gradually regulated, such as the release and metabolism of key neurotransmitters like dopamine and γ-aminobutyric acid tend to stabilize, thereby significantly alleviating a series of severe physiological reactions caused by opioid withdrawal, including muscle tremors, cold sweats, anxiety and irritability, creating a relatively stable internal environment for the body, helping patients to smoothly pass through the acute withdrawal period and gradually get rid of physiological dependence on addictive drugs. These drugs may effectively reduce the addictive behavior of patients in the early stage of treatment, but patients are often prone to relapse after stopping the drug, and it is difficult to maintain the treatment effect in the long term. Moreover, these drugs may also be accompanied by a series of side effects, such as gastrointestinal discomfort, nervous system reactions, and cardiovascular system changes. These side effects not only affect the patient's quality of life, but may also increase the risks and difficulties in the treatment process. (2) Another type of drug, naltrexone, has a completely different mechanism of action and clinical positioning. Naltrexone is an opioid receptor antagonist, which mainly acts on the key nodes of relapse prevention and control in addiction treatment. With its superior affinity for μ-opioid receptors, naltrexone can rapidly and potently bind to these receptors, forming a stable complex that competitively blocks the interaction between exogenous opioids and the receptors. Thus, even if a patient inadvertently re-experiences opioids, the receptors are now occupied by naltrexone, preventing the drugs from exerting their effects and providing no euphoria. This effectively reduces cravings and dependence on the addictive drugs psychologically, lowering the risk of relapse. Although naltrexone has shown promising results in relapse prevention, it is not immune to side effects, with gastrointestinal discomfort and cardiovascular stress occurring frequently, hindering its clinical application.

[0011] Furthermore, different types of drugs have different mechanisms of addiction. Currently used detoxification drugs target opioid GPCR receptors. These antagonists or partial agonists are effective in treating opioid addiction, but existing drug treatments are not very effective for addiction to cocaine, methamphetamine, and new synthetic drugs. Unlike opioid addiction, these psychoactive substances act on the brain through more complex mechanisms. They can strongly interfere with the normal transcription and translation processes of multiple key genes, leading to imbalances in the brain's reward system and neurotransmitter metabolism pathways. For example, abnormal metabolism and signal transmission of multiple neurotransmitters such as dopamine and serotonin in the central nervous system, and imbalances in the brain's reward circuit homeostasis, can induce addictive behavior.

[0012] Compared to traditional treatments, this application directly addresses the cellular biological essence of addiction, inhibiting cells that are overactivated by addiction-related signaling pathways. This fundamentally weakens the brain's pathological response to addictive substances, reshaping normal neural circuits and reward mechanisms. Unaffected by patient compliance or psychological fluctuations, the treatment cycle is short, and the effects are long-lasting and stable. It is expected to break the current stalemate in SUD treatment, bringing tangible and effective hope for recovery to hundreds of millions of addicts worldwide. It fills the current gap in the lack of effective treatments for multiple addictive substances, driving revolutionary progress in the field of SUD treatment. Summary of the Invention

[0013] Based on the aforementioned technical problems in the existing technology, this application provides a gene construct and its use. The gene construct can inhibit the over-excitation of neurons caused by the overuse or abuse of addictive substances through injection, and can thus be used for substance use disorders.

[0014] The specific technical solution of this application is as follows:

[0015] 1. A gene construct for inhibiting neuronal hyperexcitability caused by overuse or abuse of addictive substances, said gene construct comprising a neuronal inhibitory protein-coding gene and a promoter operatively linked to said neuronal inhibitory protein-coding gene.

[0016] 2. The gene construct according to claim 1, wherein the neuronal inhibitory protein encoding gene includes a potassium channel protein encoding gene and / or a DREADD encoding gene.

[0017] 3. The gene construct according to item 2, wherein the potassium ion channel protein encoding gene includes a gene encoding a voltage-gated potassium channel (Kv) family protein, an inward rectifier potassium channel (Kir) family protein, a functional fragment thereof, or a functional mutant thereof.

[0018] 4. The gene construct according to item 3, wherein the potassium ion channel protein encoding gene is selected from KCNA1 (Kv1.1), KCNA2 (Kv1.2), KCNA3 (Kv1.3), KCNA4 (Kv1.4), KCNA5 (Kv1.5), KCNA6 (Kv1.6), KCNA7 (Kv1.7), KCNA8 (Kv1.8), KCNA9 (Kv1.9), KCNA10 (Kv1.10), KCNB1 (Kv2.1), KCNB2 (Kv2.2), KCNB3 (Kv2.2). .3), KCNC1(Kv3.1), KCNC2(Kv3.2), KCNC3(Kv3.2), KCNC4(Kv3.4), KCND1(Kv4.1), KCND2(Kv4.2), KCND3(Kv4.3), KCNF1(K v5.1), KCNG1(Kv6.1), KCNG2(Kv6.2), KCNG3(Kv6.3), KCNG4(Kv6.4), KCNH1(Kv8.1), KCNH2(Kv11.1), KCNH3(Kv12.1), KCN H4(Kv12.2), KCNH5(Kv12.3), KCNH6(Kv12.4), KCNH7(Kv12.5), KCNH8(Kv12.6), KCNQ1(Kv7.1), KCNQ2(Kv7.2), KCNQ3(Kv 7.3), KCNQ4(Kv7.4), KCNQ5(Kv7.5), KCNV1(Kv9.1), KCNV2(Kv9.2), KCNJ1(Kir1.1), KCNJ2(Kir2.1), KCNJ3(Kir3.1), KCN The genes of any one or more proteins in the group consisting of KCNJ4 (Kir2.3), KCNJ5 (Kir3.4), KCNJ6 (Kir3.2), KCNJ8 (Kir6.1), KCNJ9 (Kir3.3), KCNJ10 (Kir4.1), KCNJ11 (Kir6.2), KCNJ12 (Kir2.2), KCNJ13 (Kir7.1), KCNJ14 (Kir2.4), KCNJ15 (Kir4.2), KCNJ16 (Kir5.1), their functional fragments, and their functional mutants.

[0019] 5. The gene construct according to item 2, wherein the DREADD encoding gene includes a gene encoding Rq(R165L), hM1Dq, hM5Dq, rM3Ds, hM2Di, hM4Di, hM3Dq, AlstR or KORD, a functional fragment thereof or a functional mutant thereof.

[0020] 6. The gene construct according to claim 5, wherein the gene construct is a gene construct administered in combination with its exogenous ligand, optionally, the exogenous ligands of hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160 or compound 21 (C21).

[0021] 7. The gene construct according to claim 6, wherein the gene construct is delivered prior to the administration of the exogenous ligand.

[0022] 8. The gene construct according to any one of items 1-7, wherein the promoter comprises a constitutive promoter, a neuron-specific promoter, or a neuron activity-dependent promoter.

[0023] 9. The gene construct according to item 8, wherein the constitutive promoter is selected from 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, herpes simplex virus HSV thymidine kinase promoter, H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa β member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin β-KIN, human ROSA 26 promoter, ubiquitin C promoter UBC, phosphoglycerate kinase-1PGK promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter or β-actin promoter, or any combination thereof.

[0024] 10. The gene construct according to item 8, wherein the neuron-specific promoter is selected from the human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, etc. One or a combination of the following promoters: 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, vesicle GABA transporter (VGAT) promoter, glutamate decarboxylase (GAD65) promoter, tyrosine hydroxylase (TH) promoter, and non-distal homeobox (Dlx) promoter.

[0025] 11. The gene construct according to item 10, wherein the neuron-specific promoter is the CaMKIIα promoter.

[0026] 12. The gene construct according to item 8, wherein the neuronal activity-dependent promoter is selected from c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter or synthetic activity-dependent promoter, such as one of PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), FRAM (Fos Robust Activity Marker), or any combination thereof.

[0027] 13. The gene construct according to item 12, wherein the neuronal activity-dependent promoter is a promoter that senses and responds to specific transcriptional regulatory dynamics within the neuron induced by first or repeated intake of an addictive substance.

[0028] 14. The gene construct according to any one of claims 1-13, wherein the addictive substance comprises a drug, narcotic drug, psychotropic drug or addictive compound, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants or hallucinogens, inhaled substances, more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

[0029] 15. The gene construct according to any one of claims 1-14, wherein the gene construct is delivered to the anterior cingulate cortex (ACC) and / or the nucleus accumbens (NAc).

[0030] 16. The gene construct according to items 1-15, wherein the gene construct is delivered to the anterior cingulate cortex and / or the nucleus accumbens by in situ injection.

[0031] 17. A recombinant vector comprising the gene construct described in any one of claims 1-16.

[0032] 18. The recombinant vector according to item 17, wherein the recombinant vector is a viral vector or a non-viral vector.

[0033] 19. The recombinant vector according to item 18, wherein the viral vector is selected from adeno-associated virus, adenovirus, retrovirus, lentivirus, vaccinia virus, herpes simplex virus and bacteriophage, preferably adeno-associated virus;

[0034] The non-viral vector is selected from one of the following: GalNac-coupled modified, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, exosomes, and peptides.

[0035] 20. The recombinant vector according to claim 19, wherein the adeno-associated virus is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10 and Anc80, or their progeny or variants.

[0036] 21. A pharmaceutical composition comprising a gene construct as described in any one of items 1-14 or a recombinant vector as described in any one of items 17-20, and optionally, a pharmaceutically acceptable vector.

[0037] 22. A method for treating substance use disorder or its symptoms or for preventing relapse of substance use disorder or its symptoms, comprising administering to a subject in need a therapeutically effective amount of any one of items 1-14, any one of items 17-20, or the pharmaceutical composition of item 21.

[0038] 23. The method according to claim 22, wherein the substance use disorder is caused by the overuse or abuse of an addictive substance, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic substances or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens or inhaled substances, more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

[0039] 24. A method for treating substance use disorder or its symptoms or for preventing relapse of substance use disorder or its symptoms, comprising administering to a subject in need a therapeutically effective amount of any one of items 1-14 of the gene construct and the exogenous ligand of the neuronal inhibitory protein.

[0040] 25. The method according to claim 24, wherein the substance use disorder is caused by the overuse or abuse of an addictive substance, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic substances or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens or inhaled substances, more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

[0041] 26. The method according to claim 24 or 25, wherein the neuronal inhibitory protein is selected from variants of hM4Di and / or hM3Dq and / or more thereof, and the exogenous ligands of hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160, or compound 21 (C21).

[0042] 27. The method according to any one of claims 24-26, wherein the gene construct is delivered prior to the administration of the exogenous ligand.

[0043] 28. The gene construct of any one of items 1-15, the recombinant vector of any one of items 17-20, or the pharmaceutical composition of item 21, for treating neuronal hyperexcitability caused by overuse or abuse of addictive substances, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic drugs, or addictive compounds, optionally, the addictive substance being tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens, or inhaled substances, preferably tobacco, alcohol, morphine, cocaine, or synthetic drugs.

[0044] 29. Use of the gene construct of any one of items 1-15, the recombinant vector of any one of items 17-20, or the pharmaceutical composition of item 21 in the preparation of a medicament for treating neuronal hyperexcitability caused by overuse or abuse of addictive substances, optionally, the addictive substance comprising a narcotic, a narcotic drug, a psychotropic drug, or an addictive compound, optionally, the addictive substance being tobacco, alcohol, opioids, cocaine, cannabis, a central nervous system depressant, a central nervous system stimulant, a hallucinogen, or an inhaled substance, preferably tobacco, alcohol, morphine, cocaine, or a synthetic drug.

[0045] The effects of the invention

[0046] The gene construct described in this application exhibits significant advantages in precise targeting, closed-loop regulation, preservation of natural reward responses, reduced side effects and improved safety, as well as potential long-term efficacy and sustainability. This strategy provides new ideas and methods for the treatment of drug use disorders and is expected to become a major breakthrough in the field of drug use disorders in the future. Attached Figure Description

[0047] Figure 1 is a flowchart of the behavioral paradigm of injecting recombinant vectors into specific brain regions of mice and self-administering drugs.

[0048] Figure 2A is a schematic diagram showing the effective and ineffective nasal touch curves of the two groups of mice over time after self-administration following injection of AAV expressing FRAM-mcherry and FRAM-mcherry T2A EKC into the NAc brain region of mice. Figure 2B is a schematic diagram showing the statistical analysis of effective and ineffective nasal touches of the two groups of mice on day 15 of the formation period after self-administration. Figure 2C is a schematic diagram showing the statistical analysis of effective and ineffective nasal touches of the two groups of mice during the cue ignition period 13 days after withdrawal.

[0049] Figure 3 shows the body weight of mice in the experimental group and the control group after injecting AAV expressing FRAM-mcherry T2A EKC into the NAc brain region of mice.

[0050] Figure 4 is a schematic diagram of the sucrose preference behavior of experimental and control mice after injecting AAV expressing FRAM-mcherry T2A EKC into the NAc brain region of mice.

[0051] Figure 5A is a schematic diagram showing the effective and ineffective nasal touch curves of mice over time after self-administration following injection of AAV expressing CamkII-mcherry and CamkII-hM4Di T2A mcherry into the ACC brain region of mice. Figure 5B is a schematic diagram showing the statistical analysis of effective and ineffective nasal touches of the two groups of mice on day 14 of the formation period after self-administration. Figure 5C is a schematic diagram showing the statistical analysis of effective and ineffective nasal touches of the two groups of mice during the cue ignition period 13 days after withdrawal.

[0052] Figure 6 is a schematic diagram showing the body weight of mice in the experimental group and the control group after injecting AAV expressing CamkII-hM4Di T2A mcherry into the ACC brain region of mice.

[0053] Figure 7 is a schematic diagram of the sucrose preference behavior of experimental and control mice after injecting AAV expressing CamkII-hM4Di T2A mcherry into the ACC brain region of mice.

[0054] Figure 8 is a schematic diagram of the ΔCPA Score of different groups of mice. Detailed Implementation

[0055] The present application will now be described in detail. While specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0056] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0057] definition

[0058] As used herein, the term "neuron" includes a neuron and one or more of its parts (e.g., neuronal cell body, axon, or dendrites). The term "neuron" as used herein refers to a cell of the nervous system, which includes a central cell body or somatic cell, and two types of extensions or projections: dendrites, through which most neuronal signals are transmitted to the cell body, and through axons from the cell body to effector cells (such as target neurons or muscles). Neurons can transmit information from tissues and organs to the central nervous system (afferent neurons or sensory neurons) and transmit signals from the central nervous system to effector cells (efferent neurons or motor neurons).

[0059] As used herein, the term "gene" refers to the deoxyribonucleotide sequence containing the coding region of a structural gene. A "gene" can also include untranslated sequences located near the 5' and 3' ends of the coding region, such that the gene corresponds to the length of full-length mRNA. A sequence located at the 5' end of the coding region and present on the mRNA is called a 5' untranslated sequence. A sequence located at or downstream of the 3' end of the coding region and present on the mRNA is called a 3' untranslated sequence. The term "gene" includes both the cDNA and genomic forms of a gene. mRNA plays a role in translation to specify the sequence or order of amino acids in the nascent polypeptide.

[0060] As used herein, the term "operably linked" refers to a functional relationship between two or more DNA segments, particularly the functional relationship between a gene sequence to be expressed and those sequences that control its expression. For example, a promoter (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable expression system. A promoter regulatory sequence operably linked to the transcribed gene sequence is physically adjacent to the transcribed sequence.

[0061] As used in this article, the term "neuronal inhibitory protein-coding gene" refers to a protein that expresses negative regulation of neuronal excitability, thereby effectively reducing the frequency, amplitude, and propagation efficiency of neuronal action potentials, weakening the intensity and range of signal transmission between neurons, and ultimately reducing neuronal excitability.

[0062] As used herein, the term "neuron-specific promoter" refers to a DNA sequence that specifically initiates gene expression in neurons. "Specific expression" means that the promoter preferentially drives gene transcription in a particular cell type, while exhibiting significantly reduced or no activity in other cell types. In this application, "specific expression" is further defined as at least 75% of gene expression activity in a target neuronal population concentrated in this cell type. For example, the neuron may be anterior cingulate cortex (ACC) neurons or nucleus accumbens (NAc) neurons. Furthermore, the specificity of the promoter can be experimentally verified, for example, quantitatively assessed through expression analysis of fluorescent reporter genes (such as GFP), RNA in situ hybridization, or single-cell RNA sequencing.

[0063] As used herein, the term "neuronal activity-dependent promoter" refers to a promoter that alters or drives the expression of a target gene in response to changes in neuronal activity within a nerve cell. Such changes in neuronal activity may be caused by nerve cells becoming overexcited.

[0064] As used herein, “DREADD” is an acronym for “designer receptor exclusively activated by a designer drug.” DREADD is a modified G protein-coupled receptor (GPCR) that can be administered or specifically introduced into a subject or their cells, such as glutamatergic neurons, using a viral vector containing a polynucleotide sequence encoding DREADD or through genetic breeding. DREADD is referred to as a chemically genetic or chemogenetic molecule that enables precise timing control of neuronal excitation and inhibition. Once expressed, DREADD can be activated by a specific ligand (or agonist) that can be administered via intraperitoneal injection, intravenous injection, or oral administration.

[0065] As used in this article, the term "exogenous receptor" refers to foreign substances such as drugs that can specifically bind to the receptor.

[0066] As used in this article, the term "functional fragment" refers to a portion of a gene or peptide that retains the full activity of the gene or peptide.

[0067] As used herein, the term "functional mutant" refers to a protein variant obtained by modifying the amino acid sequence of a wild-type protein (e.g., point mutation, insertion, deletion, or domain substitution), which retains at least some of the biological activities of the wild-type protein or exhibits enhanced biological activities compared to the wild-type protein. The "biological activities" include, but are not limited to, enzyme activity, ligand binding capacity, signal transduction function, or the ability to interact with other molecules. The activity of a functional mutant can be verified by in vitro experiments (e.g., enzyme kinetic analysis, binding affinity assays) or in vivo experiments (e.g., cell function assays, animal model behavioral analysis). 。

[0068] As used herein, the term "promoter" refers to a DNA sequence operatively linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. A promoter is typically located upstream of the nucleic acid sequence to be transcribed and provides a site for the specific binding of RNA polymerase and other transcription factors. In a particular embodiment, the promoter is typically located upstream of the DNA sequence to be transcribed, is a site of specific recognition and binding by RNA polymerase, controls the initiation of transcription, and helps generate the desired RNA molecule. Furthermore, promoters may interact with other transcription factors to jointly regulate the efficiency and specificity of transcription.

[0069] As used in this article, the term "adeno-associated virus (AAV)" is a small, non-enveloped virus belonging to the Parvoviridae family. It is commonly used as a gene delivery vector and is widely applied in gene therapy and gene editing. The AAV genome is a single-stranded linear DNA molecule, approximately 4700 bp, containing two open reading frames (ORFs): Rep and Cap, located between two inverted terminal repeats (ITRs), each consisting of 145 nucleotides. The ITRs act as the origin of viral replication and packaging signals. The Rep gene participates in viral replication and integration, encoding viral replication proteins, while the Cap gene encodes three viral capsid proteins. Compared to other commonly used viral vectors, such as lentiviruses, adenoviruses, and retroviruses, AAV has low immunogenicity and a long duration of action in vivo, making it clinically used as a gene therapy drug.

[0070] As used herein, the term "AAV serotype" refers to different variants of adeno-associated virus (AAV), which are primarily distinguished by differences in the capsid proteins of their viral particles. Different AAV serotypes possess unique capsid protein spatial structures, sequences, and tissue specificities. These characteristics determine their ability to recognize and bind to receptors on the surface of host cells, thereby affecting viral infection efficiency, tissue distribution, and immunogenicity. Currently, dozens of AAV serotypes with different capsid proteins have been identified, and they have broad application potential in gene therapy, gene delivery, and other biomedical research.

[0071] As used herein, the term "subject" means any individual or patient undergoing the methods of this application. Typically, a subject is a human being, but as those skilled in the art will understand, a subject can be an animal. Therefore, the definition of a subject also includes other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, and rabbits; farm animals including cattle, horses, goats, sheep, pigs, etc.; and primates (including monkeys, chimpanzees, orangutans, and gorillas).

[0072] As used herein, the term "treatment" refers to the administration of a pharmaceutical composition to a subject with an undesirable condition. This condition may include a disease or disorder.

[0073] As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water and emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents.

[0074] Gene construct

[0075] This application provides a gene construct for inhibiting neuronal overexcitation caused by the overuse or abuse of addictive substances, the gene construct comprising a neuronal inhibitory protein-coding gene and a promoter operatively linked to the neuronal inhibitory protein-coding gene.

[0076] This application operatively links a neuronal inhibitory protein-coding gene to a promoter operatively linked to the neuronal inhibitory protein-coding gene, thereby inhibiting the overexcitation of neurons sensitive to addictive substances, and thus treating diseases associated with neuronal overexcitation, namely substance use disorder.

[0077] In some embodiments, the neuronal inhibitory protein encoding gene includes a potassium channel protein encoding gene and / or a DREADD encoding gene. In some embodiments, the potassium channel protein encoding gene includes a gene encoding a voltage-gated potassium channel (Kv) family protein, an inward rectifier potassium channel (Kir) family protein, a functional fragment thereof, or a functional mutant thereof.In some embodiments, the potassium ion channel protein encoding gene is selected from KCNA1 (Kv1.1), KCNA2 (Kv1.2), KCNA3 (Kv1.3), KCNA4 (Kv1.4), KCNA5 (Kv1.5), KCNA6 (Kv1.6), KCNA7 (Kv1.7), KCNA8 (Kv1.8), KCNA9 (Kv1.9), KCNA10 (Kv1.10), KCNB1 (Kv2.1), KCNB2 (Kv2.2), KCNB3 (Kv2.3), KCNC1 (Kv3.1), KCNC ... 3.2), KCNC3(Kv3.2), KCNC4(Kv3.4), KCND1(Kv4.1), KCND2(Kv4.2), KCND3(Kv4.3), KCNF1(Kv5.1), KCNG1(Kv6.1), KCNG2(Kv6.2), K CNG3(Kv6.3), KCNG4(Kv6.4), KCNH1(Kv8.1), KCNH2(Kv11.1), KCNH3(Kv12.1), KCNH4(Kv12.2), KCNH5(Kv12.3), KCNH6(Kv12.4), KC NH7(Kv12.5), KCNH8(Kv12.6), KCNQ1(Kv7.1), KCNQ2(Kv7.2), KCNQ3(Kv7.3), KCNQ4(Kv7.4), KCNQ5(Kv7.5), KCNV1(Kv9.1), KCNV2( Kv9.2), KCNJ1(Kir1.1), KCNJ2(Kir2.1), KCNJ3(Kir3.1), KCNJ4(Kir2.3), KCNJ5(Kir3.4), KCNJ6(Kir3.2), KCNJ8(Kir6.1), KCNJ9 Genes of any one or more proteins from the group consisting of (Kir3.3), KCNJ10 (Kir4.1), KCNJ11 (Kir6.2), KCNJ12 (Kir2.2), KCNJ13 (Kir7.1), KCNJ14 (Kir2.4), KCNJ15 (Kir4.2), KCNJ16 (Kir5.1), their functional fragments and their functional mutants, preferably KCNA1 (Kv1.1), KCNJ2 (Kir2.1), KCNJ4 (Kir2.3), KCNJ10 (Kir4.1), their functional fragments and their functional mutants.

[0078] In this application, EKC (Engineered Potassium Channel) refers to a potassium channel protein encoding gene that is engineered and mutated based on KCNA1.

[0079] The KCNJ16 gene encodes a member of the inwardly rectifying potassium channel subfamily J, called Kir5.1 or BIR9, which is located in the chromosomal region 17q24.3. This gene plays an important role in maintaining the cell membrane potential and potassium ion transport. The protein encoded by the KCNJ16 gene belongs to the inwardly rectifying potassium channel family, specifically Kir5.1 (Potassium inwardly rectifying channel subfamily J member 16). These channels help maintain the resting membrane potential of cells and regulate the electrophysiological activities of cells by allowing potassium ions to pass through the cell membrane in an inwardly rectifying manner. Kir5.1 channels usually form heterodimers with other inwardly rectifying potassium channels (such as Kir4.1) to enhance their function and regulate the flow of potassium ions.

[0080] In some embodiments, the DREADD-encoding gene includes a gene encoding Rq (R165L), hM1Dq, hM5Dq, rM3Ds, hM2Di, hM4Di, hM3Dq, AlstR or KORD, a functional fragment thereof or a functional mutant thereof. In some embodiments, the gene construct is a gene construct administered in combination with its exogenous ligand. Optionally, the exogenous ligands for hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, dechloroclozapine (DCZ), perlapine, JHU 37152, JHU37160 or compound 21 (C21). In some embodiments, the gene construct is delivered before administering the exogenous ligand.

[0081] Compound 21, which is DREDD agonist 21, has the following chemical structural formula:

[0082] Rq (R165L) is used to enhance inhibitory protein signaling, and this specific pathway has been regarded as related to the mechanism of psychoactive drugs.

[0083] Armbruster and Roth developed a DREADD technology platform suitable for neural research based on GPCRs. By modification, receptors such as hM1Dq, hM2Di, hM3Dq, hM4Di, hM5Dq were obtained, among which hM2Di and hM4Di are inhibitory receptors, and hM1Dq, hM3Dq, hM5Dq are excitatory receptors.

[0084] hM4Di is an artificial receptor (DREADD) modified from the human muscarinic acetylcholine receptor M4 that responds only to CNO. It is no longer activated by acetylcholine, and it reduces the activation of cAMP and specific potassium channels, leading to neuronal silencing and inhibiting the release of presynaptic neurotransmitters.

[0085] hM3Dq is an artificial receptor (DREADD) modified from the human muscarinic acetylcholine receptor M3 that responds only to CNO. It is no longer activated by acetylcholine, and it increases the calcium level in cells, leading to explosive discharge.

[0086] AlstR (Allatostatin Receptor) is an insect-derived G protein-coupled receptor (GPCR) that specifically binds to the neuropeptide allatostatin (AST) and is often used as a "silencing receptor." By expressing AlstR in specific neurons through genetic engineering, and then using its ligand allatostatin or analogues, the activity of these neurons can be inhibited.

[0087] KORD (kappa-opioid receptor based DREAAD) is an artificial receptor modified from the human κ-opioid receptor. It is sensitive to SALB (serum albumin), is not activated by opioids or other ligands, and weakens or inhibits neuronal excitation and suppresses the release of presynaptic neurotransmitters.

[0088] In some embodiments, the promoter includes a constitutive promoter, a neuron-specific promoter, or a neuron activity-dependent promoter. In some embodiments, the constitutive promoter is selected from the following: cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) promoter, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) thymidine kinase promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70 kDa 5HSPA5, heat shock protein 90 kDa β member 1HSP90B1, heat shock protein 70 kDa HSP70, β-kinin β-KIN, and human ROSA. 26 promoters, ubiquitin C promoter UBC, phosphoglycerate kinase-1 PGK promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, or β-actin promoter, or combinations thereof. In some embodiments, the neuron-specific promoter is selected from human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, A The promoter may be one or a combination of the following: the dvillin promoter, the Drosophila single homologue 1 (SIM1) promoter, the oxytocin (OXT) promoter, the spiky mouse-associated protein (AgRP) promoter, the protein kinase C-δ (PKC-δ) promoter, the auxin-releasing peptide promoter, the glutamate decarboxylase (GAD1 / 2) promoter, the choline acetyltransferase (ChAT) promoter, the vesicle GABA transporter (VGAT) promoter, the glutamate decarboxylase (GAD65) promoter, the tyrosine hydroxylase (TH) promoter, and the promoter without a distal homeobox (Dlx). In some embodiments, the neuron-specific promoter is the CaMKIIα promoter.In some embodiments, the neuronal activity-dependent promoter is selected from the c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter, or synthetic activity-dependent promoters, such as PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), FRAM (Fos Robust Activity Marker), or any combination thereof.

[0089] The FRAM (Fos-dependent r Robust Activity Marking) promoter contains a common DNA-binding sequence of Fos transcription factors. Fos, as a transcription factor, functions by binding to specific DNA sequences to regulate downstream gene transcription. This design allows it to specifically respond to changes in Fos activity, making it a key cis-acting element for Fos's transcriptional regulatory function. When neurons are stimulated and activated or under stress, Fos transcription factors are rapidly activated and expressed. Activated Fos transcription factors, using their highly conserved and specific DNA-binding domains, precisely recognize and tightly bind to the AP-1 binding site in the c-fos promoter region, and bind to its DNA-binding sequence, thereby driving gene expression downstream of the FRAM promoter. In this way, FRAM can activate and express downstream signaling in Fos neurons under specific behaviors or stimuli (such as stimulation by addictive substances like cocaine).

[0090] Fos transcription factor is a nuclear protein belonging to the AP-1 transcription factor family. It mainly regulates gene transcription by binding to specific DNA sequences (such as AP-1 binding sites present in many gene promoter regions) through its DNA-binding domain.

[0091] The c-fos promoter is a DNA sequence located upstream of the c-fos gene that contains various cis-acting elements, such as the AP-1 (activator protein-1) binding site. The c-fos promoter region contains binding sites for Fos transcription factors. When cells are stimulated (e.g., by addictive substances stimulating neurons), intracellular signal transduction pathways are activated, leading to the expression and activation of Fos transcription factors. Activated Fos transcription factors can recognize and bind to the AP-1 binding site in the c-fos promoter region via their DNA-binding domains. This binding recruits other transcription-related proteins (such as RNA polymerases) to the promoter region, thereby initiating the transcription process of the c-fos gene. In other words, Fos transcription factors are a key regulator of the c-fos promoter's transcription initiation function.

[0092] Calcium-calmodulin-dependent protein kinase II (CaMK II) is abundant in the central nervous system. As a protein kinase (protein phosphorylase) that modifies the function of various proteins by phosphorylation, it is closely related to the regulation of neural activity and synaptic plasticity. Furthermore, it is considered to play an important role in higher brain functions, primarily learning and memory, in inhibiting epileptic seizures, and in suppressing brain damage during cerebral ischemia. In the central nervous system, CaMK II forms a multimeric structure composed of an α subunit (CaMK IIα) and a β subunit (CaMK IIβ). These two subunits have high homology and each possesses protein kinase activity, calcium-calmodulin binding capacity, and inter-subunit association ability.

[0093] In some embodiments, the neuronal activity-dependent promoter is a promoter that senses and responds to specific transcriptional regulatory dynamics within the neuron induced by first or repeated intake of an addictive substance.

[0094] In some embodiments, the gene construct is delivered to the anterior cingulate cortex (ACC) or the nucleus accumbens (NAc).

[0095] In this application, the anterior cingulate cortex (ACC) refers to a key brain region in the brain's emotion regulation, cognitive processing, and behavioral decision-making circuits. It integrates and processes emotional, cognitive, motivational, and sensory information in real time, laying a solid neural foundation for an individual's behavioral decisions, emotion regulation, and cognitive processing. Located on the medial surface of the frontal lobe, the ACC has extensive and close neural connections with numerous brain regions, constructing a complex and orderly neural network architecture. It primarily receives neural projections from multiple brain regions, including the prefrontal cortex, amygdala, hippocampus, and thalamus. When an individual ingests addictive substances, the emotional coordination regulation between the ACC and amygdala becomes unbalanced, amplifying the drug-seeking impulse triggered by negative emotions; weakening the cognitive coordination mechanism between the ACC and the prefrontal cortex, reducing the individual's rational judgment and inhibitory ability regarding the consequences of addictive behavior; and disrupting the memory interaction program between the ACC and the hippocampus, enhancing the consolidation and retrieval weight of addiction-related memory traces. This, in turn, works synergistically from multiple dimensions, including emotional motivation arousal, cognitive control failure, and the reinforcement of addictive memories, to foster and consolidate addictive behavior.

[0096] In this application, the nucleus accumbens (NAc) refers to a key brain region in the brain's reward circuitry, primarily involved in the regulation of various behaviors such as reward, aversion, fear, and addiction. Located in the basal forebrain, it is part of the limbic system and receives neural projections from multiple regions, including the cerebral cortex, limbic system, and thalamus. When an individual ingests addictive substances, these substances directly or indirectly activate neurons in the NAc region, releasing neurotransmitters such as dopamine, thereby producing a strong sense of pleasure and a reward effect. This reward effect is a significant driving force for drug abusers seeking drug experiences.

[0097] In some embodiments, the gene construct is delivered to the anterior cingulate cortex or nucleus accumbens by injection.

[0098] For example, by integrating the gene construct into a recombinant vector, the gene construct can be delivered to the anterior cingulate cortex or nucleus accumbens via injection. In this application, for example, injection can be performed directly using a stereotaxic device or via intravenous injection.

[0099] In some embodiments, the addictive substance comprises drugs, narcotic drugs, psychotropic drugs, or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants or hallucinogens, inhaled substances, more preferably tobacco, alcohol, cocaine, or synthetic drugs, wherein the opioids include morphine, heroin, fentanyl, oxycodone; the central nervous system stimulants include methamphetamine, cocaine, ecstasy, amphetamine; the hallucinogens include lysergic acid diethylamide, serotonin, dimethyltryptamine; the inhaled substances include paint thinner, glue, nitrous oxide; cannabis substances, mainly cannabis products containing tetrahydrocannabinol; synthetic drugs; and common nicotine and alcohol.

[0100] In this application, the synthetic drugs are a class of psychotropic substances primarily synthesized through chemical synthesis. They act directly on the human central nervous system, with some having stimulant effects, some hallucinogenic effects, and others central nervous system depressant effects. In some embodiments, the synthetic drugs may be, for example, methamphetamine, methcathinone, ecstasy, ketamine, triazolam, etc.

[0101] For example, when a subject ingests an addictive substance such as cocaine, the gene construct excites neurons in the anterior cingulate cortex or nucleus accumbens, increasing the expression level of the gene product. This gene product can then inhibit the neurons, restoring cellular activation to near-normal levels, reducing promoter activity, and returning gene product expression to baseline. Therefore, this gene therapy is applicable to both overactive neurons and blocking the dopaminergic reward circuit associated with drug abuse, thereby reducing intake.

[0102] The gene construct described above has significant advantages over current conventional drug addiction treatment drugs. For example, the expression of the potassium channel gene (EKC) in NAc is initiated by the neuronal activity-dependent promoter FRAM:

[0103] 1. Precise targeting and closed-loop regulation

[0104] Precise targeting: As an immediate early gene, FRAM is rapidly activated when neuronal activity increases, thus specifically marking NAc neurons that are overexcited due to drug intake. By driving the expression of potassium channel genes through the FRAM promoter, it can be ensured that therapeutic genes are expressed only in drug-affected neurons, achieving precise targeting.

[0105] Closed-loop regulation: Potassium channels play a crucial role in the regulation of neuronal excitability. When NAc neurons are overexcited due to drug stimulation, the FRAM promoter drives an increase in potassium channel expression, thereby reducing neuronal excitability by increasing potassium efflux, forming a negative feedback regulatory mechanism. This closed-loop regulation can automatically respond to drug stimulation and effectively inhibit addictive behavior.

[0106] 2. Preserve the natural reward response

[0107] Distinguishing between drugs and natural rewards: Due to the high specificity of FRAM activation, it primarily responds to aberrant stimuli such as drugs, while its response to natural rewards (such as food, sexual activity, etc.) is weaker. Therefore, a strategy of driving potassium ion channel gene expression through FRAM can suppress drug addiction while preserving the response to natural rewards, avoiding interference with normal physiological functions.

[0108] Protecting the integrity of the reward system: Traditional addiction medications often alleviate addiction symptoms by broadly inhibiting the function of the nervous system, which may damage the reward system and affect an individual's normal behavior and emotional responses. The FRAM-driven strategy, however, acts more precisely on addiction-related neurons, protecting the integrity and function of the reward system.

[0109] 3. Reduce side effects and improve safety

[0110] Reduced non-specific effects: Because the therapeutic gene is expressed only in NAc neurons affected by the drug, the non-specific effects of the drug throughout the body are reduced, thus lowering the risk of side effects.

[0111] Enhancing safety: Potassium ion channels, as endogenous regulators, are widely present and play an important role under physiological conditions. Therefore, the strategy of driving potassium ion channel gene expression through FRAM offers a high level of safety assurance.

[0112] 4. Potential long-term effectiveness and sustainability

[0113] Long-lasting effect: Once the FRAM-driven potassium channel gene is stably expressed in NAc neurons, its regulatory effect will persist and help to suppress drug addiction behavior in the long term.

[0114] Sustainability: This strategy achieves long-term regulation through gene therapy, avoiding the inconvenience of long-term use of traditional detoxification drugs and the potential problem of drug dependence.

[0115] Recombinant vector

[0116] This application provides a recombinant vector comprising the gene construct described above.

[0117] In this application, the recombinant vector is a viral vector or a non-viral vector. Preferably, the viral vector is selected from adeno-associated virus, adenovirus, retrovirus, lentivirus, vaccinia virus, herpes simplex virus, and bacteriophage, with adeno-associated virus being the most preferred. The adeno-associated virus may be, for example, one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, and Anc80, or its descendants or variants.

[0118] In some embodiments, the non-viral vector is selected from one of GalNac-coupled modified materials, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, exosomes, and peptides.

[0119] This application employs conventional genetic techniques to integrate a gene construct into a recombinant vector, such as AAV, and introduces it into a specific region (anterior cingulate cortex or nucleus accumbens). The neuronal inhibitory protein-coding gene is driven by a promoter, thereby inhibiting the overexcitation of neurons sensitive to addictive substances such as cocaine, for the treatment of substance-related disorders.

[0120] Pharmaceutical compositions, treatment methods and uses

[0121] This application provides a pharmaceutical composition comprising the gene construct or the recombinant vector described above, and optionally, a pharmaceutically acceptable vector.

[0122] This application operatively links a neuronal inhibitory protein-coding gene to a promoter, the expression of which is driven by the promoter, thereby inhibiting neuronal overexcitation and thus being used to treat substance-related disorders.

[0123] This application provides a method for treating substance use disorder or its symptoms, or for preventing its relapse, comprising administering to a subject in need a therapeutically effective amount of any of the gene constructs, recombinant vectors, or pharmaceutical compositions described above. In some embodiments, the substance use disorder is caused by the overuse or abuse of an addictive substance, optionally comprising narcotics, narcotic drugs, psychotropic substances, or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens, or inhaled substances, more preferably tobacco, alcohol, morphine, cocaine, or synthetic drugs.

[0124] In this application, no restrictions are placed on the method of administration. Those skilled in the art can administer it by injection, for example, by direct injection using a stereotactic instrument or by intravenous injection.

[0125] This application provides the use of the gene construct, the recombinant vector, or the pharmaceutical composition described above in the treatment of neuronal hyperexcitability caused by overuse or abuse of addictive substances, preferably, the neuronal hyperexcitability caused by overuse or abuse of addictive substances is a substance use disorder.

[0126] In some embodiments, the addictive substance comprises a drug, narcotic, psychotropic substance, or addictive compound. Optionally, the addictive substance is tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens, or inhaled substances, preferably tobacco, alcohol, morphine, cocaine, or synthetic drugs.

[0127] This application also provides the use of the gene construct, the recombinant vector, or the pharmaceutical composition described above in the preparation of a medicament for treating neuronal hyperexcitability caused by overuse or abuse of addictive substances, preferably, where neuronal hyperexcitability caused by overuse or abuse of addictive substances is a substance use disorder.

[0128] This application provides a method for treating substance use disorder or its symptoms, or for preventing relapse of substance use disorder or its symptoms, comprising administering to a subject in need a therapeutically effective amount of any one of the gene constructs described above and an exogenous ligand of the neuronal inhibitory protein. In some embodiments, the addictive substance comprises a narcotic, a narcotic drug, a psychotropic drug, or an addictive compound, preferably tobacco, alcohol, opioids, cocaine, cannabis, a central nervous system depressant, a central nervous system stimulant, a hallucinogen, or an inhaled substance, more preferably tobacco, alcohol, morphine, cocaine, or a synthetic drug. In some embodiments, the neuronal inhibitory protein is selected from functional mutants of hM4Di and / or hM3Dq and / or more than one of these proteins, and the exogenous ligands for hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160, or compound 21 (C21). In some embodiments, the gene construct is delivered prior to the administration of the exogenous ligand.

[0129] Example

[0130] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products. The experimental equipment used in the examples is shown in Table 1, and the experimental solvents used are shown in Table 2.

[0131] Table 1

[0132] Table 2

[0133] Example 1

[0134] Mice that have undergone jugular vein cannulation and cerebral stereovirus injection can be used to establish a cocaine self-administration model. After the formation and withdrawal periods, the relapse rate of mice in the experimental and control groups was compared when the cue was lit.

[0135] 1.1 Mouse jugular vein cannulation

[0136] 1.1.1 Fabrication of a jugular vein cannula

[0137] Cut the MRE-025 tubing into approximately 6.5cm sections (soak in 75% alcohol to soften it for easier subsequent fabrication), cut the top rigid tube into approximately 3cm sections, and completely cut the silicone tubing to approximately 1.5mm. Connect the MRE-025 tubing and the top rigid tube using a metal hollow tube (the length of the MRE-025 tubing and the top rigid tube inserted into the metal hollow tube should not be less than 5mm); insert the silicone tubing into the other end of the MRE-025 tubing approximately 1.3cm from the top, and secure it to the upper edge of the silicone tubing with 502 super glue. Let it dry.

[0138] 1.1.2 Performing jugular vein cannulation surgery in mice

[0139] All 7-week-old C57BL / 6J mice (purchased from Vital River) underwent jugular vein cannulation.

[0140] Disinfect the operating table with 75% alcohol and prepare a lamp, saline solution, and culture dishes. Pour saline solution into the culture dishes and soak the jugular vein cannula and sutures in the saline solution. Prepare a heparin syringe by attaching a 25G flat needle to a 1ml syringe. Anesthetize the mice by intraperitoneal injection of avertin. After the righting reflex disappears, apply erythromycin eye ointment to the mice's eyes.

[0141] The surgery begins by inserting the jugular vein cannula into the heparin needle, introducing heparin, and checking for patency. Hair is removed from the mouse's head and right chest area, and the area is disinfected with iodine. The mouse is placed in a prone position, and the skin on the head is cut open to expose the skull. The mouse is then placed in a supine position, and a small incision is made approximately 0.5 cm to the right of the midline of the neck. A curved hemostatic forceps is inserted through the chest incision, looped behind the ear, and exited through the head, gently clamping the jugular vein cannula and pulling it out. The jugular vein is dissected using curved hemostatic forceps and curved forceps, and the distal end is ligated with sutures, while a loose knot is tied at the proximal end. A small incision is made on the jugular vein using VANNAS spring shears, and a Micro-Renathane Tubing MRE-025 jugular vein cannula is inserted (inserted to the lower edge of the silicone tubing). Heparin is introduced, and aspiration is performed to check for blood return (if blood return is observed, it indicates that a blood vessel has been inserted). The proximal end is then tightly ligated. Ligate the distal end suture (at the upper edge of the silicone tubing) and the proximal end suture (at this point, the proximal end suture changes from the left and right sides to the top and bottom ends). Then ligate the top and bottom ends of the proximal end suture, cross-ligate the distal and proximal end sutures, and suture the neck skin. Clean the wound with penicillin solution, squeeze in erythromycin eye ointment (for antibacterial purposes and to prevent wound adhesion), and suture the chest wound.

[0142] Following the surgery, a stereotactic injection of nucleovirus accumbens into the brain was performed.

[0143] 1.2 Subsequently, the virus was injected into the nucleus accumbens region of the brain.

[0144] Mice were divided into two groups. The experimental group was injected with AAV9:FRAM-mcherry T2AEKC and AAV9:FRAM-mcherry viruses (n=9) into the nucleus accumbens. All other procedures were the same. The nucleotide sequence of pAAV-FRAM-mcherry T2AEKC is shown in SEQ ID NO:1, and the nucleotide sequence of pAAV-FRAM-mcherry is shown in SEQ ID NO:2.

[0145] First, the AAV preparation method: pAAV-FRAM-mcherry T2A EKC plasmid or pAAV-FRAM-mcherry plasmid, along with pHelper plasmid (its nucleotide sequence is shown in SEQ ID NO:3) and AAV9 pRC plasmid (its nucleotide sequence is shown in SEQ ID NO:4) at a molar ratio of 1:1:1, were co-transfected into HEK-293F cells using PEI transfection reagent (approximately 1 μg of plasmid per million cells). After culturing at 37°C in an incubator containing 5% carbon dioxide for 3 days, the cells were washed once with PBS buffer. After collecting the cells, they were subjected to five freeze-thaw cycles. Solid NaCl was added to bring the final concentration to 500 mM. The cells were centrifuged at 10000g for half an hour. The supernatant was filtered through a 0.45 μm filter membrane. A gradient of iodixanol solutions was then added to centrifuge tubes (5 mL 60% iodixanol, 5 mL 40% iodixanol, 6 mL 25% iodixanol, 8 mL iodixanol). Add 15% iodixanol to the top layer of the sample and centrifuge at 350,000g for 1 hour. Collect the virus layer at the 40% and 60% interface. Replace the medium by centrifugation using a 50 kDa ultrafiltration tube to dissolve the virus in 0.01% poloxamer PBS buffer. Perform qPCR to determine the virus titer and adjust the titer accordingly. The final result is a virus titer of 1 × 10^13 vg / ml.

[0146] Surgical operation method: First, on the exposed skull of the mouse, use a cotton swab dipped in physiological saline to wipe away the periosteum on the skull surface to expose the suture. Wait until the bregma area and the lambdoid suture are clear for positioning. Then, place the mouse's front teeth on the tooth rod of the adapter and tighten the ear rods on both sides to fix the mouse on the stereotaxic apparatus. Apply erythromycin eye ointment to the mouse's eyes to prevent strong light from stimulating the mouse's eyes. Use a clean cotton swab to dip in physiological saline and wipe the skull surface to keep the skull surface clean and dry. Use a positioning needle for positioning. Take the anterior fontanelle (bregma) as the origin, mark the anterior fontanelle (Bregma) on the skull surface, and the lambdoid suture as the end point. Adjust the front-back level about 4.2 mm in the front-back distance, and then adjust the left-right level 1.5 mm to the left and right (the front-back and left-right errors do not exceed 0.1 mm). Locate to the target coordinates, drill a hole in the skull with a skull drill and use a needle to pierce the dura mater. If bleeding occurs during the drilling process, clean the blood with a cotton swab, then use a clean cotton swab to dip in physiological saline and press on the bleeding point. If there is a lot of bleeding, hemostatic sponge can be used. <ooo00310><ooo00311>Inject 300 nL into each of the bilateral NAc, and the coordinates are: Medial-Lateral (ML): 1.1 mm; Anterior-Posterior (AP): 1.3 mm; Dorsal-Ventral (DV): 4.0 mm), and the injection speed is 30 nL / min. After the injection is completed, stop the needle for 10 min and slowly withdraw the needle. Place the mouse in the prone position, clean the skull surface, gently scrape the skull surface with the syringe needle to make it rough, and keep the surface dry. Bend the metal hollow tube of the cannula 90 degrees and use instant glue to vertically fix it on the skull surface, and use dental cement to fix the top of the head. <ooo00312><ooo00313>The operation is over, put the mouse back and raise it in a single cage. Before waking up, avoid the mouse from losing body temperature. Perform catheter maintenance after the operation, and inject 0.02 ml - 0.04 ml of heparin sodium solution intravenously every day to reduce infection and avoid blockage. <ooo00314><ooo00315>After 3 days of recovery, mice with unobstructed catheters can be used for cocaine self-administration training. <ooo00316><ooo00317>1.3 Cocaine self-administration in mice [[ID=一十三]]<ooo00318>[[ID=一十四]]<ooo00319>[[ID=一十五]]The training conditions and treatments of the two groups of mice are the same during the self-administration training. [[ID=一十六]]<ooo00320>[[ID=一十七]]<ooo00321>[[ID=一十八]]1.3.1 Mouse self-administration system: Manufactured by Anlai Software Technology Co., Ltd. (Ningbo, China). Each behavior box is placed in a soundproof box. The equipment used in the behavior box includes a nose-touch light, two nose-touchers, an infusion connection system, a peristaltic pump, a behavior recording system, etc. [[ID=一十九]]<ooo00322>[[ID=二十]]<ooo00323>1.3.2 Forming period program settings: In this study, the classical fixed ratio 1 (FR1, fixed ratio - 1) program was adopted, and the control group and the experimental group were treated the same. Each time a mouse made an effective nose poke, it received a drug injection, and the injection volume was calculated by the system according to the animal's body weight and cocaine concentration. The self - administration training time was 120 min / day, the cocaine injection dose was 0.5 mg / kg, and the maximum number of injections per round of experiment was 50 times. The left nose poke hole was the effective nose poke, and the right nose poke hole was the ineffective nose poke. When the mouse touched the effective nose poke hole once, the nose poke light would be on for 5 s, the pump would start to inject cocaine once, and the refractory period was 5 s; there was no response when touching the ineffective nose poke. The training lasted at least 12 days. Mice with the number of effective nose pokes greater than 20 and greater than twice the number of ineffective nose pokes were considered to have formed drug dependence.

[0154] 1.3.3 Extinction period program settings: When the mouse touched the effective nose poke, no cocaine and nose poke light were given, and the rest was the same as in the forming period. The training lasted at least one week. Ignition could be carried out when the difference in the effective nose poke data in the last three days did not exceed 20%. The specific operation process is shown in Figure 1.

[0155] 1.3.4 Recovery of cocaine self - administration behavior in mice with cue - induced reinstatement (simulating cocaine relapse)

[0156] During cue - induced reinstatement, when the mouse touched the left nose poke, the left nose poke light was on, but it was not accompanied by cocaine injection, and the rest was the same as in the forming period. The schematic diagrams of the curves of effective and ineffective nose pokes of the two groups of mice over time are shown in Figure 2A.

[0157] As can be seen from Figure 2A, in the 15 - day cocaine self - administration experiment, during the forming period of cocaine addiction, we observed a separation trend in the curves of effective and ineffective nose pokes of the mice in the control group and the experimental group over time.

[0158] After 15 days of cocaine self - administration, the data of the two groups of mice were statistically analyzed by Two - way ANNOVA test. The results are shown in Figure 2B. It was found that there was a significant difference between the effective and ineffective nose pokes of the mice in the control group (FRAM - mcherry) during the forming period. Although the curve of the experimental group (FRAM mcherry T2AEKC) showed separation, it did not reach statistical significance. This indicates that FRAM - mcherry T2A EKC gene therapy has an inhibitory effect on the formation of drug addiction.

[0159] After a 12-day withdrawal period, the differences between effective and ineffective nasal touch data in the two groups of mice gradually decreased and eventually stabilized. Subsequently, a drug-related cue ignition experiment was performed on the mice, and a t-test was conducted on the data from both groups after ignition, as shown in Figure 2C. The results showed that during the cue ignition period, the effective nasal touch value of the control group mice (FRAM-mcherry) was significantly higher than that of the ineffective nasal touch, and the difference was statistically significant. In contrast, although there was a difference between the effective and ineffective nasal touch values ​​in the experimental group mice (FRAM-mcherry T2A EKC), it did not reach a statistically significant level. Furthermore, the effective nasal touch value of the control group mice was significantly higher than that of the experimental group mice, suggesting that FRAM-mcherry T2A EKC gene therapy may have a certain inhibitory effect on cocaine addiction behavior.

[0160] The results of the cue-ignition study showed that after injecting the EKC-expressing virus into the nucleus accumbens to inhibit its neuronal activity, the number of times the mice actively touched the effective nasal touch when they were exposed to drug-related cues again was significantly lower than that of the control group, indicating that FRAM-mcherry T2A EKC gene therapy has an inhibitory effect on drug relapse.

[0161] This discovery not only reveals the crucial role of FRAM-mcherry T2A EKC in regulating the neural mechanisms of drug addiction, but also provides important molecular targets and experimental evidence for developing novel anti-addiction therapies. In the future, intervention strategies based on FRAM-mcherry T2A EKC are expected to become an important means of treating cocaine addiction and preventing relapse.

[0162] In addition, the body weight of mice in the experimental and control groups was monitored, and the results are shown in Figure 3.

[0163] As shown in Figure 3, there was no significant difference in body weight between the experimental group and the control group. This indicates that the expression of FRAM-mcherry T2A EKC in the NAc (nucleus accumbens) did not have any adverse effects on the physiological state of mice, especially basic physiological functions related to body weight.

[0164] Example 2 compares the differences in natural reward responses among neurons in brain regions overexpressing potassium ion channels and inhibiting NAc using a sucrose preference experiment.

[0165] The steps of the sugar water preference experiment are briefly described below:

[0166] Experimental preparation

[0167] Animal grouping and feeding: Healthy, age- and weight-matched mice were randomly divided into two groups: an experimental group and a control group, with several mice in each group. AAV virus injection was performed in the same manner as in Example 1. Mice were divided into two groups: the control group received AAV9:FRAM-mcherry AAV in the nucleus accumbens region, while the experimental group received AAV9:FRAM-mcherry T2A EKC AAV in the nucleus accumbens region.

[0168] Mice should be housed individually in cages and allowed free access to food.

[0169] Experimental steps

[0170] Day 1: Adaptation Period

[0171] Two identical bottles of drinking water were placed in each cage, allowing the mice to drink freely to adapt to the experimental environment and drinking device.

[0172] Day 2: Introducing sugar water

[0173] Replace one bottle of drinking water with 1% sucrose solution, while keeping the other bottle of drinking water unchanged. Be sure to record the initial volume of each bottle and mark its location for future interchanges.

[0174] Day 3: Reversal of Positions

[0175] The positions of the two bottles of water were swapped to ensure that the mice's drinking choices were based on the solution itself, not on location preference.

[0176] Day 4: Fasting and water restriction

[0177] All mice were fasted and deprived of water for 12-24 hours to stimulate their motivation to drink.

[0178] Day 5: Sugar Water Preference Test

[0179] Give the mouse a bottle of drinking water and a bottle of 1% sucrose solution, and be sure to record the initial volume of each bottle.

[0180] Day 6: Data Collection

[0181] The remaining water was collected, and the consumption of each bottle was calculated. The sugar water preference of the two groups of mice was assessed by comparing their consumption of sucrose water and drinking water, and the results are shown in Figure 4.

[0182] As shown in Figure 4, both the experimental group and the control group exhibited similar sucrose preference behaviors during the experiment, with no significant difference. This indicates that the expression of FRAM-mcherry T2A EKC in the nucleus accumbens (NAc) brain region did not have an adverse effect on the mice's function of assessing natural rewards.

[0183] Example 3 compares the effects of chemogenetic inhibition of neurons in the anterior cingulate cortex (ACC) region on drug abuse through a cocaine self-administration experiment.

[0184] 1.1 Mouse jugular vein cannulation

[0185] As in 1.1 of Example 1, after the surgery, the anterior cingulate cortex virus was injected into the brain using stereotactic localization.

[0186] 1.2 Subsequently, the virus was injected into the anterior cingulate cortex.

[0187] Mice were divided into two groups. The experimental group was injected with AAV9::CamkII-hM4Di T2A mcherry and AAV9::CamkII-mcherry viruses (n=9) into the anterior cingulate gyrus. All other procedures were the same. The nucleotide sequence of pAAV-CamkII-hM4Di T2A mcherry is shown in SEQ ID NO:5, and the nucleotide sequence of pAAV-CamkII-mcherry is shown in SEQ ID NO:6.

[0188] AAV preparation method: PAAV CamkII-hM4Di T2A mcherry plasmid or pAAV-CamkII-mcherry plasmid, along with pHelper plasmid and AAV9 pRC plasmid in a molar ratio of 1:1:1, were co-transfected into HEK-293F cells using PEI transfection reagent (approximately 1 μg of plasmid per million cells). The AAV purification procedure was the same as in Example 1 to obtain the viral solution.

[0189] The viral surgery involved bilateral ACC injection of 300 nL each, with coordinates as follows: Medial-Lateral (ML): ±0.25 mm; Anterior-Posterior (AP): 0.38 mm; Dorsal-Ventral (DV): 1.12 mm. The surgical procedure and subsequent mouse care were the same as in Example 1.

[0190] Three days after recovery, mice with patent ducts can be used for cocaine self-administration training.

[0191] 1.3 Autocaine administration in mice

[0192] Three days after recovery, mice with patent ducts can be used for cocaine self-administration training.

[0193] During the self-administration training period, the training conditions and treatments for both groups of mice were the same in both the formation and withdrawal phases, and were identical to those in Example 1. During the cue ignition phase, the experimental group received an intraperitoneal injection of 3 mg / kg CNO, while the control group received an intraperitoneal injection of the same volume of physiological saline. The training procedure was the same as in Example 1. Figure 5A shows a schematic diagram of the effective and ineffective nasal touch curves of the two groups of mice over time.

[0194] As shown in Figure 5A, during the 14-day cocaine self-administration experiment, in the period of cocaine addiction formation, we observed that the effective and ineffective nasal touch curves of the control group and the experimental group mice showed a separation trend over time.

[0195] Fourteen days after cocaine self-administration, data from two groups of mice were collected and analyzed using a two-way ANNOVA test. The results, shown in Figure 5B, revealed significant differences in effective and ineffective nasal touches between the control group (CamkII-mcherry) and the experimental group during the developmental period. This indicates that both groups of mice developed cocaine addiction.

[0196] After a 13-day withdrawal period, the differences between effective and ineffective nasal touch data in both groups of mice gradually decreased and eventually stabilized. Subsequently, the mice underwent a drug-related cue ignition experiment, and the data from the two groups after ignition were analyzed by a t-test. The results are shown in Figure 5C. The results showed that during the cue ignition period, the effective nasal touch value of the control group (CamkII-mcherry) mice was significantly higher than that of the experimental group (CamkII-hM4Di T2A mcherry) mice. This indicates that CamkII-hM4Di T2A mcherry gene therapy may have a certain inhibitory effect on cocaine relapse.

[0197] The results of the cue ignition study showed that after injecting the virus expressing hM4Di into the anterior cingulate cortex to inhibit its neuronal activity, the number of times the mice actively touched the effective nasal touch when they were exposed to drug-related cues again was significantly lower than that of the control group. This indicates that CamkII-hM4Di T2A mcherry gene therapy has an inhibitory effect on drug relapse.

[0198] This discovery not only reveals the crucial role of CamkII-hM4Di T2A mcherry in regulating the neural mechanisms of drug addiction, but also provides important molecular targets and experimental evidence for the development of novel anti-addiction therapies. In the future, intervention strategies based on CamkII-hM4Di T2A mcherry are expected to become an important means of treating cocaine addiction and preventing relapse.

[0199] In addition, the body weight of mice in the experimental and control groups was monitored, and the results are shown in Figure 6.

[0200] As shown in Figure 6, there was no significant difference in body weight between the experimental group and the control group. This indicates that the expression of CamkII-hM4Di T2A mcherry in the anterior cingulate cortex (ACC) did not adversely affect the physiological state of mice, especially basic physiological functions related to body weight.

[0201] Example 4 compares the differences in regenerative reward responses of neurons in the ACC brain region by inhibiting sucrose preference through a sucrose preference experiment.

[0202] The steps of the sugar water preference experiment are briefly described below:

[0203] Experimental preparation

[0204] Animal grouping and feeding: Healthy, age- and weight-matched mice were randomly divided into two groups: an experimental group and a control group, with several mice in each group. AAV virus injection was performed in the same manner as in Example 1. Mice were divided into two groups: the control group received an injection of AAV9::CamkII-mcherry AAV into the anterior cingulate cortex, while the experimental group received an injection of AAV9::CamkII-hM4Di T2Amcherry AAV into the anterior cingulate cortex (n=6).

[0205] The sucrose preference experiment was conducted in the same manner as in Example 3, and the results are shown in Figure 7. As can be seen from Figure 7, both the experimental and control groups exhibited similar sucrose preference behaviors during the experiment, with no significant difference. This indicates that the expression of CamkII-hM4Di T2A mcherry in the anterior cingulate cortex (ACC) does not adversely affect the mouse's function in assessing natural rewards.

[0206] Example 5

[0207] Mice that have undergone brain stereovirus injection can be used for fentanyl withdrawal experiments to compare the performance of experimental and control mice in fentanyl withdrawal-induced conditioned place aversion (CPA).

[0208] 5.1 Animal grouping and surgery

[0209] Healthy, age- and weight-matched mice were randomly divided into two groups: an experimental group and a control group, with several mice in each group. The AAV virus packaging method and AAV injection procedure were the same as in Example 1. Mice were divided into two groups: the control group received AAV9:FRAM-mcherry injected into the nucleus accumbens, while the experimental group received AAV9:FRAM Kir2.3 injected into the nucleus accumbens (n=5). The nucleotide sequence of pAAV-FRAM-mcherry is shown in SEQ ID NO:7, and the nucleotide sequence of pAAV-FRAM-Kir2.3 is shown in SEQ ID NO:8.

[0210] 4.2 Conditioned position aversion test induced by fentanyl withdrawal in mice

[0211] For the three days prior to the experiment, the mice were allowed to move freely in the hands for 5 minutes each day to familiarize them with the experimenter's scent and reduce the stress response caused by handling the mice.

[0212] Day 1: Pre-test. Mice were placed in the middle chamber of a self-made three-chamber behavior box. After the mice explored the left and right chambers and returned to the middle chamber, their movements were recorded to establish baseline positional preferences. The video recording lasted 15 minutes. MATLAB software was used to analyze the mice's movement trajectories and dwell times. Mice exhibiting strong initial preferences (preference time difference to a particular chamber exceeding 2.25 minutes) underwent additional pre-testing until the initial preferences decreased (preference time difference to a particular chamber ≤ 2.25 minutes).

[0213] Day 1-Day 4: Induction of fentanyl addiction. Mice were given intraperitoneal injections of fentanyl in their cages daily, with the injection dose increasing daily at 0.06 mg / kg, 0.12 mg / kg, 0.18 mg / kg, and 0.24 mg / kg, respectively.

[0214] Day 4: Conditioning. 0.5–1 hour after fentanyl injection on Day 4, mice were intraperitoneally injected with 5 mg / kg body weight of naloxone saline solution. Immediately afterward, the mice were confined to the preferred side of the pre-test for 20 minutes. Because fentanyl withdrawal induces strong aversion in the mice, they will associate the chamber environment with this aversion, forming a conditioned reflex.

[0215] Day 5: Testing. Same as the pre-test procedure. Use MATLAB software to analyze the mouse's movement trajectory and dwell time, and calculate the CPA Score and ΔCPA Score:

[0216] ΔCPA score = CPA score during testing phase - CPA score during pre-testing phase.

[0217] As shown in Figure 8, the ΔCPA score of the control group was negative, indicating that after conditioning training, the mice developed a strong aversion to the fentanyl withdrawal-coupled chamber, leading to avoidance behavior during the test. In contrast, the ΔCPA score of the experimental group was significantly lower in absolute value compared to the control group, indicating a reduced aversion to fentanyl withdrawal in the mice and a significant alleviation of drug withdrawal symptoms, thus having a positive impact on the treatment of drug addiction. This finding provides important molecular targets and experimental evidence for the development of novel anti-addiction therapies. In the future, intervention strategies based on FRAM Kir2.3 are expected to become an important means of treating fentanyl addiction and preventing relapse.

[0218] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A gene construct for inhibiting neuronal hyperexcitability caused by overuse or abuse of addictive substances, said gene construct comprising a neuronal inhibitory protein-coding gene and a promoter operatively linked to said neuronal inhibitory protein-coding gene.

2. The gene construct according to claim 1, wherein, The neuronal inhibitory protein encoding genes include potassium ion channel protein encoding genes and / or DREADD encoding genes.

3. The gene construct according to claim 2, wherein, The potassium channel protein encoding genes include genes encoding voltage-gated potassium channel (Kv) family proteins, inward rectifier potassium channel (Kir) family proteins, their functional fragments, or functional mutants thereof.

4. The gene construct according to claim 3, wherein, The potassium ion channel protein encoding gene is selected from KCNA1 (Kv1.1), KCNA2 (Kv1.2), KCNA3 (Kv1.3), KCNA4 (Kv1.4), KCNA5 (Kv1.5), KCNA6 (Kv1.6), KCNA7 (Kv1.7), KCNA8 (Kv1.8), KCNA9 (Kv1.9), KCNA10 (Kv1.10), KCNB1 (Kv2.1), KCNB2 (Kv2.2), KCNB3 (Kv2.3), and KCNC1 (Kv3). 1), KCNC2(Kv3.2), KCNC3(Kv3.2), KCNC4(Kv3.4), KCND1(Kv4.1), KCND2(Kv4.2), KCND3(Kv4.3), KCNF1(Kv5.1), KCNG1 (Kv6.1), KCNG2(Kv6.2), KCNG3(Kv6.3), KCNG4(Kv6.4), KCNH1(Kv8.1), KCNH2(Kv11.1), KCNH3(Kv12.1), KCNH4(Kv12.2 ), KCNH5(Kv12.3), KCNH6(Kv12.4), KCNH7(Kv12.5), KCNH8(Kv12.6), KCNQ1(Kv7.1), KCNQ2(Kv7.2), KCNQ3(Kv7.3), KC NQ4(Kv7.4), KCNQ5(Kv7.5), KCNV1(Kv9.1), KCNV2(Kv9.2), KCNJ1(Kir1.1), KCNJ2(Kir2.1), KCNJ3(Kir3.1), KCNJ4(Ki The genes of any one or more proteins in the group consisting of r2.3), KCNJ5(Kir3.4), KCNJ6(Kir3.2), KCNJ8(Kir6.1), KCNJ9(Kir3.3), KCNJ10(Kir4.1), KCNJ11(Kir6.2), KCNJ12(Kir2.2), KCNJ13(Kir7.1), KCNJ14(Kir2.4), KCNJ15(Kir4.2), KCNJ16(Kir5.1), their functional fragments, and their functional mutants.

5. The gene construct according to claim 2, wherein, The DREADD encoding genes include genes encoding Rq(R165L), hM1Dq, hM5Dq, rM3Ds, hM2Di, hM4Di, hM3Dq, AlstR or KORD, their functional fragments or functional mutants thereof.

6. The gene construct according to claim 5, wherein the gene construct is a gene construct administered in combination with its exogenous ligand, optionally, the exogenous ligands of hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160 or compound 21 (C21).

7. The gene construct according to claim 6, wherein, The gene construct is delivered prior to the administration of the exogenous ligand.

8. The gene construct according to any one of claims 1-7, wherein the promoter comprises a constitutive promoter, a neuron-specific promoter, or a neuron activity-dependent promoter.

9. The gene construct according to claim 8, wherein the constitutive promoter is selected from the following: cytomegalovirus (CMV) immediate early promoter, virosarcomavirus 40 (SV40) promoter, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus RSV LTR, herpes simplex virus HSV thymidine kinase promoter, H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1EGR1, ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase GAPDH, eukaryotic translation initiation factor 4A1 EIF4A1, heat shock 70kDa protein 5HSPA5, heat shock protein 90kDa β member 1HSP90B1, heat shock protein 70kDa HSP70, β-kinin β-KIN, human ROSA26 promoter, ubiquitin C promoter UBC, phosphoglycerate kinase-1PGK promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter or β-actin promoter, or any combination thereof.

10. The gene construct according to claim 8, wherein the neuron-specific promoter is selected from human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 (TUBA1A) promoter, methylated CpG-binding protein 2 (Mecp2) promoter, neuron-specific enolase (NSE) promoter, Nms promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, and Nav1. The following are promoters, or combinations thereof: 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, vesicle GABA transporter (VGAT) promoter, glutamate decarboxylase (GAD65) promoter, tyrosine hydroxylase (TH) promoter, and promoter without a distal homeobox (Dlx).

11. The gene construct according to claim 10, wherein the neuron-specific promoter is the CaMKIIα promoter.

12. The gene construct according to claim 8, wherein the neuronal activity-dependent promoter is selected from c-fos promoter, CREB promoter, SRE promoter, Egr1 promoter, Arc promoter, mArc promoter, Homer1a promoter, Bdnf promoter, Mef2 promoter, Fosb promoter, Npas4 promoter, AP1 promoter, or a synthetic activity-dependent promoter, such as one of PRAM (Promoter Robust Activity Marker), ESARE, NRAM (NPAS4 Robust Activity Marker), FRAM (Fos Robust Activity Marker), or any combination thereof.

13. The gene construct according to claim 12, wherein the neuronal activity-dependent promoter is a promoter that senses and responds to specific transcriptional regulatory dynamics within the neuron induced by first or repeated intake of an addictive substance.

14. The gene construct according to any one of claims 1-13, wherein the addictive substance comprises a drug, narcotic drug, psychotropic drug or addictive compound, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants or hallucinogens, inhaled substances, and more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

15. The gene construct according to any one of claims 1-14, wherein, The gene construct was delivered to the anterior cingulate cortex (ACC) and / or the nucleus accumbens (NAc).

16. The gene construct according to claims 1-15, wherein, The gene construct was delivered to the anterior cingulate cortex and / or nucleus accumbens via in situ injection.

17. A recombinant vector comprising the gene construct according to any one of claims 1-16.

18. The recombinant vector according to claim 17, wherein the recombinant vector is a viral vector or a non-viral vector.

19. The recombinant vector according to claim 18, wherein the viral vector is selected from adeno-associated virus, adenovirus, retrovirus, lentivirus, vaccinia virus, herpes simplex virus and bacteriophage, preferably adeno-associated virus; The non-viral vector is selected from one of the following: GalNac-coupled modified, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, exosomes, and peptides.

20. The recombinant vector of claim 19, wherein the adeno-associated virus is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10 and Anc80, or their descendants or variants.

21. A pharmaceutical composition comprising a gene construct according to any one of claims 1-14 or a recombinant vector according to any one of claims 17-20, and optionally, a pharmaceutically acceptable vector.

22. A method for treating substance use disorder or its symptoms or for preventing relapse of substance use disorder or its symptoms, comprising administering to a subject in need a therapeutically effective amount of the gene construct of any one of claims 1-14, the recombinant vector of any one of claims 17-20, or the pharmaceutical composition of claim 21.

23. The method of claim 22, wherein the substance use disorder is caused by the overuse or abuse of an addictive substance, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic substances or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens or inhaled substances, more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

24. A method for treating substance use disorder or its symptoms or for preventing relapse of substance use disorder or its symptoms, comprising administering to a subject in need a therapeutically effective amount of any one of claims 1-14 of the gene construct and the exogenous ligand of the neuronal inhibitory protein.

25. The method of claim 24, wherein the substance use disorder is caused by the overuse or abuse of an addictive substance, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic substances or addictive compounds, preferably tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens or inhaled substances, more preferably tobacco, alcohol, morphine, cocaine or synthetic drugs.

26. The method according to claim 24 or 25, wherein the neuronal inhibitory protein is selected from variants of hM4Di and / or hM3Dq and / or more than the above proteins, and the exogenous ligands of hM4Di and hM3Dq include clozapine, clozapine N-oxide (CNO), olanzapine, desclozapine (DCZ), perlapine, JHU 37152, JHU37160, or compound 21 (C21).

27. The method according to any one of claims 24-26, wherein, The gene construct is delivered prior to the administration of the exogenous ligand.

28. The gene construct of any one of claims 1-15, the recombinant vector of any one of claims 17-20, or the pharmaceutical composition of claim 21, for treating neuronal hyperexcitability caused by overuse or abuse of addictive substances, optionally, the addictive substance comprising narcotics, narcotic drugs, psychotropic drugs, or addictive compounds, optionally, the addictive substance being tobacco, alcohol, opioids, cocaine, cannabis, central nervous system depressants, central nervous system stimulants, hallucinogens, or inhaled substances, preferably tobacco, alcohol, morphine, cocaine, or synthetic drugs.

29. Use of the gene construct of any one of claims 1-15, the recombinant vector of any one of claims 17-20, or the pharmaceutical composition of claim 21 in the preparation of a medicament for treating neuronal hyperexcitability caused by overuse or abuse of addictive substances, wherein optionally, the addictive substance comprises a narcotic, a narcotic drug, a psychotropic drug, or an addictive compound, wherein optionally, the addictive substance is tobacco, alcohol, opioids, cocaine, cannabis, a central nervous system depressant, a central nervous system stimulant, a hallucinogen, or an inhaled substance, preferably tobacco, alcohol, morphine, cocaine, or a synthetic drug.

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