Pharmaceutical composition for long-acting treatment of STXBP1-related nervous system diseases, nucleic acid construct and application

Abstract

The invention discloses a pharmaceutical composition for treating STXBP1-related nervous system diseases, a nucleic acid construct and application of the pharmaceutical composition and the nucleic acid construct. Aiming at the defects that existing gene therapy is poor in targeting, unstable in expression, insufficient in safety verification, incomprehensive in neurological function improvement and the like, targeted intervention on nervous system abnormality caused by STXBP1 gene function deletion or mutation is realized by accurately regulating and controlling sequence composition, a packaging system and a drug delivery scheme of a nucleic acid construct; therefore, STXBP1 protein expression is effectively recovered, and synaptic delivery dysfunction and related behavioral anomaly phenotypes are improved for a long time.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and genetic engineering technology, specifically to a method for long-acting treatment. STXBP1 Drug compositions, nucleic acid constructs and applications related to nervous system diseases. Background Technology

[0002] STXBP1 (Syntaxin Binding Protein 1) gene mutations are a known pathogenic factor leading to a class of neurodevelopmental disorders, manifesting as abnormal electroencephalograms, epilepsy, language disorders, motor delays, and cognitive deficits. Current technologies lack specific treatments targeting this condition. STXBP1 Effective interventions for gene function defects, the few reports in the early stages of animal research, have the following problems or shortcomings: (1) Limited target selection: Most existing technologies focus on symptomatic regulation of epilepsy or motor development disorders, lacking specific targets for gene function defects. STXBP1 (1) Direct intervention as the core pathogenic mechanism; (2) Instability of the expression system: some studies have not targeted the core pathogenic mechanism. STXBP1 Specific expression in neurons requires promoter optimization, which can easily lead to non-specific expression or insufficient expression levels; (3) Insufficient viral titer control and biosafety verification: There is a lack of systematic long-term studies to assess the long-term safety of different doses of virus in terms of behavior, EEG, neurotoxicity and liver and kidney function; (4) Incomplete assessment of the overall functional recovery of the nervous system: Existing studies are mostly limited to protein expression or local behavioral tests, and lack systemic mechanism verification such as synaptic function, autophagy / apoptosis pathway, and oxidative stress.

[0003] Therefore, existing technology in STXBP1 Gene therapy still has significant shortcomings in terms of targeting, comprehensive functional recovery, and long-term safety, and cannot yet meet the needs of preclinical translational research. In particular, there is a lack of a gene therapy product that can effectively improve abnormal neural electrical activity and neural developmental function over a long period of time.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] In order to solve at least some of the technical problems in the prior art, such as existing STXBP1 The present invention addresses the lack of effective and specific treatments for gene-related encephalopathy, as well as the shortcomings of existing gene therapies such as poor targeting, unstable expression, insufficient safety verification, and incomplete improvement of neurological function. It provides a long-acting treatment... STXBP1The nucleic acid constructs and pharmaceutical compositions for related neurological diseases have the following objectives: (1) effectively restoring STXBP1 protein expression; (2) targeted delivery to the central nervous system, preferentially expressed in neurons; (3) long-term improvement of synaptic transmission dysfunction and related behavioral abnormalities; and (4) maintaining good tissue biocompatibility and systemic safety. In some embodiments, the present invention achieves the following effects by precisely controlling the sequence composition of the nucleic acid construct, the packaging system, and the dosing regimen: STXBP1 This invention provides targeted interventions for nervous system abnormalities caused by gene dysfunction or mutation. Specifically, the invention includes the following:

[0006] A first aspect of the present invention provides a method for long-term treatment or improvement STXBP1 Nucleic acid constructs for related neurological diseases, wherein the nucleic acid constructs include or are capable of producing the following polynucleotides (1) and / or (2): (1) Polynucleotides with sequences as shown in SEQ ID No. 2; (2) The polynucleotides in (1) above that have been modified with nucleotides and have the same function.

[0007] A second aspect of the present invention provides a method for long-term treatment or improvement STXBP1 A pharmaceutical composition for related neurological diseases, wherein the pharmaceutical composition comprises the nucleic acid construct described in the first aspect.

[0008] In some embodiments, the method according to the invention for long-acting treatment or improvement STXBP1 A pharmaceutical composition for a related neurological disease, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0009] In some embodiments, the method according to the invention for long-acting treatment or improvement STXBP1 A pharmaceutical composition for related neurological diseases, wherein the pharmaceutical composition is an injectable preparation.

[0010] In some embodiments, the method according to the invention for long-acting treatment or improvement STXBP1 A pharmaceutical composition for related neurological diseases, wherein the pharmaceutical composition is administered via intracerebral injection.

[0011] In some embodiments, the method according to the invention for long-acting treatment or improvement STXBP1 A pharmaceutical composition for related neurological diseases, wherein, the STXBP1 Related neurological disorders refer to those in which the subject is diagnosed with the presence of STXBP1 1 E549V / - mutation.

[0012] A third aspect of the invention provides nucleic acid constructs for the preparation of long-acting therapeutic or therapeutic agents. STXBP1 Application in medications for related neurological diseases.

[0013] A fourth aspect of the invention provides a method for determining the effectiveness of a test drug in treating or improving [the condition]. STXBP1 The method for effective treatment of related neurological diseases includes the following steps: (1) Provide a cell model or animal model and detect relevant indicators including the expression level of STXBP1 protein to obtain the first parameter, wherein the cell model or animal model contains STXBP1 E549V / - mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including the expression level of STXBP1 protein are detected to obtain the second parameter; (3) Compare the first parameter and the second parameter.

[0014] In some embodiments, the determination of the test drug for treatment or improvement according to the present invention is performed... STXBP1 Methods for the effectiveness of treatment for related neurological diseases, wherein the indicators further include STXBP1 E549V / - Mutation-related neuropathological markers, wherein the neuropathological markers include at least one of the following: expression of synaptic function-related proteins, number of neurons, activation status of nervous system cells, oxidative stress level, total dendritic length, number of dendritic branches, and dendritic spine density; Preferably, when an animal model is selected, the neuropathological indicators also include at least one of cognitive function, social response, and anxiety phenotype.

[0015] In some embodiments, the determination of the test drug for treatment or improvement according to the present invention is performed... STXBP1 A method for assessing the effectiveness of the drug in relation to related neurological diseases, wherein the aforementioned indicators are detected at least 1.5 months after administration of the test drug to the animal model.

[0016] This invention provides a method for long-term treatment or improvement STXBP1 Nucleic acid constructs related to neurological diseases, pharmaceutical compositions containing them, and their applications in regulating nervous system function, particularly in improving... STXBP1 Application in gene-related brain dysfunction. This technical solution has the following significant advantages and beneficial effects compared to existing technologies: I. Technical Aspects: 1. Highly targeted and efficient delivery: This invention utilizes a nucleic acid construct containing a neuron-specific promoter (hSyn), which has been experimentally proven to be efficiently expressed in neurons of the central nervous system, thus achieving exogenous... STXBP1 The gene is stably expressed in multiple key brain regions (including the hippocampus, prefrontal cortex, cerebellum, striatum, etc.), and has good brain targeting and tissue affinity.

[0017] 2. Significant recovery of protein expression: exist STXBP1 E549V / - In the mutation model, the nucleic acid construct of the present invention can significantly restore the expression of STXBP1 protein, thereby improving the expression levels of multiple synaptic structural proteins (such as SYN1, SYP, PSD95, etc.) and comprehensively improving synaptic plasticity and transmission efficiency.

[0018] 3. Neuroprotective and antioxidant functions: Experiments showed that the number of neurons in the brain tissue of the animal model increased significantly after treatment, and the level of oxidative stress decreased (such as a decrease in MDA and an increase in the activity of SOD and GSH-Px), suggesting that the present invention has excellent neuroprotective and anti-free radical functions, which is beneficial to neuronal survival and functional maintenance.

[0019] 4. Good long-term safety: After treatment, the serum levels of liver and kidney function indicators such as ALT, AST, TBIL, BUN, and CREA in the animal model remained within the normal range, and no glial cell activation or inflammatory response was observed, verifying that the long-term use of this invention has good safety in terms of systemic and neuroimmune aspects.

[0020] II. Application Level: 1. Overcoming the limitations of traditional treatment methods: In response to the current situation STXBP1 Given the current lack of effective molecularly targeted therapies for related brain diseases, this invention provides a precise treatment strategy that directly restores the function of pathogenic genes, thus offering a new approach. STXBP1 This has opened up new intervention pathways for related brain diseases (including but not limited to early-onset epilepsy encephalopathy, neurodevelopmental disorders, etc.).

[0021] 2. Highly scalable and widely applicable: The vector platform of this invention is highly modular. For example, by changing the promoter or target sequence, it can be applied to gene therapy for other central nervous system diseases, and has broad prospects for clinical application and industrial transformation.

[0022] 3. Aligns with clinical translation trends: The technical solution of this invention is easy to scale up and prepare for clinical use, which aligns with the development trend of modern precision medicine and personalized treatment. Attached Figure Description

[0023] Figure 1 The recombinant vector structure constructed according to the present invention is shown.

[0024] Figure 2 The STXBP1-AAV pair is shown. STXBP1 E549V / - Long-term corrective effects on social and anxiety-like behaviors in mice. (A) STXBP1 E549V / - (B) Schematic diagram of the gene locus and construction of point mutant mice. Experimental procedure: Mice received intraventricular injections of control AAV or different doses of STXBP1-AAV at approximately 0.75 months of age, and completed three rounds of behavioral tests at 1.5, 6, and 12 months of age. (C) Schematic diagram of the three-compartment socialization experimental setup, including the positions of the empty cage, unfamiliar mouse 1, and newly added unfamiliar mouse 2. (D, E) Results of the three-compartment socialization experiment at 1.5 months of age. (F, G) Results of the three-compartment socialization experiment at 6 months of age. (H, I) Results of the three-compartment socialization experiment at 12 months of age. STXBP1 E549V / - The sniffing time in both the social preference and social novelty preference stages of the group continued to decrease, while those who received STXBP1-AAV in early childhood... STXBP1 E549V / - Mice maintained enhanced olfactory responses to both new and unfamiliar individuals in stages two and three, indicating that STXBP1-AAV has a long-term corrective effect on social function. (J) Schematic diagram of the elevated cross maze (EPM) apparatus. (K) EPM behavioral results at 1.5 months of age. (L) EPM behavioral results at 6 months of age. (M) EPM behavioral results at 12 months of age. STXBP1 E549V / - Mice continued to exhibit open arm avoidance and anxiety-like behavior during long-term follow-up, while the STXBP1-AAV group maintained near-wild-type levels in terms of open arm dwell time and entry frequency. (N) Representative EPM trajectory plots of 12-month-old mice, showing... STXBP1 E549V / - The group mainly stayed in the closed arms, while the STXBP1-AAV group explored more in the open arms. (O) Schematic diagram of the zero maze device. (P) Results of the zero maze at 1.5 months of age: there was no significant difference in the time spent in the open area and the number of entries among the groups. (Q) Results of the zero maze at 6 months of age. (R) Results of the zero maze at 12 months of age. STXBP1 E549V / - The avoidance behavior of mice towards open areas became more entrenched at this time point, while the STXBP1-AAV group continued to show a higher level of open area exploration, indicating that gene therapy has a lasting inhibitory effect on anxiety-like behavior.

[0025] Figure 3 This demonstrates the long-term improvement of STXBP1-AAV. STXBP1E549V / - Learning and memory in mice. (A) Schematic diagram of the Y-maze apparatus. (BD) Results of Y-maze tests at 1.5, 6, and 12 months of age: Compared with wild-type mice from the same littermate, STXBP1 E549V / - The percentage of spontaneous alternation in mice decreased continuously at all time points. STXBP1-AAV treatment significantly increased the spontaneous alternation rate without affecting the total number of entries, suggesting long-term correction of working memory. (E) Schematic diagram of the novel object recognition device. (FH) Novel object recognition results at 1.5, 6, and 12 months of age: STXBP1 E549V / - Mice exhibited decreased olfactory detection time and discrimination index for novel objects, but STXBP1-AAV restored preference for novel objects at all three time points. (IN) Results of Morris water maze learning and detection experiments at different time points: STXBP1 E549V / - Mice exhibited a slower decrease in escape latency, fewer platform crossings, and less time spent in the target quadrant. STXBP1-AAV accelerated the learning curve and improved spatial memory metrics in the detection test. (OQ) Representative swimming trajectories at each time point show increased search concentration in the target quadrant after STXBP1-AAV treatment. (RT) Latency at the first arrival at the platform position in the detection test: STXBP1 E549V / - The latency period in mice was prolonged, but STXBP1-AAV significantly shortened this latency period at all three follow-up time points.

[0026] Figure 4 This demonstrates the improvement of STXBP1-AAV. STXBP1 E549V / - Abnormal oxidative stress in the hippocampus, prefrontal cortex, and serum of mice. (A, B) NO levels in the hippocampus, mPFC, and serum. STXBP1 E549V / - NO levels in mice were significantly elevated, but decreased markedly after administration of medium and high doses of STXBP1-AAV. (C, D) MDA levels in the hippocampus, mPFC, and serum. STXBP1 E549V / - MDA was increased in mice, and treatment with STXBP1-AAV significantly reduced MDA. (EH) SOD and CAT activities in the hippocampus, mPFC, and serum. STXBP1 E549V / - The activity of antioxidant enzymes in mice decreased, while STXBP1-AAV increased the activity of SOD and CAT. (IL) GSH-Px activity and T-AOC levels in the hippocampus, mPFC, and serum. STXBP1 E549V / - GSH-Px and T-AOC were decreased in mice, and STXBP1-AAV restored these indicators.

[0027] Figure 5STXBP1-AAV recovery is shown STXBP1 E549V / - Expression of STXBP1 and STX1A in multiple brain regions of mice. (A, B) Representative images of STXBP1 and STX1A protein expression in the hippocampus, medial prefrontal cortex (mPFC), brainstem (BS), basal ganglia (BG), and cerebellum (CB). (CG) Quantitative analysis of STXBP1 protein in each brain region: compared with littermate wild-type, STXBP1 E549V / - STXBP1 protein levels were decreased in all five brain regions of mice, with the most significant decreases observed in the hip and mPFC. High-dose STXBP1-AAV significantly restored STXBP1 levels in all brain regions, medium-dose showed a significant recovery primarily in the hip and mPFC, while low-dose showed no significant improvement. (HL) Quantitative analysis of STX1A protein in each brain region: STXBP1 E549V / - STX1A levels decreased in all five brain regions of mice, with the largest decrease observed in hip and BS. High-dose STXBP1-AAV significantly restored STX1A expression in hip, mPFC, BS, and BG, while medium and low doses showed no significant changes. (M) STXBP1 mRNA expression levels in each brain region. STXBP1 E549V / - mice STXBP1 mRNA levels decreased in all five brain regions, and both medium and high doses of STXBP1-AAV significantly increased them. STXBP1 mRNA levels showed a trend consistent with protein results.

[0028] Figure 6 This demonstrates the improvement of STXBP1-AAV. STXBP1 E549V / - Abnormalities in mature and immature neurons in the mouse dentate gyrus. (A) Representative image of co-stained immunofluorescence of STXBP1 and MAP2 in the dentate gyrus (DG) of the hippocampus. (B) Quantitative analysis of the number of MAP2-positive neurons in the DG region: STXBP1 E549V / - The number of MAP2 neurons in mice was significantly reduced, and significantly increased after STXBP1-AAV treatment. (C) Quantitative analysis of the mean fluorescence intensity of STXBP1 in the DG region: STXBP1 E549V / - STXBP1 signaling was weakened in mice, but significantly increased after STXBP1-AAV treatment. (D) Quantitative analysis of the number of DCX-positive cells in the DG region: STXBP1 E549V / - The number of DCX-positive cells in mice was significantly reduced, while STXBP1-AAV significantly increased the number of DCX-positive cells. (E) Representative image of DCX immunofluorescence in the DG region, showing the reduction of DCX-positive cells in mutant mice and the recovery after STXBP1-AAV treatment.

[0029] Figure 7 STXBP1-AAV recovery is shown STXBP1 E549V / - Mouse hippocampus and mPFC BDNF-TrkB-AKT Axial and synapse-related proteins. (A) Representative immunoblot images of p-TrkB, TrkB, PSD95, SYN-1, p-AKT, AKT, SYP, and BDNF in the hippocampus. (BG) Quantitative analysis of hippocampal-related proteins: STXBP1 E549V / - In mice, BDNF was downregulated, the p-TrkB / TrkB and p-AKT / AKT ratios decreased, and PSD95, SYN-1, and SYP levels were significantly reduced. High-dose STXBP1-AAV significantly restored these indicators, medium-dose mainly improved p-TrkB / TrkB, SYN-1, and BDNF, while low-dose showed no significant changes. (H) Representative immunoblot images of the same group of proteins in mPFC. (IN) Quantitative analysis of mPFC-related proteins: STXBP1 E549V / - In mouse mPFC, BDNF, p-TrkB / TrkB, p-AKT / AKT, SYN-1, and SYP were all downregulated; high-dose STXBP1-AAV could fully restore the expression of the above proteins, and medium-dose also showed significant recovery in p-TrkB / TrkB, p-AKT / AKT, SYN-1, SYP, and BDNF.

[0030] Figure 8 AAV-STXBP1 rescue was demonstrated STXBP1 E549V / - The patient has defects in dendrites and dendritic spines from forebrain neurons. (A) From carriers STXBP1 E549V / - A schematic diagram illustrating the process of obtaining peripheral blood from mutant patients and normal controls, reprogramming it into iPSCs, and then sequentially inducing differentiation into NSCs and forebrain neurons. (B) NC and STXBP1 E549V / - Immunofluorescence staining of both groups of NSCs showed positive results for Sox2 and Nestin. (C) Immunofluorescence staining of differentiated neurons showed positive results for MAP2 and FOXG1, indicating successful acquisition of forebrain-like neurons. (D) MAP2 / STXBP1 double-labeled images of three groups of forebrain neurons showed a reduction in dendrites and dendritic spines in the mutant group, which was significantly improved after AAV treatment.

[0031] Figure 9 The STXBP1-AAV rescue was demonstrated. STXBP1 E549V / - Synaptic protein expression in patient-derived neurons and BDNF- TrkB-AKT-CaMKII Signaling pathway. (AO) NC+ empty virus, STXBP1E549V / - +empty virus and STXBP1 E549V / - Examples and statistical results of Western blot bands of STXBP1, STX1A, SYN-1, SYP, PSD95, GRIN2A, GRIN2B, BDNF, TrkB and p-TrkB, AKT and p-AKT, GluR1, p-GluR1, GluR2, p-GluR2, CaMKIIα, and p-CaMKIIα in the three groups of forebrain neurons +STXBP1-AAV. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Experimental methods not specified in the specific embodiments are generally performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0036] Nucleic acid structure One aspect of the present invention provides a treatment STXBP1 Nucleic acid constructs for related neurological diseases, said nucleic acid constructs comprising or capable of producing the following polynucleotides (1) and / or (2): (1) Polynucleotides with sequences as shown in SEQ ID No. 2; (2) The polynucleotides in (1) above that have been modified with nucleotides and have the same function.

[0037] As used in this invention, the term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct includes a vector backbone, i.e., an expression vector and an expression frame. In a preferred embodiment, the nucleic acid construct can be a plasmid or a viral vector.

[0038] In this invention, "polynucleotide," "nucleic acid molecule," "polynucleotide," or any grammatically equivalent description refers to a polymeric form of nucleotides or nucleic acids of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified forms, for example, by methylation and / or by capping, as well as unmodified forms of polynucleotides. The term also means including molecules containing non-naturally occurring or synthetic nucleotides and nucleotide analogues.

[0039] In this invention, chemical modification of nucleic acid molecules to improve their stability, activity, or half-life is known in the art. The nucleic acid molecules of this invention use nucleotide groups (or nucleotide residues) as basic structural units, wherein the nucleotide groups contain phosphate groups, ribose groups, and bases. Preferably, the nucleic acid molecule contains at least one modified nucleotide group. The modified nucleotide group does not cause the function of the nucleic acid molecule to be inhibited or lost.

[0040] In a preferred embodiment, the nucleic acid construct of the present invention is a recombinant adeno-associated virus vector, comprising an operably linked enhancer, intron, promoter, and human... STXBP1 Complementary DNA (coding sequence for overexpression of STXBP1), a terminator sequence, and an ITR sequence flanking the above sequence, preferably an ITR from AAV2 or AAV9. In a specific implementation, it is used to correct the presence of [unclear - possibly a specific substance or component] in the subject. STXBP1 E549V / - The coding sequence of the mutation is an optimized sequence, as shown in SEQ ID No. 1. Although various serotypes of AAV have been reported to have advantages in delivering target genes, the use of AAV as a vector in clinical settings (especially in encephalopathy) still faces significant challenges due to difficulties in efficiently delivering target genes to target tissues, achieving long-term expression of corrective transgenes, and avoiding harmful effects on the host immune system.

[0041] In this invention, "operably connected" is used to describe the connection between a regulatory element and a gene or its coding region. That is, gene expression is usually under the control of certain regulatory elements, and "operably connected" a gene or coding region to a regulatory element means that the gene or coding region is controlled or influenced by the regulatory element.

[0042] In one specific embodiment, the nucleic acid construct of the present invention is rAAV-hSyn- as shown in SEQ ID No. 2. STXBP1 -WPRE-hGH polyA.

[0043] In this invention, the preparation method of the above-mentioned nucleic acid constructs is not particularly limited. Those skilled in the art can obtain the corresponding nucleic acid constructs using known methods based on the sequences disclosed herein. For example, they can be obtained through in vitro chemical synthesis or through biotechnology or bioengineering methods (such as genetic engineering). Specific methods are also known in the art.

[0044] For treatment STXBP1 Drug compositions for related neurological diseases One aspect of the present invention provides a treatment STXBP1 A pharmaceutical composition for a related neurological disease, the pharmaceutical composition comprising the nucleic acid construct and optionally a pharmaceutically acceptable carrier or excipient.

[0045] In this invention, pharmaceutically acceptable carriers or excipients participate in the delivery or transport of nucleic acid constructs from one organ or part of the body to another organ or part of the body. Each carrier or excipient is "acceptable," meaning it is compatible with other components of the formulation and does not harm the patient. In this invention, the pharmaceutically acceptable carriers or excipients include at least one of diluents, absorbents, wetting agents, sweeteners, preservatives, and antioxidants. Pharmaceutically acceptable carriers are preferably those administered via injection, examples of which include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media.

[0046] In this invention, pharmaceutically acceptable excipients may also include one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming antiions, such as sodium, and nonionic surfactants, such as Tween and polyethylene glycol (PEG).

[0047] In a preferred embodiment, it is used for treatment STXBP1The composition for the relevant neurological diseases is an injectable preparation, particularly suitable for administration to subjects in need via intracerebral injection.

[0048] application One aspect of the present invention provides nucleic acid constructs for use in preparing treatment or improvement in subjects in need. STXBP1 Application in medications for related neurological diseases.

[0049] The term "subject" as used in this invention refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, and the like, who are about to receive a specific treatment. Generally, "subject" and "patient" are used interchangeably in this invention, both referring to the subject of the study. In a specific embodiment, the subject has been diagnosed with... STXBP1 E549V / - Subjects with mutations, especially those with... STXBP1 E549V / - Subjects suffering from encephalopathy due to mutations. In some embodiments, the subjects are pediatric patients under the age of 18. In some embodiments, the subjects are adult patients.

[0050] As used herein, the term "effective amount" refers to the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeutic effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function. Generally, the effective amount as used herein varies depending on various factors, such as the given drug or composition, pharmaceutical preparation, route of administration, type of disease or symptom, subject being treated, etc., but can still be routinely determined by those skilled in the art.

[0051] The term "treatment" as used in this invention refers to improvement of a condition before or after the onset of a disease or dysfunction. This degree of relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete).

[0052] The therapeutic dosage of this invention can vary widely. Generally, the dosage of the nucleic acid construct or pharmaceutical composition containing it in this invention is well known to those skilled in the art. For a person weighing 60 kg, the daily dose of the nucleic acid construct or pharmaceutical composition containing it is typically 0.1 μg-50 μg. Preferably, the dose used for administration is 0.2-10 μg / kg. It can be administered as a single dose once daily, multiple times daily, or at intervals. The dose can be administered as a single dose or divided into several doses, such as two, three, or four doses. The administered dose is within the range expected by clinicians or laboratory personnel, and the optimal dosage can be obtained by appropriately adjusting the dose, for example, through efficacy and safety testing.

[0053] In this invention, "treatment or improvement" STXBP1 "Related neurological disorders" refers to conditions that can be improved or treated due to the presence of... STXBP1 E549V / - Neurodevelopmental abnormalities caused by mutations, wherein the improvement or treatment includes at least one of the following: (1) Improve anxiety-like behaviors; (2) Improve memory, learning, communication and social participation abilities; (3) Improve abnormal oxidative stress, such as reducing NO and MDA levels, increasing SOD and CAT activities, and restoring GSH-Px activity and T-AOC levels; (4) Restore or increase STXBP1 expression levels; (5) Improve dendrites and dendritic spine defects; (6) Effectiveness in restoring synaptic function and improving neurobehavior, including upregulating the expression level of at least one of STXBP1, STX1A, SYN-1, SYP, PSD95, GRIN2A, GRIN2B, BDNF, TrkB, p-TrkB, AKT, p-AKT, GluR1, p-GluR1, GluR2, p-GluR2, CaMKIIα, and p-CaMKIIα; (7) Promotes the repair and functional recovery of the nervous system; (8) Increase the number of neurons.

[0054] In a preferred embodiment, the composition of the present invention is preferably administered to the subject in need by intracerebral injection.

[0055] Used to determine the effectiveness of a test drug in treating or improving [the condition]. STXBP1 Effective methods for treating related neurological diseases One aspect of the present invention provides a method for determining the effect of a test drug on treating or improving [the condition / condition]. STXBP1 Effective methods for treating related neurological diseases include: (1) Provide a cell model or animal model and detect relevant indicators including the expression level of STXBP1 protein to obtain the first parameter, wherein the cell model or animal model contains STXBP1 E549V / - mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including the expression level of STXBP1 protein are detected to obtain the second parameter; (3) Compare the first parameter and the second parameter.

[0056] In a preferred embodiment, the indicator further includes STXBP1 E549V / - Mutation-related neuropathological markers, including but not limited to at least one of the following: expression of synaptic function-related proteins, number of neurons, activation state of nervous system cells, oxidative stress level, total dendritic length, number of dendritic branches, and dendritic spine density. Preferably, when an animal model is selected, the neuropathological markers also include at least one of cognitive function, social response, and anxiety phenotype.

[0057] In this invention, the animal model is not particularly limited; for example, it can be any model animal, such as rat, mouse, chicken, rabbit, etc. Regarding the animal model... STXBP1 Methods for performing p.E549V (GAG to GTG) mutations are known in the art; for example, gene editing techniques can be used to introduce the aforementioned point mutations.

[0058] In this invention, when improvements in behavioral indicators (including improvements in cognitive function, social response, and anxiety phenotype) are observed in the second parameter, and / or changes in the expression levels of related genes are detected (increased levels of STXBP1, and / or increased levels of at least one of STXBP1, STX1A, SYN-1, SYP, PSD95, GRIN2A, GRIN2B, BDNF, TrkB, p-TrkB, AKT, p-AKT, GluR1, p-GluR1, GluR2, p-GluR2, CaMKIIα, p-CaMKIIα), and / or improvements in oxidative stress abnormalities (e.g., reduced NO and MDA levels, increased SOD and CAT activity, and restored GSH-Px activity and T-AOC levels), and / or changes in synaptic structure (improvement in dendritic and dendritic spine defects), and / or an increase in the number of neurons), it indicates that the drug under test can effectively treat [the condition]. STXBP1 Related neurological disorders. If no improvement is observed in the second parameter, it indicates that the tested drug is not effectively treating these conditions. STXBP1 Related neurological disorders.

[0059] In some embodiments, the method for testing the drug to be tested according to the present invention is described. STXBP1 A method for assessing the effects of the drug on related neurological diseases, wherein the above indicators are measured in an animal model 1.5 months after administration of the drug to be tested (preferably after 2 months, such as after 3, 6, 9, or 12 months).

[0060] In the specific implementation plan, construct STXBP1 Heterozygous point mutant mice for use STXBP1 Evaluation of the treatment effects of related developmental and epileptic encephalopathy.

[0061] Example I. Construction of AAV Vector Design and build portable STXBP1 Gene recombination vectors ( Figure 1 Its core structure is: rAAV-hSyn- STXBP1 -WPRE-hGH polyA, where rAAV is a recombinant adeno-associated virus, a non-enveloped, non-integrating virus with low immunogenicity and neural tissue affinity; hSyn is a human synapsin promoter, a neuron-specific promoter that can drive the efficient expression of transgenes in neurons. STXBP1 : Encodes Syntaxin BindingProtein 1, a cDNA sequence for prokaryotic or eukaryotic expression optimization; WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element, which enhances mRNA stability and transcription efficiency; hGH polyA: human growth hormone gene polyadenylation signal sequence, used to terminate transcription.

[0062] The vector was constructed using standard molecular cloning techniques, and then packaged into viruses in HEK293T cells using a three-plasmid system (pHelper, pAAV-RC, and the plasmid of this invention). The viruses were then purified using PEG precipitation and iodine gradient ultracentrifugation. Viral titers were quantified by qPCR at a concentration of 1.25E. 13 vg / mL.

[0063] II. Preparation of experimental animal and cell models and drug administration regimens Creating by precisely introducing point mutations through homology-guided repair. STXBP1 E549V / - Mutant mice.

[0064] To investigate whether AAV-STXBP1 could rescue the behavioral phenotype of point mutant mice, this study performed behavioral testing after bilateral lateral ventricle overexpression of AAV-STXBP1. The AAV-STXBP1 vector was injected into the brain three weeks after the mutation was diagnosed using a stereotactic injection method. STXBP1 E549V / - Lateral ventricles of the mouse brain. Mice were anesthetized with 1.5-2% isoflurane and placed on the mouse adapter of a stereotaxic apparatus. Hair was removed from the head, and the skin was incised to expose the skull. The stereotaxic apparatus was leveled anteriorly, posteriorly, laterally, and laterally using Bregma as the zero point to maintain the mouse's head balance. The injection coordinates were 0.5 mm posterior to the anterior fontanelle and 0.8 mm lateral to both sides, with a needle insertion of 2.0 mm. The location of the micro-injection needle was determined and marked based on the coordinates of the lateral ventricles. A hole was drilled at this location using a skull drill; a cavity was felt, indicating success. AAV virus was slowly injected (50 nL / min) into the bilateral LV brain regions and allowed to remain and diffuse for 10 min. Mouse grouping: At 3 weeks of age, mice were randomly divided into 5 groups of 8 mice each, with half males and half females. The low-dose (3.75 × 10^7 vg / mouse) AAV showed only slight improvement in some indicators, with significant overall fluctuation; while the medium- and high-dose (3.75 × 10^8 and 3.75 × 10^9 vg / mouse) AAV showed… a) Wild-type mice from the same littermate were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of AAV-NC 3.75×10^8 vg / mouse (NC group); b) STXBP1 E549V / - Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of 3.75 × 10^8 vg / mouse. STXBP1 E549V / - Group); c) STXBP1 E549V / - Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of 13.75 × 10^7 vg / mouse (AAV-L group); d) STXBP1 E549V / - Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of 13.75 × 10^8 vg / mouse (AAV-M group); e) STXBP1 E549V / - Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of 13.75 × 10^9 vg / mouse (AAV-H group).

[0065] Use suture needles to suture the scalp. Determine the number of sutures based on the size of the opening. After suturing, wipe the scalp again with 75% water to prevent infection. Once completed, remove the animal from the adapter and place it in a water bath incubator until it awakens. Afterward, place the animal in its enclosure and provide it with ample food and water.

[0066] For cell experiments, this embodiment extracted pluripotent stem cells from patients and healthy individuals, which then differentiated into forebrain neurons. Therefore, neurons from a healthy control group (CTR group), neurons from a patient-specific point mutation group (STXBP1 group), and point mutation neurons treated with AAV (AAV group) were defined. The AAV infection multiplicity was set at 1×10^4, the infection time point was day 7 of neuronal differentiation, and the sample collection time point was day 14.

[0067] III. Experimental Results 1. STXBP1-AAV pair STXBP1 E549V / - Long-term corrective effects on social, emotional, and cognitive behaviors in mice To assess early one-time STXBP1 Can gene supplementation provide stable improvement over a long timescale? STXBP1 E549V / - Related behavioral abnormalities, in this embodiment, were detected at 0.75 months of age. STXBP1 E549V / - Mice were injected with STXBP1-AAV or control AAV into the lateral ventricle, and their social behavior, anxiety-like behavior, learning and memory, and cognitive function were systematically monitored at 1.5, 6, and 12 months of age. Figure 2 A, B). Overall, STXBP1 E549V / - Mice exhibit neuropsychiatric phenotypes such as weakened social response and cognitive decline from a young age, which gradually solidify with age; while a single STXBP1-AAV intervention can continuously, broadly and relatively dose-dependently alleviate the above abnormalities over a follow-up period of at least 12 months.

[0068] In terms of social behavior, the three-room social experiment showed STXBP1 E549V / - Mice exhibited stable social preference and social novelty preference deficits from 1.5 months of age. Compared to littermate wild-type controls, STXBP1 E549V / - In the second stage, the mice exhibited significantly reduced sniffing time towards unfamiliar companions, and in the third stage, their exploration of newly introduced unfamiliar mice further weakened, suggesting that their spontaneous approach motivation to social stimuli and their ability to distinguish "social novelty" were both impaired. After treatment with STXBP1-AAV during infancy, at 1.5 months of age, the sniffing time in both the second and third stages of three-compartment social interaction was significantly prolonged, and social preference and social novelty preference partially recovered. Figure 2CE). In repeat tests at 6 months and 12 months of age, untreated STXBP1 E549V / - Mice consistently exhibited a pattern of low social response, while the STXBP1-AAV group maintained sniffing time at near-wild-type levels throughout both phases, suggesting that early gene substitution can produce a lasting social behavioral benefit spanning adolescence to adulthood. Figure 2 FI).

[0069] In terms of emotion-related behaviors, longitudinal results from the elevated cross maze (EPM) and zero maze revealed... STXBP1 E549V / - Age-dependent evolution of anxiety-like behavior in mice. At 1.5 months of age, there were no significant differences among the groups in terms of time spent in the open arm / open area and number of entries, suggesting that the anxiety-like phenotype was not yet prominent at this point. Figure 2 K, P). Until 6 months of age, STXBP1 E549V / - Mice exhibited significantly reduced dwell time and entry frequency in the open arms during the EPM and showed persistent avoidance of the open area in the zero maze, reflecting the gradual dominance of anxiety-like behavior as development progresses and its further solidification at 12 months of age. Figure 2 L, M, Q, R). In contrast, early adopters of STXBP1-AAV STXBP1 E549V / - In the EPM and zero maze tests at 6 and 12 months of age, mice showed a significant increase in open arm / open area exploration and a decreased preference for closed arms. Their anxiety-like behavior was generally close to that of the wild type and remained consistent throughout the 12 months. Figure 2 JR) showed that STXBP1-AAV not only prevented the progression of anxiety-like behavior, but also had a long-term maintenance effect.

[0070] Follow-up studies on learning, memory, and cognitive behavior further indicate that... STXBP1 E549V / - Mutations cause persistent impairment across multiple cognitive dimensions, while STXBP1-AAV provides stable protection over time. In the Y-maze, STXBP1 E549V / - The percentage of spontaneous alternation in mice from 1.5 months of age was significantly lower than that in their littermates (wild-type). This working memory deficit persisted at 6 and 12 months of age, while the total number of arm entries did not decrease significantly, indicating that exploration motivation and baseline activity levels were relatively preserved. STXBP1-AAV treatment significantly increased the spontaneous alternation rate in mutant mice at all three time points, bringing it close to wild-type levels, suggesting that early gene therapy can provide long-term and stable improvement in working memory function. Figure 3 AD). In the novel object recognition experiment, STXBP1 E549V / -Mice at 1.5, 6, and 12 months of age showed a persistent decrease in olfactory detection time and discrimination index for novel objects, indicating a difficulty in developing a preference for novel stimuli and reflecting widespread impairment in recognition memory and novelty preference. STXBP1-AAV significantly prolonged olfactory detection time and increased the novelty preference index in mutant mice at all time points, demonstrating a long-term corrective effect on recognition memory impairment. Figure 3 EH).

[0071] Morris Water Maze further reveals STXBP1 E549V / - Persistent deficits in spatial learning and reference memory in mice. Throughout the training phases at different age levels, STXBP1 E549V / - The escape latency curve in mice shifted upward and decreased slowly overall, indicating reduced spatial learning efficiency. In the corresponding detection experiment, mutant mice crossed the original platform position less frequently, had a longer latency to the platform for the first time, and spent less time in the target quadrant. Their swimming trajectories showed insufficient clustering in the target quadrant, suggesting impaired spatial reference memory consolidation. Figure 3 (IN, RT). A single injection of STXBP1-AAV during juvenile stage can continuously improve water maze performance at three time points: 1.5, 6 and 12 months of age. The treatment group mice showed a faster decrease in escape latency during the learning phase, enhanced target quadrant preference, increased platform crossings, and shortened time to first platform arrival in the detection test. The spatial learning and long-term memory indicators were generally close to wild-type levels.

[0072] In conclusion, STXBP1 E549V / - Mice exhibited early-onset, progressive, and long-term functional impairments across multiple behavioral dimensions, including social behavior, anxiety-like emotions, working memory, recognition memory, and spatial learning memory. A single administration of STXBP1-AAV at 0.75 months of age consistently and broadly corrected these social and emotional abnormalities and stably restored multidimensional cognitive function within a follow-up window of 1.5–12 months, highlighting… STXBP1 Gene therapy in STXBP1 It has significant and long-lasting behavioral benefits in related brain diseases.

[0073] 2. STXBP1-AAV reversal STXBP1 E549V / - Oxidative stress phenotypes in the brain and periphery of mice In order to evaluate STXBP1 E549V / - The effects of point mutations on redox homeostasis in the brain and serum were investigated in this study. Several oxidative stress-related indicators in the hippocampus and prefrontal cortex were examined, including the activities of reactive oxygen species (ROS), the lipid peroxidation product malondialdehyde (MDA), and the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).

[0074] Compared with the wild-type control, STXBP1 E549V / - NO levels were significantly elevated in the hippocampus and prefrontal cortex tissues of mice, as well as in serum, and decreased significantly with treatment of medium and high levels of AAV. Figure 4 AB). STXBP1 E549V / - -MDA levels also increased significantly. After AAV treatment, medium and high concentrations showed a good improvement trend, suggesting that mutations lead to increased accumulation of reactive oxygen species and aggravated lipid peroxidation damage in brain tissue. Figure 4 Correspondingly, the antioxidant defense system was imbalanced: in both brain regions, the activities of SOD, CAT, and GSH-Px showed an overall decreasing trend, and the enzyme activities of the mutant group were significantly lower than those of the WT group (CD). Figure 4 The presence of free radicals (FL) indicates an impaired ability to scavenge endogenous free radicals, making it difficult to counteract excessive oxidative attack. Based on this, this example compares the effects of different doses of STXBP1-AAV on the aforementioned indicators. Low-dose (3.75 × 10^7 vg / animal) AAV showed only slight improvement in some indicators, with significant overall fluctuations; while medium and high doses (3.75 × 10^8 and 3.75 × 10^9 vg / animal) showed relatively consistent rescue effects in both the hippocampus and prefrontal cortex.

[0075] In summary, STXBP1 E549V / - Mice exhibited significant oxidative stress and impaired antioxidant defense in the brain. STXBP1-AAV, especially at medium to high doses, dose-dependently alleviated this abnormality, supporting the view that oxidative stress imbalance is a contributing factor. STXBP1 One of the important downstream mechanisms of functional deficits, and gene replacement therapy has a clear protective effect in restoring redox homeostasis in the brain. Figure 4 ).

[0076] 3. STXBP1-AAV enhances the expression of STXBP1 and STX1A in key brain regions. STXBP1 is a necessary "chaperone" in the synaptic vesicle fusion process, mainly maintaining the assembly and stability of the SNARE complex by binding to presynaptic t-SNAREs such as STX1A. Therefore, in order to determine... STXBP1 E549V / - To investigate whether mutations not only lead to downregulation of STXBP1 itself but also affect its downstream STX1A axis, this embodiment examined the protein and mRNA expression of STXBP1 and STX1A in multiple functionally related brain regions (hippocampus, medial prefrontal cortex mPFC, brainstem BS, basal ganglia BG, and cerebellum CB) and evaluated the dose-dependent correction effect of STXBP1-AAV.

[0077] At the protein level, compared with the wild-type control from the same litter, STXBP1 E549V / - STXBP1 protein expression was significantly decreased in all five brain regions of mice, with the most significant decrease observed in the hippocampus. Figure 5 A, CG). STXBP1-AAV treatment showed a clear dose-dependent effect: high doses fully restored STXBP1 protein levels in five brain regions: hip, mPFC, BS, BG, and CB; medium doses mainly showed significant recovery in the hip and mPFC. Further analysis of STX1A revealed that its protein expression was also reduced to varying degrees in all five brain regions, with the most significant decreases in the hippocampus and brainstem (BS). Figure 5 HL), prompt STXBP1 E549V / - The related pathology has affected the presynaptic SNARE assembly. After administration of high-dose STXBP1-AAV, the levels of STX1A protein in hip, mPFC, BS, and BG all significantly increased, approaching the wild-type range, while medium and low doses did not show clear correction, indicating that effective restoration of STXBP1 may affect the reconstruction of the downstream STX1A axis.

[0078] At the transcriptional level, the trends in STXBP1 mRNA changes in different brain regions were highly consistent with the protein results: STXBP1 E549V / - STXBP1 mRNA expression was generally decreased in mouse hip, mPFC, BS, BG, and CB. Both medium and high doses of STXBP1-AAV significantly increased STXBP1 mRNA levels overall, while low doses had limited effects. Figure 5 M). In summary, STXBP1 E549V / - Mutations lead to widespread downregulation of STXBP1 expression in multiple brain regions, accompanied by a secondary reduction in its key chaperone protein STX1A. STXBP1-AAV, especially at medium and high doses, can restore STXBP1 at both the transcriptional and protein levels and partially reconstruct the STX1A axis, providing a basis for subsequent structural and molecular correction of synaptic function.

[0079] 4. STXBP1-AAV improves abnormalities in mature and immature neurons in the dentate gyrus. Based on the aforementioned abnormalities in STXBP1 expression and synapse-related proteins, this embodiment further examines... STXBP1 E549V / - Changes in mature and immature neurons within the dentate gyrus (DG) of the mouse hippocampus. To this end, confocal immunofluorescence staining was performed on STXBP1 and MAP2 in the DG region, and DCX was used to label immature neurons and neuroblasts.

[0080] In the DG area, STXBP1E549V / - The number of MAP2-positive neurons in mice was significantly reduced, and the dendritic structure became sparse, indicating damage to the dendritic structure of mature granule cells. Simultaneously, the mean fluorescence intensity of STXBP1 was significantly decreased, reflecting an overall downregulation of STXBP1 protein levels within local neurons. After treatment with STXBP1-AAV, the number of MAP2-positive neurons in the DG region significantly increased, the mean fluorescence intensity of STXBP1 significantly recovered, and the overall staining morphology resembled that of the wild-type control. Figure 6 AC). Furthermore, DCX immunostaining results showed that... STXBP1 E549V / - The number of DCX-positive cells in the DG region of mice was significantly reduced, suggesting that the neurogenic process in adults is suppressed; STXBP1-AAV treatment significantly increased the number of DCX-positive cells and partially restored the neurogenic level of the dentate gyrus. Figure 6 DE). The above results indicate that STXBP1 E549V / - Mutations not only weaken the structural integrity of mature neurons in the DG region, but also inhibit the generation of immature neurons. STXBP1-AAV can improve the abnormalities of both mature and immature neurons in this key brain region.

[0081] 5. STXBP1-AAV improves hippocampus and mPFC BDNF-TrkB-AKT Abnormalities in axon and synapse-related proteins The aforementioned results show that STXBP1 E549V / - Downregulation of STXBP1 in mice was particularly pronounced in the hippocampus and medial prefrontal cortex (mPFC), two brain regions closely related to learning, memory, and emotion regulation. As a synaptic vesicle fusion regulator, STXBP1 participates in maintaining the efficiency of presynaptic neurotransmitter release; impaired function may subsequently affect neurotrophic factor signaling and the homeostasis of presynaptic and postsynaptic structural proteins. Therefore, we examined BDNF, the receptor TrkB and its phosphorylated form p-TrkB, the downstream effector molecule AKT / p-AKT, as well as PSD95 representing the postsynaptic compacta, and synapsin-1 (SYN-1) and synaptophysin (SYP) representing presynaptic vesicle reserve and release in the hippocampus and mPFC.

[0082] In the seahorse, compared with the wild-type control from the same litter... STXBP1 E549V / - Mouse BDNF protein expression was significantly decreased, and the p-TrkB / TrkB ratio was downregulated, while the total TrkB level remained essentially unchanged, suggesting that TrkB receptor activation was suppressed; the p-AKT / AKT ratio decreased simultaneously, indicating that classical TrkB receptor activation was suppressed. BDNF-TrkB-AKTThe plasticity signaling pathway was generally at a low activity level. Simultaneously, PSD95 was significantly downregulated, and the expression of SYN-1 and SYP was also significantly reduced, reflecting disruption of the postsynaptic receptor anchoring platform and presynaptic vesicle reserve and release-related proteins. STXBP1-AAV showed dose-dependent correction in the hippocampus: high doses significantly restored the expression of p-TrkB / TrkB, p-AKT / AKT, PSD95, SYN-1, SYP, and BDNF; medium doses mainly showed statistically significant improvements in p-TrkB / TrkB, SYN-1, and BDNF; and low doses showed no significant overall change. Figure 7 AG).

[0083] In mPFC, the overall trend of change is similar to that of the hippocampus. STXBP1 E549V / - Mice also showed downregulation of BDNF, decreased p-TrkB / TrkB and p-AKT / AKT ratios, while total TrkB and total AKT levels remained relatively stable; PSD95, SYN-1, and SYP expression were all decreased to varying degrees, suggesting that the prefrontal synaptic network was damaged in both neurotrophic factor signaling and synaptic structure. After STXBP1-AAV treatment, high doses significantly restored the above indicators in the mPFC; medium doses, in addition to correcting the decreases in p-TrkB / TrkB and p-AKT / AKT, also showed significant increases in SYP, SYN-1, and BDNF; low doses did not show a stable corrective effect. Overall, STXBP1 E549V / - Mutations caused in the hippocampus and mPFC BDNF-TrkB-AKT Decreased signaling activity and downregulation of presynaptic and postsynaptic marker proteins, particularly at medium and high doses of STXBP1-AAV, can rebuild the aforementioned signaling and structural basis in both brain regions, providing molecular support for the aforementioned improvements in emotion and cognitive behavior. Figure 7 HN).

[0084] 6. AAV-STXBP1 rescue STXBP1 E549V / - Defects in dendrites and dendritic spines of forebrain neurons originating from the patient To construct an in vitro model that matches clinical genotypes, this embodiment uses samples from carriers... STXBP1 E549V / - Peripheral blood was collected from children with heterozygous mutations, reprogrammed to obtain iPSCs, and then sequentially induced into neural stem cells (NSCs) and forebrain neurons (iPSCs). Figure 8 A). Immunofluorescence showed that the normal control (NC) and STXBP1 E549V / - Both groups of NSCs highly expressed Sox2 and Nestin, suggesting comparable neural stem characteristics. Figure 8B); the neurons obtained from further differentiation all expressed MAP2 and the forebrain marker FOXG1, proving that forebrain-like neurons could be successfully obtained in both groups. Figure 8 C).

[0085] Based on this, quantification of dendrites and dendritic spines of patient-derived neurons was performed using double-labeled staining with MAP2 and STXBP1. Compared with the NC group, STXBP1 E549V / - The neuronal dendrites became thinner and shorter, with fewer branches and a significantly reduced density of dendritic spines, suggesting a marked absence of excitatory synaptic structures. This morphological pattern is consistent with the aforementioned... STXBP1 The reduction in dendrites and synapses observed in the mPFC of loss-of-function mice is consistent with this. Following transfection with STXBP1-AAV (1×10^4 vg / mL), STXBP1 E549V / - The total dendritic length, number of branches, and dendritic spine density of neurons all significantly increased, reaching levels close to those of the NC group, indicating that AAV-mediated... STXBP1 Supplementation can reconstruct structural synaptic phenotypes parallel to in vivo experiments in patient-derived forebrain neurons. Figure 8 D).

[0086] 7. STXBP1-AAV rescue STXBP1 E549V / - Synaptic proteins in neurons from patients and BDNF-TrkB-AKT- CaMKII Signal exist Figure 9 In the experiment shown, further experiments were conducted on NC+ empty virus, STXBP1 E549V / - +empty virus and STXBP1 E549V / - In the forebrain neurons derived from the +STXBP1-AAV group of patients, presynaptic, postsynaptic, and neurotrophic / plasticity signaling-related proteins were systematically detected to explore... STXBP1 E549V / - Molecular pathology of mutations and AAV mediation STXBP1 The basis for its supplementary role.

[0087] At the presynaptic level, STXBP1 (Munc18-1) and STX1A are key factors involved in synaptic vesicle docking and membrane fusion, synapsin-1 (SYN-1) is involved in maintaining the vesicle reservoir, and synaptophysin (SYP) reflects the number of synaptic vesicles. STXBP1 E549V / - + In the empty viral group, STXBP1, STX1A, SYN-1 and SYP were all significantly downregulated, indicating a reduction in the number of synaptic vesicles available for release, decreased vesicle docking and fusion efficiency, and impaired overall neurotransmitter release capacity. STXBP1E549V / - In the +STXBP1-AAV group, the levels of the above proteins all rebounded to varying degrees, indicating AAV-mediated... STXBP1 The supplement not only restored STXBP1 Its own expression also reconstructs, to some extent, the molecular basis required for synaptic vesicle mobilization and release.

[0088] At the postsynaptic level, PSD95 is the core scaffold protein of the excitatory postsynaptic density, used to anchor NMDA and AMPA receptors; GRIN2A and GRIN2B are key subunits of the NMDA receptor, determining Ca^2+ conduction and plasticity characteristics; and the phosphorylation status of GluR1 and GluR2 (p-GluR1 / GluR1, p-GluR2 / GluR2) reflects the functional activation of AMPA receptors and the regulation of synaptic strength. STXBP1 E549V / - In the empty viral genome, PSD95, GRIN2A, and GRIN2B were significantly downregulated, and the p-GluR1 / GluR1 and p-GluR2 / GluR2 ratios were significantly reduced, indicating a decrease in mature excitatory postsynaptic structures and low levels of NMDA / AMPA receptor-mediated glutamate transfer and activity-dependent plasticity. This overall pattern of reduced postsynaptic scaffold and receptor plasticity corroborates the previously observed reduction in dendritic spines. STXBP1 E549V / - In the +STXBP1-AAV group, the expression of PSD95 and GRIN2A / GRIN2B significantly increased, and p-GluR1 / GluR1 and p-GluR2 / GluR2 also recovered to levels close to those of the NC+ empty viral group, suggesting that the excitatory postsynaptic density structure and glutamate receptor function were substantially corrected.

[0089] At the level of neurotrophic factors and signal transduction, BDNF is an activity-dependent neurotrophic factor. Activation of the TrkB receptor can initiate downstream pathways such as AKT and CaMKIIα, thereby maintaining synaptic stability and plasticity. STXBP1 E549V / - + The BDNF level was decreased in the empty viral genome, and the phosphorylation ratios of TrkB (p-TrkB / TrkB), p-AKT / AKT, and p-CaMKIIα / CaMKIIα were all significantly reduced, suggesting BDNF-TrkB-AKT-CaMKI The overall activity of the signal axis was weakened, neurotrophic support was insufficient, and synaptic plasticity signaling was suppressed, which is consistent with the results of low BDNF pathway activity in previous animal models. STXBP1 E549V / - In the +STXBP1-AAV group, BDNF expression was upregulated, and the ratios of p-TrkB / TrkB, p-AKT / AKT, and p-CaMKIIα / CaMKIIα were significantly restored, indicating that the correction STXBP1While exhibiting defects, AAV also reactivates key signaling pathways that maintain excitatory synaptic stability and plasticity. Combining these results, STXBP1 E549V / - + Empty viral mass release presynaptically, postsynaptic receptor-scaffold complex, and BDNF-TrkB-AKT-CaMKI All three levels of the signal exhibit consistent functional impairment, while STXBP1 E549V / - The +STXBP1-AAV group showed synergistic recovery in these three aspects, which corresponds in vivo and in vitro with the improvement in dendritic spine density and the aforementioned animal experimental results.

[0090] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for long-term treatment or improvement STXBP1 A pharmaceutical composition for related neurological diseases, characterized in that, The invention comprises a nucleic acid construct, said nucleic acid construct including or capable of producing the following polynucleotides (1) and / or (2): (1) Polynucleotides with sequences as shown in SEQ ID No. 2; (2) The polynucleotides in (1) above that have been modified with nucleotides and have the same function.

2. The method for long-term treatment or improvement according to claim 1 STXBP1 A pharmaceutical composition for related neurological diseases, characterized in that, It further includes pharmaceutically acceptable excipients.

3. The method for long-term treatment or improvement according to claim 1 STXBP1 A pharmaceutical composition for related neurological diseases, characterized in that, The pharmaceutical composition is an injectable preparation.

4. The method for long-term treatment or improvement according to claim 1 STXBP1 A pharmaceutical composition for related neurological diseases, characterized in that, The pharmaceutical composition can be administered via intracerebral injection.

5. A method for long-term treatment or improvement STXBP1 Nucleic acid constructs related to neurological diseases, characterized in that, The nucleic acid construct includes or is capable of producing the following polynucleotides (1) and / or (2): (1) Polynucleotides with sequences as shown in SEQ ID No. 2; (2) The polynucleotides in (1) above that have been modified with nucleotides and have the same function.

6. The pharmaceutical composition according to any one of claims 1-4, or the nucleic acid construct according to claim 5, characterized in that, The STXBP1 Related neurological disorders refer to those in which the subject is diagnosed with the presence of STXBP1 E549V / - mutation.

7. The nucleic acid construct of claim 5 in the preparation of a treatment or improvement STXBP1 Application in medications for related neurological diseases.

8. A method for determining the effectiveness of a test drug in treating or improving [the condition]. STXBP1 The method for the effectiveness of treatment of related neurological diseases is characterized by, Includes the following steps: (1) Provide a cell model or animal model and detect relevant indicators including the expression level of STXBP1 protein to obtain the first parameter, wherein the cell model or animal model contains STXBP1 E549V / - mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including the expression level of STXBP1 protein are detected to obtain the second parameter; (3) Compare the first parameter and the second parameter.

9. The method for determining the effectiveness of a test drug in treating or improving [the condition] according to claim 8. STXBP1 The method for the effectiveness of treatment of related neurological diseases is characterized by, The indicators further include neuropathological indicators, which include at least one of the following: expression of synaptic function-related proteins, number of neurons, activation status of nervous system cells, oxidative stress level, total dendritic length, number of dendritic branches, and dendritic spine density. Preferably, when an animal model is selected, the neuropathological indicators also include at least one of cognitive function, social response, and anxiety phenotype.

10. The method for determining the effectiveness of a test drug in treating or improving [the condition] according to claim 8. STXBP1 The method for the effectiveness of treatment of related neurological diseases is characterized by, The above indicators were measured at least 1.5 months after the animal model was administered the test drug.

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