Pharmaceutical composition for treating KCNQ2 developmental epileptic encephalopathy, nucleic acid construct and application

Abstract

The invention discloses a pharmaceutical composition for treating KCNQ2 developmental epileptic encephalopathy, a nucleic acid construct and application of the pharmaceutical composition and the nucleic acid construct. According to the invention, by introducing a nucleic acid construct carrying the hKCNQ2 gene into a KCNQ2A294V / + developmental epileptic encephalopathy animal model and a human forebrain neuron model, the effectiveness of the nucleic acid construct in improving the expression level of the KCNQ2 and improving related neuropathological mechanisms is verified. Not only is a new hope provided for treatment of KCNQ2-related developmental epileptic encephalopathy provided, but also a new direction is opened up for application of gene therapy in neurodevelopmental diseases.

Description

Technical Field

[0001] This invention relates to the fields of medical technology and gene therapy, specifically to a method for treating... KCNQ2 Drug compositions, nucleic acid constructs and applications for developmental epileptic encephalopathy. Background Technology

[0002] Epilepsy is a chronic disease caused by highly synchronized abnormal electrical discharges in the brain, resulting in temporary brain dysfunction. According to WHO data, more than 50 million people worldwide suffer from epilepsy, with approximately 10 million in China, and this number is increasing by 600,000 cases annually, resulting in an annual economic burden exceeding 20 billion RMB. Developmental and epileptic encephalopathy (DEE) is one of the most severe types of epilepsy. It is a serious neurodevelopmental disorder characterized by frequent seizures and significant developmental delays or intellectual disabilities. DEE can occur at any age, but it is most common and severe in infancy and early childhood. Infancy and early childhood are critical periods for the maturation and development of brain morphology, neural connections, and synaptic plasticity. DEE not only has underlying causes leading to developmental delays but also progressive functional impairment caused by uncontrollable seizures. This is different from the sequelae caused by simple status epilepticus and the temporary damage caused by occasional epileptic seizures. It will irreversibly affect the child's brain development, aggravate the damage to the child's existing functions and the development of age-related new functions, and ultimately seriously affect the child's motor, language, intellectual development and social function.

[0003] for KCNQ2 -DEE( KCNQ2 For developmental epileptic encephalopathy caused by pathogenic mutations, the first-line symptomatic treatment includes drugs such as sodium channel blockers. Common sodium channel blockers include oxcarbazepine, carbamazepine, phenytoin sodium, and lamotrigine. Although these drugs are effective in controlling epileptic seizures, they have limited therapeutic effects. KCNQ2 The neurodevelopmental improvement and long-term prognosis of DEE remain limited. Furthermore, some patients may develop resistance to conventional antiepileptic drugs, leading to poor treatment outcomes.

[0004] Therefore, there is an urgent need for a treatment or improvement method. KCNQ2 Drugs for developmental epileptic encephalopathy, thereby reversing the condition through a long-term mechanism of action. KCNQ2 The resulting irreversible impact on brain development.

[0005] 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

[0006] To address at least some of the technical problems in the prior art, the present invention provides a method for salvage... KCNQ2 Gene therapy regimens for mutation-induced developmental epileptic encephalopathy. In some implementations, recombinant vectors (pFD-rAAV-ITR-hSyn-) are used. hKCNQ2 -WPRE-BGHpA), with KCNQ2 Gene expression to correct due to KCNQ2 Neurological dysfunction caused by mutations. The recombinant vector developed in this invention has high transduction efficiency in the central nervous system, and the hSyn neuron-specific promoter used can achieve better therapeutic effects. This invention fundamentally improves cognitive impairment in developmental epileptic encephalopathy by regulating synaptic plasticity in the brain. Specifically, this invention includes the following:

[0007] A first aspect of the present invention provides a treatment KCNQ2 Nucleic acid constructs for related developmental epileptic encephalopathy, 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.

[0008] A second aspect of the present invention provides a treatment KCNQ2 A pharmaceutical composition for developmental epileptic encephalopathy, wherein the pharmaceutical composition comprises the nucleic acid construct described in the first aspect.

[0009] In some embodiments, the treatment according to the present invention KCNQ2 A pharmaceutical composition for developmental epileptic encephalopathy, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0010] In some embodiments, the treatment according to the present invention KCNQ2 A pharmaceutical composition for developmental epileptic encephalopathy, wherein the pharmaceutical composition is an injectable preparation.

[0011] In some embodiments, the treatment according to the present invention KCNQ2 A pharmaceutical composition for developmental epileptic encephalopathy, wherein the pharmaceutical composition is administered via intracerebral injection.

[0012] In some embodiments, the treatment according to the present invention KCNQ2 A pharmaceutical composition for related developmental epileptic encephalopathy, wherein, the KCNQ2Related developmental epileptic encephalopathy refers to a condition in which a subject is diagnosed with epilepsy. KCNQ2 A294V / + Missense mutation.

[0013] A third aspect of the invention provides nucleic acid constructs for the preparation of drugs for improving or treating diseases. KCNQ2 Application in medications for developmental epileptic encephalopathy.

[0014] A fourth aspect of the invention provides a method for determining the effectiveness of a test drug in treating or improving [the condition]. KCNQ2 An effective method for treating developmental epileptic encephalopathy includes the following steps: (1) Provide a cell model or animal model, and detect relevant indicators including KCNQ2 protein expression level and neuropathological markers to obtain the first parameter, wherein the cell model or animal model has KCNQ2 A294V / + Missense mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including KCNQ2 protein expression level and neuropathological indicators are detected to obtain the second parameter; (3) Compare the first parameter and the second parameter.

[0015] In some embodiments, the determination of the test drug for treatment or improvement according to the present invention is performed... KCNQ2 A method for assessing the effectiveness of the test drug in developmental epileptic encephalopathy, wherein the above-mentioned indicators are detected in the animal model after 3 weeks of administration of the test drug, and in the cell model after 1 week of administration of the test drug.

[0016] In some embodiments, the determination of the test drug for treatment or improvement according to the present invention is performed... KCNQ2 A method for the effectiveness of developmental epileptic encephalopathy, wherein, when an animal model is selected, the neuropathological indicators include at least one of social competence, anxiety indicators, cognitive competence, frequency or duration of spontaneous epileptic seizures, and dendritic spine density.

[0017] This invention by providing KCNQ2 A294V / + Introducing carriers into animal models of developmental epileptic encephalopathy and human forebrain neuronal cell models hKCNQ2 Gene recombination vectors have been used to verify their effectiveness in improving... KCNQ2 The effectiveness of expression levels and improvement of related neuropathological mechanisms. This not only provides for KCNQ2 This provides new hope for the treatment of developmental epileptic encephalopathy and opens up new directions for the application of gene therapy in neurodevelopmental diseases.

[0018] Furthermore, the technical effects of the present invention also include: (1) Targeted treatment: This invention provides a targeted treatment... KCNQ2 A specific treatment for neurological diseases caused by genetic defects involves precise gene replacement to directly target the cause of the disease.

[0019] (2) Highly efficient gene transduction: This invention utilizes the high efficiency of the vector and introduces the neuron-specific hSyn promoter to achieve specific and efficient gene delivery to neurons, thereby improving the therapeutic effect while reducing the potential impact on non-target tissues.

[0020] (3) Comprehensive behavioral improvement: This invention significantly improves KCNQ2 The point mutant mice exhibited a variety of behavioral phenotypes, including anxiety-like behavior, social interaction, cognitive abilities, and the frequency and duration of spontaneous epileptic seizures, demonstrating the comprehensiveness and effectiveness of the treatment.

[0021] (4) Synaptic function recovery: This invention reveals that by restoring dendritic spine density, the recombinant vector promotes the repair and functional recovery of the nervous system by improving synaptic function.

[0022] (5) Potential clinical application prospects: This invention provides a method suitable for human use. KCNQ2 Gene therapy strategies for related diseases provide a scientific basis and therapeutic potential for developing new clinical treatments.

[0023] (6) Scientific and social benefits: This invention not only provides scientific benefits for... KCNQ2 A deeper understanding of related diseases is expected to reduce patient suffering and medical costs at the societal level, and improve patients' quality of life. Attached Figure Description

[0024] Figure 1 The recombinant vector structure constructed according to the present invention is shown.

[0025] Figure 2 It shows KCNQ2 A294V / + Three weeks after gene therapy, the time mice spent in the elevated zero maze open arm was measured.

[0026] Figure 3 It shows KCNQ2 A294V / + Three weeks after gene therapy in mice, the interaction time and interaction index of social interaction and social preference in each group of mice were compared.

[0027] Figure 4 It shows KCNQ2 A294V / + Three weeks after gene therapy in mice, the spontaneous alternation rate in the water maze and Y-maze was measured in each group of mice.

[0028] Figure 5 It shows KCNQ2 A294V / + Three weeks after gene therapy in mice, the frequency and duration of spontaneous epileptic seizures were monitored by electroencephalography (EEG) in each group of mice.

[0029] Figure 6 It shows KCNQ2 A294V / + Dendritic spine density was measured in the prefrontal cortex and hippocampus of mice after gene therapy.

[0030] Figure 7 It shows KCNQ2 A294V / + Dendritic spine density of neurons in each group was measured one week after gene therapy for mutant neurons.

[0031] Figure 8 It shows KCNQ2 A294V / + After gene therapy in mice, the levels of KCNQ2 protein and RNA in the prefrontal cortex and hippocampus of mice in each group were detected. 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 constructs One aspect of the present invention provides a treatment KCNQ2 Nucleic acid constructs for related developmental epileptic encephalopathy, 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," "nucleotide," "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... KCNQ2 Complementary DNA (overexpression) KCNQ2The encoding sequence), the termination sequence, and the ITR sequence adjacent to the above sequences, preferably an ITR from AAV2 or AAV9. In a specific implementation, it is used to correct the presence of [unclear] in the subject. KCNQ2 A294V / + The coding sequence for the missense 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) remains extremely challenging due to difficulties in efficiently delivering the target gene to the target tissue, 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 pFD-rAAV-ITR-hSyn- as shown in SEQ ID No. 2. hKCNQ2 -WPRE-BGHpA.

[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 KCNQ2 Pharmaceutical compositions for developmental epileptic encephalopathy One aspect of the present invention provides a treatment KCNQ2 A pharmaceutical composition for developmental epileptic encephalopathy, 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, the composition for treating developmental epileptic encephalopathy is an injectable preparation, particularly suitable for administration to a subject 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. KCNQ2 Its application in drugs for related developmental epileptic encephalopathy.

[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... KCNQ2 A294V / + Subjects with missense mutations, especially those with [missing information], KCNQ2 A294V / + Subjects suffering from developmental epileptic encephalopathy due to missense 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" KCNQ2 "Related developmental epileptic encephalopathy" refers to conditions that can be improved or treated due to the presence of... KCNQ2 A294V / + Neurodevelopmental disorders caused by missense mutations, wherein the treatment or improvement includes at least one of the following: (1) Improve cognitive impairment and adaptive behavior deficits; (2) Improve learning, communication and social participation skills; (3) Reduce the frequency and duration of spontaneous epileptic seizures; (4) Restore or improve KCNQ2 Level of expression; (5) Restore or reduce dendritic spine density; (6) Effectiveness in restoring synaptic function and improving neurobehavioral function; (7) Promotes the repair and functional recovery of the nervous system; (8) Reduce the power of delta waves and theta brain waves.

[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]. KCNQ2 Effective methods for developmental epileptic encephalopathy One aspect of the present invention provides a method for determining the effect of a test drug on treating or improving [the condition / condition]. KCNQ2 Effective methods for treating developmental epileptic encephalopathy include: (1) Provide a cell model or animal model, and detect relevant indicators including KCNQ2 protein expression level and neuropathological markers to obtain the first parameter, wherein the cell model or animal model has KCNQ2 A294V / + Missense mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including KCNQ2 protein expression level and neuropathological indicators are detected to obtain the second parameter; (3) Compare the first parameter and the second parameter.

[0056] In a preferred embodiment, the indicator is KCNQ2 A294V / + Missense mutation-related neuropathological markers, including but not limited to at least one of social skills, anxiety index, cognitive ability, frequency or duration of spontaneous epileptic seizures, and dendritic spine density.

[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... KCNQ2 conduct KCNQ2 A294V / + Methods for heterozygous point 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 are observed in the second parameter (including improvements in cognitive impairment and adaptive behavior deficits; improvements in learning, communication, and social participation abilities; and a reduction in the frequency and duration of spontaneous epileptic seizures), and / or changes in the expression levels of related genes are detected ( KCNQ2 An increase in the amount of [something] and / or changes in synaptic structure (decreased dendritic spine density) indicates that the test drug is effective in treating developmental epileptic encephalopathy. If no improvement is observed in the above indicators in the second parameter, it indicates that the test drug is not effective in treating [something]. KCNQ2 Developmental epileptic encephalopathy.

[0059] In some embodiments, the method for testing the drug to be tested according to the present invention is described. KCNQ2 A method for assessing the efficacy of the drug in developmental epileptic encephalopathy, wherein the above-mentioned indicators are detected in animal models 3 weeks after administration of the drug to be tested, and in cells 1 week after administration of the drug to be tested.

[0060] Example I. Construction of AAV Vector Design and build portable KCNQ2 AAV2 vector for the gene: AAV- KCNQ2 Contains people KCNQ2 Complementary DNA, driven by the potent neuron-specific promoter hSyn KCNQ2 Gene expression. The final overexpressed AAV virus is: pFD-rAAV-ITR-hSyn- hKCNQ2 -WPRE-BGHpA (Fiter: 3.77E+13 vg / ml), abbreviated as AAV- KCNQ2 . KCNQ2 Control virus: pFD-rAAV-ITR-hSyn-WPRE-BGHpA (Fiter: 1.8E+13 vg / ml), abbreviated as AAV-NC.

[0061] II. Preparation of Experimental Animals and Cell Models Through homology comparison, it was found that patients with developmental epileptic encephalopathy carry [the virus / carrier]. KCNQ2 A294V / + A missense mutation was used, changing the amino acid from alanine A to valine V. This led to the creation of a corresponding mutant mouse, which was then created by precisely introducing a point mutation via homology-guided repair. KCNQ2 A294V / + Mice.

[0062] III. AAV Vector Drug Delivery To explore AAV- KCNQ2 Whether the behavioral phenotype of point mutant mice can be salvaged was investigated in this embodiment by overexpressing AAV- in the bilateral lateral ventricles. KCNQ2 Behavioral testing was then conducted. AAV- was injected into the brain using a stereotactic injection method. KCNQ2 The vector was injected into both 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 used to level the head anteriorly, posteriorly, laterally, and laterally using Bregma as the zero point. The injection coordinates were 0.5 mm posterior to the anterior fontanelle and 0.8 mm lateral to both sides, with a needle insertion depth 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. The AAV virus was slowly injected (50 nL / min) into the bilateral LV brain regions and allowed to remain and spread for 10 min. Mouse grouping: At 3 weeks of age (equivalent to approximately 6 months in humans), mice were randomly divided into 5 groups of 12 mice each, half male and half female. 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 4.5 × 10⁻⁶ AAV-NC. 10 vg(WT group); b) KCNQ2 A294V / + Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of 4.5 × 10⁴ AAV-NC. 10 vg(Mut group); c) KCNQ2 A294V / + Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of AAV- KCNQ2 3.75×10 9 vg(AAV-L group); d) KCNQ2 A294V / + Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of AAV- KCNQ2 1.3×10 10 vg(AAV-M group); e) KCNQ2 A294V / + Mutant mice were injected with 2 μL of diluted virus into each of their bilateral ventricles, for a total injection of AAV- KCNQ2 4.5×10 10 vg(AAV-H group).

[0063] 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.

[0064] This embodiment uses commercially available pluripotent stem cells from healthy individuals, which are then differentiated into forebrain neurons. The cell model is divided into three groups: neurons from a healthy control group (HC group), neurons from a patient-specific point mutation group (Mut group), and point-mutated neurons treated with AAV (AAV group). The AAV infection multiplicity was set to 1 × 10⁻⁶. 5 The infection occurred on day 7 of neuronal differentiation, and the samples were collected on day 14.

[0065] IV. Behavioral Assessment 1. AAV- KCNQ2 right KCNQ2 A294V / + Behavioral rescue assessment of mice KCNQ2 A294V / + Behavioral assessments were performed on mice three weeks after viral injection, and the results showed that gene therapy in all three dose groups effectively rescued the virus. Figures 2-4This embodiment first examined the effect in the elevated zero maze, showing that compared to the WT group mice, the Mut group mice had significantly less time spent in the open arm, indicating that the mutant mice exhibited an anxiety phenotype. After low, medium, and high doses of AAV- KCNQ2 After rescue, the mutant mice spent significantly more time in the open arm, indicating that their anxiety was significantly improved. Figure 2 Next, AAV- was explored. KCNQ2 The effects on the social abilities of mice were analyzed using a three-box social study. KCNQ2 Mutations lead to a lack of social interaction and reduced social preference in mice, while AAV treatment can significantly correct the social abnormalities in mice. Figure 3 Finally, the cognitive abilities of each group of mice were tested. Figure 4 In the Y-maze, AAV treatment significantly increased the mutation rate. KCNQ2 A294V / + The circulation index of mice. Similarly, in a 6-day water maze experiment, wild-type mice showed a gradual decrease in the time required to find the platform as the number of training days increased, demonstrating the effectiveness of the training and indicating normal learning and memory abilities. However, the mutant group mice took significantly longer to find the platform than the wild-type group. On day 6 of the water maze, the platform was removed in this example, and it was found that the Mut group had a shorter exploration time in the original platform quadrant compared to the WT group. These results reveal impaired learning and memory abilities in point mutant mice. Subsequently, the point mutant mice underwent AAV- KCNQ2 After treatment, the plateau finding time for AAV-M / H was shortened in the first 5 days, while the exploration time for AAV-L / H on the original plateau was significantly increased on day 6 compared to the Mut group. These results indicate that the system constructed in this embodiment... KCNQ2 A294V / + The mice exhibited a learning and memory impairment phenotype, and AAV- KCNQ2 This phenotype can be rescued in mice, with high doses of AAV- KCNQ2 The best rescue effect is achieved.

[0066] 2. KCNQ2 -AAV reduction KCNQ2 A294V / + Frequency and duration of spontaneous epileptic seizures in mutant mice In this embodiment, cortical electroencephalography (EEG) was performed on mice in each group. Figure 5 Compared to the WT group, mice in the Mut group exhibited spontaneous epilepsy. Figure 5 AD). Spontaneous epileptic seizures in mice manifest in various forms, including frequent facial twitching (such as blinking and whisker twitching), and falling or generalized convulsions. However, point mutant mice treated with low, medium, and high doses of AAV- KCNQ2 Treatment reduced the total number of seizures in all groups of mice. Furthermore, the average seizure duration was significantly shortened in the AAV-M / H group mice. Figure 5 E, F).

[0067] This embodiment also statistically analyzed the delta-wave and theta-wave power of mice in each group, finding that the power of the Mut group mice was significantly higher than that of the WT group mice, while the power of the AAV-M / H group mice was decreased to varying degrees compared to the Mut group mice. Figure 5 G, H). These results reveal that after a seizure in point-mutant mice, the power of delta and theta waves increases accordingly, reflecting an imbalance in the excitation-inhibition balance within the mouse brain. After AAV- KCNQ2 After treatment, the imbalance between excitation and inhibition in the mouse brain was improved to some extent. These results indicate that the [treatment method / mechanism] constructed in this embodiment... KCNQ2 A294V / + The mice exhibited a spontaneous epileptic phenotype. AAV- KCNQ2 It can reduce the frequency and duration of spontaneous epileptic seizures in point mutant mice.

[0068] V. Dendritic spine density detection Dendritic spines are the primary receivers of excitatory synaptic input and the basic units of neural computation in the mammalian brain. Changes in the density, size, and shape of mature dendritic spines, or defects in the formation of dendritic spines and synapse establishment during brain development, can lead to neuronal dysfunction and trigger cognitive and / or behavioral disorders. In this study, Golgi staining was performed on the prefrontal cortex and hippocampus of mice in the WT, Mut, and AAV-L / M / H groups to observe the structure of neuronal dendritic spines. The results showed that the density of neuronal dendritic spines in the prefrontal cortex and hippocampus of mice in the Mut group was significantly increased compared to that in the WT group. Furthermore, intraventricular injection of low, medium, and high doses of AAV-L / M / H... KCNQ2 Subsequently, the density of dendritic spines in the prefrontal cortex and hippocampus of mice in the AAV-L / M / H groups almost returned to normal, and was close to that of the WT group mice. Figure 6 This embodiment observed the dendritic spine density of neurons in each group. It was found that compared to the HC group, the dendritic spine density in the Mut group was significantly increased, while the dendritic spine density in the AAV group returned to the level of the HC group. Figure 7 These results reveal AAV- KCNQ2 save KCNQ2 A294V / + The mutant mice and their neurons exhibited an abnormal increase in dendritic spines.

[0069] VI. Biochemical Indicator Testing This embodiment tested mice in each group. KCNQ2 Overexpression verification, and comparison with KCNQ2 The pathological mechanisms associated with the mutations were explored. Results showed that in the prefrontal cortex and hippocampus, the Mut group mice showed significantly higher mutation rates compared to the WT group mice. KCNQ2Protein levels decreased, while mRNA levels did not differ significantly. Then, low, medium, and high doses of AAV- were injected via the lateral ventricle. KCNQ2 Subsequently, in the prefrontal cortex and hippocampus of mice in each of the AAV-L / M / H groups... KCNQ2 Both mRNA and protein levels were significantly elevated. Furthermore, the elevated levels showed a dose-dependent relationship with AAV dosage. Figure 8 ).

[0070] 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 treatment or improvement KCNQ2 The pharmaceutical composition for related developmental epileptic encephalopathy is characterized by, 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 treatment or improvement according to claim 1 KCNQ2 The pharmaceutical composition for related developmental epileptic encephalopathy is characterized by, It further includes pharmaceutically acceptable excipients.

3. The treatment or improvement according to claim 1 KCNQ2 The pharmaceutical composition for related developmental epileptic encephalopathy is characterized by, The pharmaceutical composition is an injectable preparation.

4. The treatment or improvement according to claim 1 KCNQ2 The pharmaceutical composition for related developmental epileptic encephalopathy is characterized by, The pharmaceutical composition can be administered via intracerebral injection.

5. A treatment or improvement KCNQ2 Nucleic acid constructs for related developmental epileptic encephalopathy, 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 KCNQ2 Related developmental epileptic encephalopathy refers to a condition in which a subject is diagnosed with epilepsy. KCNQ2 A294V / + Missense mutation.

7. The nucleic acid construct of claim 5 in the preparation of a treatment or improvement KCNQ2 Application in medications for developmental epileptic encephalopathy.

8. A method for determining the effectiveness of a test drug in treating or improving [the condition]. KCNQ2 A method for the effectiveness of developmental epileptic encephalopathy, characterized in that, Includes the following steps: (1) Provide a cell model or animal model, and detect relevant indicators including KCNQ2 protein expression level and neuropathological markers to obtain the first parameter, wherein the cell model or animal model has KCNQ2 A294V / + Missense mutation; (2) The test drug is administered to the cell model or animal model, and then relevant indicators including KCNQ2 protein expression level and neuropathological indicators 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. KCNQ2 A method for the effectiveness of developmental epileptic encephalopathy, characterized in that, The neuropathological indicators include at least one of the following: social skills, anxiety index, cognitive ability, frequency or duration of spontaneous epileptic seizures, and dendritic spine density.

10. The method for determining the effectiveness of a test drug in treating or improving [the condition] according to claim 8. KCNQ2 A method for the effectiveness of developmental epileptic encephalopathy, characterized in that, The above indicators were detected in the animal model after 3 weeks of administration of the test drug, and in the cell model after 1 week of administration of the test drug.

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