Use of a recombinant adeno-associated viral vector containing the TBC1D3 gene in the preparation of an epilepsy drug

Abstract

This invention discloses the application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of an epilepsy drug, belonging to the field of biomedical technology. The recombinant adeno-associated virus vector contains a nucleotide sequence capable of expressing the human TBC1D3 protein, and a promoter element for driving transcription of the nucleotide sequence in mammalian cells; the drug contains a therapeutically effective amount of the recombinant adeno-associated virus vector. This invention reduces the subject's susceptibility to epilepsy, prolongs the latency of epileptic seizures, and inhibits epileptic seizures caused by abnormal neuronal discharges in the central nervous system by overexpressing the TBC1D3 protein in the subject's central nervous system through the drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of epilepsy drugs. Background Technology

[0002] Epilepsy is a chronic neurological disorder caused by abnormal, excessive, or highly synchronized discharges of neurons in the brain, ranking second among the most common neurological diseases worldwide. According to clinical epidemiological statistics, more than 70 million people worldwide are affected by epilepsy. Recurrent seizures not only severely impact patients' neurobiological function, cognitive abilities, and psychosocial adaptation, but also impose a heavy medical and economic burden on individuals, families, and society as a whole. Currently, clinical treatment for epilepsy mainly relies on traditional antiepileptic drugs (ASMs). Although there are more than 30 types of antiepileptic drugs available, most of these drugs can only temporarily control seizure symptoms by inhibiting neuronal excitability, and cannot fundamentally repair damaged neuronal networks or reverse disease progression. More seriously, about one-third of patients gradually develop drug resistance during long-term drug intervention, eventually developing drug-resistant epilepsy. Therefore, in-depth exploration of the pathogenesis of epilepsy and the search for novel molecular targets that can regulate neuroplasticity and inhibit abnormal discharges are urgent technical challenges that need to be addressed in the fields of neuroscience and clinical medicine.

[0003] TBC1D3 is a human-specific gene located on chromosome 17. Current research indicates that TBC1D3 can regulate gene expression by activating downstream signaling pathways, thereby participating in the occurrence and development of various tumors, including renal cell carcinoma and breast cancer. In recent years, the biological functions of TBC1D3 in non-tumor fields have gradually attracted attention; for example, it has been shown to participate in tissue repair, macropinocytosis, and the regulation of insulin-related functions. In basic research on the central nervous system, some researchers have found that TBC1D3 can promote the generation of basal neural progenitor cells in mice and induce cerebral cortex folding. It is also believed to promote neuronal proliferation and prolong synaptic plasticity. However, current techniques and theoretical research only reveal the basic physiological functions of TBC1D3. Regarding the mechanism of highly synchronized epileptic seizures, how to effectively deliver TBC1D3 to specific brain regions and leverage its advantages in regulating neuroplasticity remains a challenge, lacking mature application exploration. Currently, there is still a lack of effective means to safely, accurately, and stably utilize this gene to intervene in and treat epilepsy. Summary of the Invention

[0004] This invention provides an application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of epilepsy drugs, which solves the problem that traditional antiepileptic drugs are prone to drug resistance and cannot fundamentally cure the disease.

[0005] This invention provides the application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of an epilepsy drug. The recombinant adeno-associated virus vector contains a nucleotide sequence capable of expressing the human TBC1D3 protein and a promoter element for driving the transcription of the nucleotide sequence in mammalian cells. The drug contains a therapeutically effective amount of the recombinant adeno-associated virus vector. The drug is used to overexpress the TBC1D3 protein in the central nervous system of a subject, thereby reducing the subject's susceptibility to epilepsy, prolonging the latency of epileptic seizures, and inhibiting epileptic seizures caused by abnormal neuronal discharges in the central nervous system.

[0006] In one optional implementation, the recombinant adeno-associated virus vector is an AAV viral particle carrying a TBC1D3 gene expression cassette, which includes, from the 5' end to the 3' end, a 5' inverted terminal repeat sequence, the promoter element, the nucleotide sequence capable of expressing human TBC1D3 protein, a polyadenylate signal sequence, and a 3' inverted terminal repeat sequence.

[0007] In one alternative implementation, the drug is formulated as a liquid injection dosage form, which further includes at least one pharmaceutically acceptable carrier selected from sterile phosphate buffer and physiological saline.

[0008] In one alternative implementation, the drug targets the cerebral cortex or the CA1 region of the hippocampus within the central nervous system.

[0009] In one alternative implementation, the epilepsy includes temporal lobe epilepsy, epilepsy with generalized tonic-clonic seizures, or drug-resistant epilepsy.

[0010] In one alternative implementation, the drug is used to reduce the frequency of seizures in the subject and to alleviate the clinical severity of seizures.

[0011] In one alternative implementation, the drug is a formulation suitable for stereotactic microinjection into the brain.

[0012] In one alternative implementation, the recombinant adeno-associated virus vector is used to specifically infect the neurons within the central nervous system and to prolong neuronal synaptic plasticity by translating and overexpressing the TBC1D3 protein in the neurons.

[0013] In one alternative implementation, the drug is used to specifically express the TBC1D3 protein via the recombinant adeno-associated virus vector, thereby inhibiting highly synchronized firing of the neurons and thus exerting a neuroprotective effect.

[0014] In one alternative implementation, the drug is formulated as a single-dose pre-filled syringe or vial containing a dose of recombinant adeno-associated virus vector sufficient to maintain continuous expression of the TBC1D3 protein in the central nervous system for at least 30 days.

[0015] Beneficial effects: This invention provides the application of a recombinant adeno-associated virus (AAV) vector containing the TBC1D3 gene in the preparation of epilepsy drugs. By utilizing the recombinant AAV vector as a highly efficient gene delivery tool, the nucleotide sequence capable of expressing the human TBC1D3 protein is targeted and delivered to the central nervous system (CNS) of the subject, achieving stable overexpression of the TBC1D3 protein in brain tissue. This effectively overcomes the limitations of existing technologies, such as the lack of neurally targeted delivery methods for the TBC1D3 gene and the difficulty in fundamentally curing refractory epilepsy. By targeting and overexpressing the TBC1D3 protein in the central nervous system of the subject, this invention can effectively prolong neuronal synaptic plasticity, fundamentally inhibit epileptic seizures caused by abnormal, highly synchronized neuronal discharges, reduce the subject's susceptibility to epilepsy, and significantly prolong the latency period of epileptic seizures, providing a novel gene therapy strategy for drug-resistant epilepsy. More importantly, the technical solution of this invention avoids the systemic off-target toxicity, cognitive impairment and drug resistance problems caused by traditional systemic ASMs. It can exert a long-lasting and stable anti-epileptic and neuroprotective effect in the targeted brain region with only a single or small dose, thus improving the clinical treatment effect and drug safety of epilepsy. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0017] Figure 1 This is an immunofluorescence verification result of the specific expression of the TBC1D3 gene in neurons of the targeted brain region (CA1 region of the hippocampus) in mice, as described in this embodiment of the invention. Figure 2This is a statistical analysis result of the effect of recombinant AAV-TBC1D3 drug on the seizure behavior and Racine score of ka-induced epilepsy model mice in the embodiments of the present invention.

[0018] Figure 3 This is a statistical analysis result of the effect of recombinant AAV-TBC1D3 on the seizure behavior and Racine score of a ptezil-induced epilepsy model mouse in this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope thereof, and all such modifications and variations fall within the scope of protection of this invention.

[0020] This invention provides the application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of an epilepsy drug. Specifically, the recombinant adeno-associated virus vector comprises a nucleotide sequence capable of expressing the human TBC1D3 protein and a promoter element for driving the transcription of the nucleotide sequence in mammalian cells. The drug comprises a therapeutically effective amount of the recombinant adeno-associated virus vector, and is used to overexpress the TBC1D3 protein in the central nervous system of a subject, thereby reducing the subject's susceptibility to epilepsy, prolonging the latency of epileptic seizures, and inhibiting epileptic seizures caused by abnormal neuronal discharges in the central nervous system.

[0021] To further optimize and refine the above technical solutions, the present invention also provides the following optional implementation methods, which can be used individually or in combination without logical conflict.

[0022] In one optional implementation, the recombinant adeno-associated virus vector is an AAV viral particle carrying a TBC1D3 gene expression cassette. The TBC1D3 gene expression cassette, from its 5' end to its 3' end, comprises: a 5' inverted terminal repeat (ITR), the promoter element, the nucleotide sequence capable of expressing the human TBC1D3 protein, a polyadenylation signal (PolyA) sequence, and a 3' inverted terminal repeat. The 5' and 3' inverted terminal repeat sequences are cis-acting elements essential for AAV viral genome replication and packaging. The promoter element can be selected from cytomegalovirus (CMV) promoters, elongation factor-1 alpha (EF-1α) promoters, or neuron-specific synapsin (Syn) promoters, to ensure efficient transcription of the target gene in target cells. The polyadenylation signal sequence is used to terminate transcription and maintain the stability of messenger RNA (mRNA).

[0023] In one alternative implementation, the drug is formulated as a liquid injectable dosage form, which further includes at least one pharmaceutically acceptable carrier selected from sterile phosphate-buffered saline (PBS) and physiological saline. The pharmaceutically acceptable carrier not only maintains the three-dimensional structural stability of the recombinant AAV virus particles and preserves their infectivity, but also regulates the osmotic pressure and pH of the injection solution to match the physiological environment of cerebrospinal fluid (CSF), thereby minimizing tissue irritation and inflammatory responses induced during intracerebral injection.

[0024] In one alternative implementation, the drug targets the cerebral cortex or the CA1 region of the hippocampus within the central nervous system. The hippocampus and cerebral cortex are core brain regions where epileptic abnormal discharges most frequently originate and spread. The CA1 region, in particular, with its dense neuronal network and abundant synaptic connections, is a key target for various epileptogenic factors. By precisely delivering the drug to these specific brain regions, an intervention can be directly exerted at the source of epilepsy.

[0025] In one alternative implementation, the epilepsy includes temporal lobe epilepsy (TLE), epilepsy with generalized tonic-clonic seizures (GTCS), or drug-resistant epilepsy (DRE). This indicates that the drug provided by the present invention has broad clinical application prospects, especially for subtypes of refractory epilepsy that cannot be effectively controlled by current traditional ASMs, providing a fundamental alternative to gene intervention.

[0026] In one alternative implementation, the drug is used to reduce the frequency of seizures in the subject and alleviate the clinical severity of seizures. Clinical severity can be objectively assessed using medically recognized behavioral scales (such as the Racine scoring system), and reducing seizure frequency and severity are core evaluation indicators for measuring the clinical efficacy of antiepileptic drugs.

[0027] In one alternative implementation, the drug is a formulation suitable for stereotactic microinjection into the brain. Stereotactic surgery utilizes a three-dimensional coordinate system to precisely and specifically inject minute amounts of drug formulations into tiny nuclei or cortical regions within the brain. This localized physical delivery method can cross the blood-brain barrier (BBB), ensuring high drug concentrations at the lesion site while effectively avoiding off-target toxicity and systemic side effects associated with systemic intravenous or oral administration.

[0028] In one alternative implementation, the recombinant adeno-associated virus vector is used to specifically infect the neurons within the central nervous system and prolong neuronal synaptic plasticity by translating and overexpressing the TBC1D3 protein in the neurons. Synaptic plasticity is the ability of neurons to modulate the intensity of information transmission between them. Epilepsy is often accompanied by pathological remodeling of synaptic structures. The drug of this invention corrects abnormal synaptic connections through molecular mechanisms, restoring the homeostasis of the neural network at both structural and functional levels.

[0029] In one alternative implementation, the drug is used to specifically express the TBC1D3 protein via the recombinant adeno-associated virus vector, thereby inhibiting highly synchronized firing of neurons and exerting a neuroprotective effect. Highly synchronized firing is the direct electrophysiological basis of epileptic seizures. Expression of the TBC1D3 protein can regulate ion channel function or inhibit the excessive release of excitatory neurotransmitters, disrupting this pathological synchronization network and protecting neurons from excitotoxic-induced apoptosis.

[0030] In one alternative implementation, the drug is formulated as a single-dose pre-filled syringe or vial containing a dose of recombinant adeno-associated virus (AAV) vector sufficient to maintain continuous TBC1D3 protein expression in the central nervous system for at least 30 days. The AAV vector possesses the biological property of maintaining an episome state and stably expressing exogenous genes in non-dividing cells (such as mature neurons) for extended periods. This implementation ensures that patients achieve a durable anti-epileptic therapeutic effect after a single minimally invasive administration, improving patient compliance.

[0031] The following section provides a detailed and step-by-step verification and explanation of the above-mentioned optional implementation methods through specific experimental materials, methods, and results. Healthy male C57BL / 6 mice, aged 8 weeks and weighing approximately 22 to 25 grams, were used in the experiment. All mice were placed in a standard specific pathogen-free (SPF) animal facility for at least one week for acclimatization before the experiment. The room temperature was controlled at 22°C to 24°C, and the relative humidity was maintained at 50% to 60%. A 12-hour light-dark cycle was used, and the mice had free access to sufficient sterile, complete nutritional feed and filtered pure water.

[0032] Human Embryonic Kidney 293T cells (HEK293T cells) were used in the experiment, which are standard in this field. This cell line was routinely passaged in Durbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture.

[0033] The main biochemical reagents used in the experiment included kainic acid (KA), pentylenetetrazole (PTZ), iodixanol, restriction endonucleases EcoRI and BamHI, T4 DNA ligase, polyethylenimine (PEI) transfection reagent, primary antibodies, and secondary antibodies with fluorescent groups. All of the above reagents are commercially available products commonly used in this field.

[0034] The following details the entire process of preparing a recombinant adeno-associated virus vector containing the TBC1D3 gene, which is the core active ingredient of the drug of this invention.

[0035] Step S101: Obtaining the target gene sequence and preparing the vector backbone. Based on the complete coding sequence (CDS) of the human TBC1D3 gene published in the National Center for Biotechnology Information (NCBI) database, the entire gene was artificially synthesized. Specific restriction endonuclease recognition sites (EcoRI and BamHI) were introduced at both ends of the synthesized sequence to facilitate subsequent targeted cloning. Simultaneously, a shuttle plasmid backbone (named pAAV-Syn-MCS-mCherry) containing the AAV2 serotype inverted terminal repeat (ITR) was selected. This backbone incorporates the neuron-specific Synapsin (Syn) promoter and the cherry red fluorescent protein (mCherry) gene as an expression indicator. The TBC1D3 gene and the mCherry gene are linked by a P2A self-cleaving peptide sequence to ensure that the two proteins can be co-expressed proportionally under the same transcript.

[0036] Step S102: Construction and identification of recombinant expression plasmids. The TBC1D3 target gene fragment and the pAAV shuttle plasmid backbone were double-digested using EcoRI and BamHI restriction endonucleases in a 37°C water bath for two hours. The digestion reaction system included restriction endonucleases, matching buffers, and an appropriate amount of nuclease-free purified water. The digestion products were separated by 1% agarose gel electrophoresis and purified using a gel extraction kit. Subsequently, the purified target gene fragment was ligated to the linearized plasmid backbone overnight at 16°C using T4 deoxyribonuclease. The ligation product was transformed into competent *Escherichia coli* DH5α using a heat shock method and plated on ampicillin-resistant agar plates. The next day, single colonies were selected, inoculated into liquid medium, and cultured in a shaker at 37°C and 220 rpm. After plasmid extraction, the recombinant plasmid (named pAAV-Syn-TBC1D3-P2A-mCherry) was sequenced using Sanger sequencing to ensure that the TBC1D3 gene sequence was accurate and the reading frame was completely correct.

[0037] Step S103: Packaging and harvesting of AAV virus particles. Since the central nervous system has a high affinity for AAV9 serotype, this experiment selected packaging AAV9 virus. Healthy HEK293T cells were seeded in 15 cm cell culture dishes and transfected when cell confluence reached 70-80%. Using PEI transfection reagent, the constructed recombinant expression plasmid (pAAV-Syn-TBC1D3-P2A-mCherry), helper plasmid (pHelper), and packaging plasmid (pRep2Cap9) were mixed in equal mass ratios, thoroughly complexed with PEI reagent, and then added dropwise to HEK293T cells. After transfection, the cells were cultured at 37°C in a cell culture incubator containing 5% carbon dioxide for 72 hours. During this period, a large number of recombinant AAV virus particles were assembled and generated within the cells. After 72 hours, the cells and their culture supernatant were collected. The cells were then completely ruptured by repeated freeze-thaw cycles (alternating between liquid nitrogen flash freezing and thawing in a 37°C water bath three times) to release the AAV virus particles encapsulated within the cells.

[0038] Step S104: Purification and titer determination of recombinant AAV virus particles. The cell lysate containing the virus was centrifuged at low speed (4°C, 3000 rpm for 15 minutes) to remove cell debris, and the virus-rich crude supernatant was collected. Subsequently, high-purity purification was performed using iodixanol density gradient ultracentrifugation. Different concentration gradients of iodixanol solution (e.g., 10%, 25%, 40%, and 60%) were sequentially layered in ultracentrifuge tubes. The crude supernatant was added very slowly to the top layer, and the tubes were ultracentrifuged at 60,000 rpm and 18°C ​​for two hours. Virus particles accumulated in a specific density layer, forming visible bands. These virus-rich bands were carefully aspirated with a syringe and desalted and concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. Finally, the virus particles were resuspended in PBS, aliquoted, and stored at -80°C for later use. Viral titer was determined using quantitative real-time PCR (qRT-PCR). Purified viral genomic DNA was extracted, and specific primers were designed using the conserved ITR sequence in the viral vector as the amplification target. A series of plasmids with known copy numbers were prepared as standards. The test viral sample and standards were subjected to PCR amplification under the same reaction system and cycling conditions. After amplification, the instrument software automatically fitted a standard curve based on the cycling threshold (Ct value) of the standards and calculated the physical concentration value (unit: vector genome per milliliter, vg / mL) of the test sample on the standard curve by comparing it with the cycling threshold. This process was completed using a built-in algorithm, without relying on manual mathematical calculations. The final titer of the recombinant AAV-TBC1D3 virus solution prepared in this experiment was determined to be 2.5 × 10⁻⁶. 12 The concentration of vg / mL met the requirements for subsequent in vivo animal experiments. As a control group, a virus expressing only the empty mCherry vector (named AAV-Vector) was also packaged and prepared.

[0039] The following details how stereotactic injection of drugs into the brain was performed on laboratory animals, and how a KA-induced epilepsy model was established. Animal experiments were conducted in strict accordance with laboratory animal ethics and welfare requirements.

[0040] Step S201: Stereotaxic prophylactic injection of recombinant AAV drug. Before inducing seizures, the aforementioned prepared AAV drug needs to be delivered into the mouse brain to allow sufficient time for TBC1D3 protein expression. Mice are anesthetized via intraperitoneal injection using a 1% sodium pentobarbital solution at a dose of 50 mg per kilogram of body weight. After the righting reflex disappears and the response to pain stimuli is sluggish, confirming stable anesthesia, the mouse's head is shaved clean using an animal-specific shaver, and its head is securely fixed to a precision stereotaxic apparatus. Ensure the fixation pins are accurately inserted into both external auditory canals, the incisor hooks are fixed to the palate, and the apparatus is adjusted to ensure the mouse's skull is completely horizontal. The surgical area on the mouse head was routinely disinfected three times with medical iodine-soaked cotton balls. After the iodine dried naturally, the skin was longitudinally incised along the midline of the skull (approximately 1.0 to 1.5 cm in length) with a scalpel. The subcutaneous connective tissue and periosteum were gently separated using micro-forceps. The skull surface was then thoroughly exposed using sterile hydrogen peroxide. Gentle manipulation was essential during the procedure to avoid damaging cerebral blood vessels and underlying brain tissue. Under a microscope, the anterior fontanelle (Bregma), an anatomical landmark where cranial sutures meet, was located and its coordinates were set as the zero point of the three-dimensional coordinate system. Based on standard mouse brain anatomy atlases, the spatial three-dimensional coordinates of the right hippocampal CA1 region were precisely calculated and located. In this step, the specific coordinate parameters of the target area were set as follows: anterior-posterior (AP) coordinate of -2.0 mm, lateral-lateral (ML) coordinate of +1.5 mm, and dorsoventral (DV) coordinate (i.e., subdural depth) of -1.7 mm. After localization, mark the corresponding location on the skull surface and gently drill a tiny hole at the marked location using a dental micro-drill. Strict control of the pressure and depth is crucial during drilling; stop immediately upon penetrating the skull to avoid damaging the delicate brain tissue beneath the meninges. Secure the microsyringe (a glass microelectrode needle from a microinjection pump) pre-loaded with recombinant AAV-TBC1D3 viral solution to the stereotactic unit's operating arm, align it with the center of the hole, and slowly lower the operating arm until the needle penetrates the dura mater to a predetermined dorsoventral depth of -1.7 mm. Start the microinjection pump and inject 1.0 μL of viral solution into the CA1 region of the hippocampus at a very stable rate of 0.1 μL per minute. Animals serving as the control group receive the same volume of AAV-Vector viral solution under the same coordinates and conditions. After injection, to prevent backflow of the drug along the injection path due to excessive pressure within the brain tissue, leave the needle in place for 10 minutes.Subsequently, the operating arm was rotated and lifted very slowly to withdraw the injection needle. The skull cavity and surgical area were disinfected again with povidone-iodine. The scalp was intermittently sutured with surgical sutures containing suture needles, and antibiotic ointment was applied to the sutured incision and surrounding skin. Postoperatively, the mice were placed on a constant-temperature heated recovery pad set at 37°C and closely observed. Once their heartbeat and respiration were stable, they had recovered from anesthesia, and were able to move independently, they were returned to their original cages for routine care.

[0041] Step S202: Chemical induction of the epilepsy animal model. To evaluate the antiepileptic efficacy of the TBC1D3 gene in vivo, epilepsy was induced 3 weeks after intracerebral injection of the AAV virus and routine feeding (allowing sufficient time for TBC1D3 protein to reach steady-state overexpression levels in neurons of the target brain region). Anesthesia, skin preparation, fixation, disinfection, incision, and drilling procedures were performed using the same procedures as in step S201. The target area was repositioned to the right hippocampal CA1 region (coordinates remained -2.0 mm anterior-posterior, +1.5 mm lateral, and -1.7 mm dorsoventrally). A pre-prepared chemical epileptogenic agent, lycine (concentration 0.6 μg / μL), prepared with sterile saline, was loaded into a microsyringe. A total volume of 0.5 μL of KA solution was injected into the target site at a very stable rate. This chemical agent activates glutamate receptors in the central nervous system, inducing neuronal hyperexcitability and synchronized abnormal discharge, thereby constructing the classic TLE animal model. After the injection, the needle is left in place for 5 minutes to prevent reflux. After slowly withdrawing the needle, the scalp is sutured, disinfected, and postoperative constant temperature recovery care is performed.

[0042] The following details the technical effects of this invention in "reducing the susceptibility of subjects to epilepsy, prolonging the latency period of epileptic seizures, and inhibiting epileptic seizures caused by abnormal discharges".

[0043] Step S301: Visual verification of TBC1D3 gene expression at the target site. To confirm whether the injected drug successfully overexpressed at the target site, 3 weeks after AAV injection and before KA induction, a random number of mice were selected for brain tissue morphology and immunofluorescence (IF) detection. Mice were deeply anesthetized, and their blood was washed away by perfusion of physiological saline, followed by tissue fixation with 4% paraformaldehyde (PFA) fixative. The brain was completely removed, subjected to gradient dehydration in sucrose solution, and then cryopreserved using an optimal sectioning temperature complex. Coronal hippocampal brain slices (approximately 30 micrometers thick) were cut using a cryostat. After routine washing and blocking, primary antibodies specifically recognizing TBC1D3 protein and neuron-specific nucleoprotein primary antibodies were added, and the slices were incubated overnight at 4°C. The next day, after washing, secondary antibodies with different fluorescent groups were added, and the slices were incubated at room temperature in the dark for two hours. The slide was then mounted with mounting medium and observed under a laser confocal microscope. Figure 1 As shown, in the CA1 region of the hippocampus of mice injected with AAV-TBC1D3, significant TBC1D3-specific red fluorescence signals and mCherry-indicated green fluorescence signals were observed in the cell bodies and dendrites of neurons, and these signals highly overlapped with the neuronal localization labeled with NeuN. This confirms that the drug prepared in this invention can be targeted and delivered into the target neurons of the central nervous system of the subject, achieving stable overexpression of the target protein.

[0044] Step S302: Continuous monitoring and severity assessment of epileptic behavior. Immediately after KA injection to induce epilepsy, mice in each group were individually housed in specially designed transparent glass observation cages. High-resolution infrared night vision cameras were installed above and to the sides of the cages to continuously record the daily behavior and seizure status of all mice 24 hours a day for 30 days from multiple angles. To scientifically and quantitatively assess the clinical severity of seizures, two professional researchers unaware of the mice's experimental groupings conducted a double-blind scoring of the seizure behavior recorded in the video footage, based on the internationally recognized Racine behavioral scoring system. The specific behavioral definitions of the Racine scoring criteria are detailed as follows: Grade 0: The test animal behaves completely normally, with no abnormal behavioral manifestations related to epilepsy; Grade 1: Mild facial muscle twitching, frequent chewing, or involuntary rhythmic tremors of the whiskers occur; Grade 2: In addition to Grade 1 symptoms, rhythmic and frequent head nodding occurs; Grade 3: Sustained clonic twitching occurs in one or both forelimbs; Grade 4: The animal stands on its hind limbs with severe twitching of the forelimbs, adopting a kangaroo-like upright posture; Grade 5: Loss of balance while upright, falling heavily backward or sideways, accompanied by generalized tonic and clonic seizures; this is a severe level of epileptic seizure. Data from the experiment were summarized and statistically analyzed, and one-way ANOVA was used to compare differences between groups. Figure 2 As shown, the statistical results indicate that compared with the control group injected with empty vector virus, the experimental group mice pre-injected with AAV-TBC1D3 showed a statistically significant difference in the time point (i.e., latency period) at which they first experienced a grade 3 or higher severe seizure. Throughout the 30-day monitoring period, the average daily seizure frequency in the experimental group mice decreased. Furthermore, according to the Racine scoring system, most mice in the control group reached a grade 4 or 5 seizure level; while in the experimental group mice pre-treated with TBC1D3, seizure severity was suppressed, with most mice exhibiting mild non-convulsive symptoms limited to grades 1 to 2, and very few mice progressing to grade 5 falls. This demonstrates that the drug possesses the core efficacy of reducing the severity of clinical seizures and protecting subjects.

[0045] Step S303: Electroencephalogram (EEG) monitoring of abnormal synchronized discharges. To verify the inhibitory effect of the drug on abnormal neuronal discharges from an electrophysiological perspective, multi-channel miniature EEG recording electrodes were precisely implanted into the lesion brain region (i.e., the CA1 region of the hippocampus) and the corresponding cortical region of mice during stereotactic injection surgery. The electrodes were connected to a multi-channel neurophysiological signal acquisition system for animals via flexible insulated wires and miniature interfaces. High-sampling-rate continuous recording of changes in local field potentials (LFP) after KA induction was performed using a dedicated hardware signal amplifier and software filtering and noise reduction system. The EEG recording results showed that the control group animals that did not receive effective drug intervention frequently exhibited high-amplitude, high-frequency pathological spikes, spike waves, and continuous bursts of spike-and-slow-wave complexes in their EEG spectra. These abnormal waveforms were characterized by long duration and frequent occurrences, all typical epileptic-like abnormal EEG signals generated by the synchronized and bursty discharges of a large number of neurons. Conversely, in subjects treated with the AAV-TBC1D3 drug of this invention, the frequency of the aforementioned pathological abnormal discharge waveforms in their electroencephalograms decreased, the duration of each abnormal discharge was shortened, and the overall background rhythm of the electroencephalogram stabilized, gradually approaching the electroencephalographic characteristics of normal healthy mice. This fully demonstrates at the physical signal level that the drug can inhibit abnormal synchronous discharge of neurons at its source by intervening in the central nervous system.

[0046] The following details the pharmacodynamic validation based on a PTZ-based chronic epilepsy model. To further demonstrate that the drug of this invention has both therapeutic and protective effects on epilepsy induced by different pathological mechanisms (especially epilepsy with generalized tonic-clonic seizures and chronic ignition models), this experiment used another classic chemical epileptogen—PTZ—to construct a chronic epilepsy ignition model.

[0047] Step S401: Modeling Method and Drug Administration Strategy. Healthy mice were selected, and the AAV-TBC1D3 liquid formulation of this invention was precisely injected into the hippocampus and cerebral cortex of the mice using stereotactic microinjection technology. After a 3-week waiting period for sufficient expression, PTZ chronic ignition induction was initiated. PTZ powder was dissolved in sterile saline to prepare an injection solution of a specific concentration. Mice were injected intraperitoneally at a subconvulsive dose of 35 mg PTZ per kilogram of mouse body weight, once every other day at a fixed time, for approximately 30 days (a total of about 15 injections). The so-called chronic "ignition" process refers to the gradual reduction of the excitability threshold of the brain's neuronal network through repeated administration of low doses of epileptogenic chemical stimulation, ultimately leading to irreversible spontaneous generalized tonic-clonic seizures in response to originally non-epileptic stimuli. This model effectively simulates the pathological progression of refractory chronic epilepsy in humans.

[0048] Step S402, Result Evaluation. For control mice that received only empty vector virus injection, the Racine seizure score induced after each PTZ injection showed a steady upward trend with each subsequent injection. Typically, around the 10th to 12th injection, most control mice were successfully "ignited," exhibiting a typical and severe grade 5 generalized tonic-clonic seizure. In contrast, the experimental group mice, which received prior intervention with the recombinant AAV-TBC1D3 drug of this invention, showed higher tolerance to PTZ chemical stimulation (i.e., reduced susceptibility to epilepsy). Under completely identical dosing frequency and dosage, the experimental group mice were extremely difficult to be completely ignited, and the highest seizure grade throughout the entire modeling period was generally suppressed to grade 2 or 3 or lower, with short seizure duration. This confirms that the drug of this invention has definite interventional value in preventing the formation of chronic epileptogenic foci and blocking the progressive deterioration of the epilepsy network.

[0049] The following details the in-depth mechanism by which TBC1D3 gene-modulating drugs regulate synaptic plasticity and exert neuroprotective effects. To support the technical characteristics of drugs "by prolonging neuronal synaptic plasticity" and "exerting neuroprotective effects" at the microscopic molecular and cellular levels, this experiment conducted in-depth investigations using ex vivo brain slice patch-clamp electrophysiological techniques and molecular biology techniques.

[0050] Step S501: Ex vivo electrophysiological recording of synaptic plasticity. Synaptic plasticity is a core foundation for learning, memory, and maintaining neural network homeostasis in the brain. Long-term potentiation (LTP) is a key electrophysiological indicator for measuring the strength of synaptic plasticity. Acute hippocampal tissue sections from mice after KA-induced epilepsy were prepared and rapidly incubated in a constant-temperature artificial cerebrospinal fluid (ACSF) continuously perfused with a mixture of 95% pure oxygen and 5% carbon dioxide. ACSF contains precisely proportioned sodium chloride, potassium chloride, calcium chloride, and glucose to maintain cell viability. After incubation, the tissue sections were transferred to a microscope electrophysiological recording chamber fixed on a vibration-damping table, maintaining continuous ACSF perfusion. High-frequency stimulation (HFS) was applied to the Schaffer collaterals of the hippocampus using concentric bipolar electrodes. Simultaneously, glass microelectrodes perfused with intracellular fluid were precisely inserted into the stratum radiatum of the CA1 region of the hippocampus. The slope and amplitude changes of the field excitatory postsynaptic potential (fEPSP) were recorded. Experimental results showed that in epileptic model mice untreated with TBC1D3, due to repeated abnormal epileptic discharges leading to synaptic structural damage and impaired neurotransmitter release function, both the amplitude and duration of LTP after high-frequency stimulation induction were significantly reduced, indicating pathological impairment of synaptic plasticity. In contrast, brain slices from mice receiving the AAV-TBC1D3 drug of this invention showed that neurons in the CA1 region maintained a more robust and sustained LTP response. This indicates that after TBC1D3 protein is translated and expressed at the synapse of target neurons, it can effectively regulate the release probability of synaptic vesicles or stabilize the density of postsynaptic membrane receptors, thereby prolonging and improving the synaptic plasticity of damaged neurons.

[0051] Step S502: Neuronal morphology and apoptosis marker detection. Recurrent epilepsy inevitably leads to irreversible apoptosis of hippocampal neurons and the loss of dendritic spines responsible for information reception. This study used Golgi staining to silver-stained mouse brain tissue to clearly delineate the complete three-dimensional morphology of neurons. Microscopic morphological observation revealed that the TBC1D3 treatment group showed a significantly increased number of dendritic branches in the hippocampus, a more complex dendritic network, and a higher overall density of dendritic spines (especially the proportion of mature mushroom-shaped dendritic spines responsible for homeostatic information transmission) compared to the untreated epilepsy control group. Furthermore, total protein was extracted from the lesioned hippocampal tissue, and the expression levels of neuronal apoptosis-related proteins were detected by Western blotting (WB). First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate protein bands of different molecular weights. These bands were then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with skim milk, and then bound with specific antibodies. Finally, chemiluminescent imaging was performed. Analysis showed that after treatment with the gene therapy drug of this invention, the expression levels of pro-apoptotic proteins (such as Bax and Caspase-3 protein in a cleavage-activated state) in the lesion tissue were significantly inhibited and downregulated, while the expression levels of proteins with anti-apoptotic effects (such as Bcl-2 protein) were significantly upregulated. Simultaneously, the expression levels of postsynaptic density protein 95 (PSD-95) and synaptophysin I, which directly reflect the degree of synaptic density, were also significantly restored. This confirms, from two different evaluation dimensions—microscopic molecular expression and macroscopic cellular morphology—that the drug of this invention possesses neuroprotective functions in preventing pathological apoptosis of neurons and maintaining the integrity of neuronal synaptic structure.

[0052] The following details the preparation of a single-dose stereotactic microinjection formulation for the brain and its long-term efficacy and stability studies. To meet the requirements of the preclinical evaluation system and the practical needs of future translation into human clinical applications, the physical dosage form and packaging scheme of this gene therapy were optimized and explained in this phase.

[0053] Step S601: Preparation of a single-dose stereotactic micro-injection formulation. Based on the requirements of micro-volume and high precision that must be met when administering drugs invasively to the brain using stereotactic technology, this nucleic acid drug is specially formulated as a liquid micro-injection preparation. Under the strictly aseptic conditions of a laminar flow hood, the purified and concentrated recombinant AAV-TBC1D3 virus solution is serially diluted using sterile phosphate buffer filtered through terminal pores for sterilization. To avoid inducing cerebral edema or osmotic shock, the pH of the diluent is adjusted to between 7.2 and 7.4 to match the physiological pH and ionic environment of the subject's cerebrospinal fluid. Because the volume that the central nervous system can accommodate is extremely limited, the volume of the drug solution injected per dose must be strictly controlled. In small rodent models, this is typically controlled at the level of several μL (e.g., 1.0 μL); in clinical applications for human patients, the volume can be scaled up to tens or hundreds of μL depending on the volume calculated by MRI of the specific target nucleus, but it is still considered a micro-volume. The prepared liquid formulation, with a therapeutically effective concentration, is aseptically filled and sealed into vials made of medical-grade borosilicate neutral glass, or pre-filled into single-dose pre-filled syringes with integrated micro-gradient markings. This single-dose, individual packaging helps eliminate the risk of cross-contamination that may occur during multi-dose extraction in clinical settings, ensuring the safety of gene therapy drugs when they enter the central nervous system.

[0054] Step S602: Long-term expression and efficacy stability verification. The recombinant AAV viral vector used in this invention has its internal genome artificially cleansed of all pathogenic gene fragments related to viral self-replication. Therefore, after entering neurons, it typically does not randomly integrate into the host's chromosomal genome, thus avoiding the risk of insertional mutations. Instead, it exists stably in the cell nucleus as an independent, free episome, thereby relying on the host cell's transcriptional machinery to achieve long-term, continuous exogenous gene expression. At long-term milestones such as day 30, day 60, and even up to 120 days after the stereotactic single-dose microinjection into the test mice, researchers again performed pathological sampling and epilepsy chemical induction tests. The results showed that not only was high-abundance continuous expression of TBC1D3 protein still detectable in the target brain region (exceeding the clinical maintenance time limit of at least 30 days), but the subject also maintained anti-epileptic seizure, anticonvulsant, and resistance to abnormal neuronal synchronization discharge when faced with chemical epileptogenic stimuli such as KA or PTZ. This long-term observational data demonstrates that, through the single-dose micro-injection technology of this invention, coupled with a specially designed carrier formulation, an innovative gene therapy drug can be provided for refractory epilepsy caused by congenital genetic defects or drug resistance resulting from long-term oral medication. This drug is effective for a long time with a single dose and can reshape the homeostasis of the neural network in the brain.

[0055] In summary, this invention, through the scientific construction of a recombinant AAV viral vector specifically overexpressing the TBC1D3 gene and its formulation into a specific micro-liquid dosage form for targeted and precise delivery to specific regions of the central nervous system in subjects, has achieved clear and objective superior technical effects across multiple evaluation levels, including effectively prolonging neuronal synaptic plasticity, inhibiting abnormal synchronized discharges, exerting deep neuroprotective effects, and reducing the severity of clinical epileptic seizures. These experimental data collectively and fully validate the various technical features and optional implementation methods of this invention, demonstrating the application value of this gene-based drug in the preparation of innovative clinical antiepileptic drugs.

[0056] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of a recombinant adeno-associated virus vector containing the TBC1D3 gene in the preparation of epilepsy drugs, characterized in that, The recombinant adeno-associated virus vector comprises a nucleotide sequence capable of expressing the human TBC1D3 protein and a promoter element for driving the transcription of the nucleotide sequence in mammalian cells; the drug comprises a therapeutically effective amount of the recombinant adeno-associated virus vector, the drug being used to overexpress the TBC1D3 protein in the central nervous system of a subject, thereby reducing the subject's susceptibility to epilepsy, prolonging the latency of epileptic seizures, and inhibiting epileptic seizures caused by abnormal neuronal discharges in the central nervous system.

2. The application according to claim 1, characterized in that, The recombinant adeno-associated virus vector is an AAV virus particle carrying a TBC1D3 gene expression cassette. The TBC1D3 gene expression cassette includes, from the 5' end to the 3' end, the following in sequence: a 5' inverted terminal repeat sequence, the promoter element, the nucleotide sequence capable of expressing human TBC1D3 protein, a polyadenylate signal sequence, and a 3' inverted terminal repeat sequence.

3. The application according to claim 1, characterized in that, The drug is formulated as a liquid injection dosage form, which further includes at least one pharmaceutically acceptable carrier selected from sterile phosphate buffer and physiological saline.

4. The application according to claim 1 or 3, characterized in that, The drug targets the cerebral cortex or the CA1 region of the hippocampus within the central nervous system.

5. The application according to claim 1, characterized in that, The epilepsy includes temporal lobe epilepsy, epilepsy with generalized tonic-clonic seizures, or drug-resistant epilepsy.

6. The application according to claim 1, characterized in that, The drug is used to reduce the frequency of epileptic seizures in the subject and to alleviate the clinical severity of epileptic seizures.

7. The application according to claim 4, characterized in that, The drug is a formulation suitable for stereotactic micro-injection into the brain.

8. The application according to claim 1, characterized in that, The recombinant adeno-associated virus vector is used to specifically infect the neurons in the central nervous system and to prolong neuronal synaptic plasticity by translating and overexpressing the TBC1D3 protein in the neurons.

9. The application according to claim 1, characterized in that, The drug is used to specifically express the TBC1D3 protein through the recombinant adeno-associated virus vector, thereby inhibiting highly synchronized firing of the neurons and exerting a neuroprotective effect.

10. The application according to claim 3, characterized in that, The drug is formulated in the form of a pre-filled syringe or vial in a single-dose package containing a dose of recombinant adeno-associated virus vector sufficient to maintain continuous expression of the TBC1D3 protein in the central nervous system for at least 30 days.

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