Potassium channel gene treatment method for treating parkinson's disease

Abstract

A potassium channel gene treatment method for treating Parkinson's disease. A recombinant adeno-associated virus (AAV) nucleotide sequence comprises a nucleotide sequence encoding an inwardly rectifying potassium channel Kir. An AAV vector is used to deliver an inwardly rectifying potassium channel (Kir) gene to the subthalamic nucleus, such that excitability of neurons of the subthalamic nucleus is reduced, so as to alleviate symptoms such as motor deficits caused by excessive excitation of the subthalamic nucleus in patients with Parkinson's disease.

Description

A potassium channel gene therapy for treating Parkinson's disease Technical Field

[0001] This invention belongs to the field of gene therapy technology for Parkinson's disease, specifically relating to a potassium channel gene therapy method for treating Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a neurological disorder characterized primarily by movement disorders. Patients often exhibit symptoms such as resting tremor, bradykinesia, and muscle rigidity, specifically a significant reduction in the amplitude and speed of movement, as well as difficulty initiating movement.

[0003] Globally, the prevalence of Parkinson's disease is approximately 100 to 300 people per 100,000, with the incidence increasing significantly with age. According to the Parkinson's Disease and Movement Disorders Society International (MDS), the incidence rate in people over 65 years of age is approximately 10 to 20 new cases per 100,000 people per year. The incidence rate is generally higher in men than in women, with a sex ratio of approximately 2:1. Furthermore, the prevalence of Parkinson's disease is relatively high in Europe, the Americas, and some high-income countries, which is related to the aging population, healthcare levels, and early diagnosis capabilities in these regions. The peak age of onset for Parkinson's disease is typically over 60, although there are also some early-onset cases (i.e., onset before age 40).

[0004] The core lesion in Parkinson's disease (PD) is generally considered to be the progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNc). Dopamine deficiency leads to a severe imbalance in motor regulation in the basal ganglia, particularly in the indirect pathway. In a healthy brain, the indirect pathway of the basal ganglia finely regulates movement by inhibiting the activity of the globus pallidus (GPe) and thalamus through GABAergic neurons. Due to the death of dopaminergic neurons in the SNc, insufficient dopamine supply impairs the function of D2 receptors, causing GABAergic neurons to lose their normal regulatory function. As a result, the inhibitory function of GABAergic neurons weakens, leading to overexcitation of signals in the indirect pathway, which in turn exacerbates symptoms such as bradykinesia and rigidity. Furthermore, neurons in the indirect pathway also exhibit abnormal potassium channel function. Potassium channels play a crucial role in neuronal electrical activity, regulating neuronal excitability. In Parkinson's disease, impaired function of these potassium channels leads to abnormal potassium ion flow, increasing neuronal excitability and triggering overexcitation of neural circuits. This overexcitation of neural circuits further exacerbates motor inhibition, leading to more pronounced bradykinesia and rigidity symptoms in patients. In summary, weakened GABAergic signaling and abnormal potassium channel function in indirect pathways are important pathological changes in Parkinson's disease. These changes reveal the crucial role of this pathway in motor control and provide potential targets for developing novel therapeutic strategies for Parkinson's disease.

[0005] Currently, the main treatments for Parkinson's disease include medication and surgery to help slow disease progression. Medication is the most common treatment for Parkinson's disease, aiming to alleviate symptoms by supplementing or regulating the dopamine system. For example, levodopa is a prodrug that can be converted into dopamine, supplementing the dopamine deficiency in the brain. A drawback is that patients are prone to developing drug tolerance, and long-term use may lead to dyskinesia. The most common surgical treatment is deep brain stimulation (DBS), which involves implanting electrodes into specific brain regions to provide electrical stimulation and alter the patient's motor symptoms. It is particularly effective for patients in the middle and late stages who experience tremors, bradykinesia, and those who do not respond well to medication. DBS uses high-frequency electrical stimulation (130-180Hz) to stimulate the subthalamic nuclei (STN) or the globus pallidus internal segment (GPi), reducing the excitability of these overexcited nuclei and decreasing abnormal neural activity. This inhibitory effect restores the dynamic balance of neural circuits, thereby alleviating the patient's motor symptoms. However, in clinical applications, deep brain stimulation may produce stimulation-induced side effects, such as anxiety or depression.

[0006] While current drug treatments (such as levodopa) and surgical interventions (such as deep brain stimulation) can alleviate some Parkinson's disease symptoms, they cannot effectively slow down or reverse disease progression. In recent years, with the rapid development of gene editing and gene delivery technologies, gene therapy has shown great potential in the treatment of Parkinson's disease and may become a transformative approach for future treatments. The core objective of gene therapy is to introduce compensatory genes via viral vectors to compensate for functional losses caused by gene deletions or mutations. Currently, several gene therapy strategies primarily target dopaminergic neurons. For example, AADC (aromatic L-amino acid decarboxylase) is a key enzyme in dopamine synthesis; however, in PD patients, AADC expression is significantly reduced, leading to decreased dopamine synthesis. AADC gene replacement therapy delivers the AADC gene to the patient's brain via an adeno-associated virus (AAV) vector, thereby promoting dopamine synthesis and improving the patient's motor symptoms. Furthermore, GDNF (glial-derived neurotrophic factor) is a neurotrophic factor that promotes the survival and function of dopaminergic neurons. GDNF gene therapy introduces the GDNF gene into the substantia nigra or striatum via a viral vector, aiming to protect and restore the function of dopaminergic neurons. Although animal experiments have shown significant efficacy, the treatment effect has not yet met expectations in early clinical trials. With continuous improvements in vector technology and administration methods, recent studies have shown more ideal therapeutic effects, indicating a broader prospect for gene therapy in the treatment of Parkinson's disease.

[0007] Existing gene therapy approaches primarily focus on alleviating Parkinson's disease symptoms by enhancing dopamine synthesis or inhibiting dopaminergic neuron death. However, these methods have significant limitations. First, the limited recovery of the dopamine system cannot fully compensate for neuronal damage during disease progression, resulting in incomplete symptom relief. Second, current treatments fail to adequately target excitatory abnormalities in the basal ganglia neural circuits, particularly the dysfunction of nuclei in indirect pathways (such as the STN, GPe, and GPi), thus failing to fundamentally regulate the imbalance in motor control. In contrast, our approach precisely modulates the electrical activity of these circuits by targeting abnormally excitatory nuclei in the basal ganglia, directly improving motor dysfunction. This method does not rely on the recovery of the dopamine system, effectively bypassing the limitations of dopaminergic neuron degeneration, and exhibits higher specificity and sustainability of therapeutic effects. Through sophisticated gene editing and targeted delivery technologies, our treatment method reduces side effects and improves therapeutic precision. Therefore, our approach opens new avenues for the treatment of Parkinson's disease, possessing significant advantages and promising potential, especially in early or mid-stage patients, where it may provide more durable efficacy. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant adeno-associated virus (AAV-Kir), wherein the recombinant adeno-associated virus is an adeno-associated virus with the Kir gene recombined on an expression vector.

[0009] To address the aforementioned technical issues, this application utilizes an adeno-associated virus (AAV) vector to deliver inward rectifier potassium channel genes (Kir2.1, Kir3.2, Kir4.1) to the subthalamic nucleus, thereby reducing the excitability of subthalamic nucleus neurons and rescuing symptoms such as decreased motor function caused by overexcitation of the subthalamic nucleus in Parkinson's disease patients.

[0010] This application also provides a pharmaceutical composition comprising the above-described recombinant adeno-associated virus AAV-Kir and a pharmaceutically acceptable carrier or excipient.

[0011] This application also provides the use of the above-mentioned recombinant adeno-associated virus AAV-Kir or pharmaceutical composition in the preparation of products having one or more of the following functions: 1) prevention and / or treatment of Parkinson's disease; 2) reduction of excitability of neurons in the subthalamic nucleus; 3) improvement of motor ability; 4) increase of motor distance and speed.

[0012] This application also provides a method for treating Parkinson's disease, the method comprising administering to an individual with Parkinson's disease a preventive and / or therapeutically effective amount of the above-described recombinant adeno-associated virus AAV-Kir, pharmaceutical composition, or product used herein.

[0013] This application also provides a nucleic acid construct containing a gene sequence for packaging the above-mentioned recombinant adeno-associated virus AAV-Kir.

[0014] The beneficial effects of this application include, but are not limited to: the adeno-associated virus (AAV-Kir) prepared in this application can be used for 1) prevention and / or treatment of Parkinson's disease; 2) reduction of excitability of neurons in the subthalamic nucleus; 3) improvement of motor ability; and 4) increase of motor distance and speed. Attached Figure Description

[0015] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0016] Figure 1 shows the polyA spectra of plasmid rAAV-EF1α-Kir2.1-P2A-EGFP-WPRE-hGH as illustrated in some examples of this application.

[0017] Figure 2 shows the polyA spectra of plasmid rAAV-EF1α-hKir4.1-P2A-EGFP-WPRE-hGH as illustrated in some examples of this application.

[0018] Figure 3 shows the polyA spectra of plasmid rAAV-EF1α-hKir3.2-P2A-EGFP-WPRE-hGH as shown in some examples of this application.

[0019] Figure 4 shows the action potential current-frequency curves and passive membrane characteristics of neurons in the subthalamic nucleus in an acute brain slice (coronal) of a mouse, as illustrated in some examples of this application.

[0020] Figure 5 shows the action potential current-frequency curves and passive membrane characteristics of neurons in the subthalamic nucleus in acute brain slices (sagittal) of mice, as illustrated in some examples of this application.

[0021] Figure 6 shows the motor function, anxiety, and depression levels of mice after injection of AAV-ef1α-Kir2.1 / Kir3.2 / Kir4.1 into the subthalamic nucleus according to some embodiments of this application, assessed by behavioral tests.

[0022] Figure 7 shows the firing patterns of subthalamic nucleus neurons under 200 pA stimulation according to some embodiments of this application.

[0023] Figure 8 shows the action potential current-frequency curves and passive membrane properties of neurons in the subthalamic nucleus after injection of AAV-ef1α-Kir2.1 / Kir3.2, according to some embodiments of this application.

[0024] Figure 9 shows the firing frequency-input current curves and passive membrane characteristics of subthalamic nucleus neurons in ACSF extracorporeal solution / Baclofen extracorporeal solution / Saclofen extracorporeal solution according to some embodiments of this application.

[0025] Figure 10 shows the firing frequency-input current curves and passive membrane characteristics of subthalamic nucleus neurons in 6OHDA-induced mouse models according to some embodiments of this application in ACSF extracorporeal solution / Baclofen extracorporeal solution / Saclofen extracorporeal solution.

[0026] Figure 11 shows the modeling and detection of blood biochemical levels in the Kir2.1 rescue group according to some embodiments of this application. Detailed Implementation

[0027] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0028] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0029] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0030] This application provides a recombinant adeno-associated virus (AAV-Kir), wherein the recombinant adeno-associated virus is an adeno-associated virus with the Kir gene recombined on an expression vector.

[0031] In some embodiments, the serotype of the recombinant adeno-associated virus may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In some embodiments, preferably, the serotype of the recombinant adeno-associated virus may be AAV9.

[0032] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives.

[0033] In some embodiments, the Kir gene may include any one or more of the Kir2.1, Kir3.2, or Kir4.1 genes.

[0034] In some embodiments, the expression vector of the recombinant adeno-associated virus contains a promoter.

[0035] In some embodiments, the promoter is located upstream of the Kir gene.

[0036] In some embodiments, the promoter may be the ef1α promoter.

[0037] In some embodiments, the nucleotide sequence of the ef1α promoter may be as shown in SEQ ID NO.1.

[0038] In some embodiments, the nucleotide sequence of the Kir2.1 gene may be as shown in SEQ ID NO.2.

[0039] In some embodiments, the nucleotide sequence of the Kir4.1 gene may be as shown in SEQ ID NO.3.

[0040] In some embodiments, the nucleotide sequence of the Kir3.2 gene may be as shown in SEQ ID NO.4.

[0041] This application also provides a pharmaceutical composition comprising the above-described recombinant adeno-associated virus AAV-Kir and a pharmaceutically acceptable carrier or excipient.

[0042] The term “pharmaceutically acceptable” as used in this article generally means a compound, material, composition, and / or dosage form that is suitable, within reasonable medical judgment, for contact with human and animal tissues, organs, and / or body fluids without excessive toxicity, irritation, allergic response, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0043] The excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When used in an aqueous solution for injection, the excipients are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50).

[0044] In the pharmaceutical composition provided in this application, the recombinant adeno-associated virus AAV-Kir can be a single active ingredient, or it can be combined with one or more other active ingredients that are useful for the treatment of diseases to form a combined formulation.

[0045] The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the recombinant adeno-associated virus and the active ingredient in the pharmaceutical composition depends on the patient's weight, the type of application, the condition and severity of the disease.

[0046] This application also provides the use of the above-mentioned recombinant adeno-associated virus AAV-Kir or pharmaceutical composition in the preparation of products having one or more of the following functions: 1) prevention and / or treatment of Parkinson's disease; 2) reduction of excitability of neurons in the subthalamic nucleus; 3) improvement of motor ability; 4) increase of motor distance and speed.

[0047] The term "prevention and / or treatment" (and its grammatical variations) refers to an attempt to alter the natural course of disease in an individual being treated, and can be a clinical intervention performed for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis.

[0048] In some embodiments, the product does not cause anxiety or depression in individuals with Parkinson's disease.

[0049] In some embodiments, the product may be a drug administered via the subthalamus.

[0050] In some embodiments, the product may be an injectable drug administered via the subthalamic nucleus.

[0051] This application also provides a method for treating Parkinson's disease, the method comprising administering to an individual with Parkinson's disease a preventive and / or therapeutically effective amount of the above-described recombinant adeno-associated virus AAV-Kir, pharmaceutical composition, or product used herein.

[0052] The term "effective amount" refers to the quantity or dose of the formulation of this application that, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific formulation administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0053] In some embodiments, the recombinant adeno-associated virus (AAV-Kir), pharmaceutical composition, or product may be administered to the subthalamus.

[0054] In some embodiments, preferably, the administration site of the recombinant adeno-associated virus AAV-Kir, pharmaceutical composition, or product may be the subthalamic nucleus.

[0055] In some embodiments, the method involves administering the recombinant adeno-associated virus (AAV-Kir), pharmaceutical composition, or product to the subthalamus of an individual with Parkinson's disease using a microinjector.

[0056] In some embodiments, the method may involve administering the recombinant adeno-associated virus (AAV-Kir), a pharmaceutical composition, or a product to the subthalamic nucleus of an individual with Parkinson's disease using a microinjector.

[0057] In some embodiments, the method may cause the subthalamus of the Parkinson's disease individual to overexpress the Kir gene.

[0058] In some embodiments, the method may cause the subthalamic nucleus of the Parkinson's disease individual to overexpress the Kir gene.

[0059] This application also provides a nucleic acid construct containing a gene sequence for packaging the above-mentioned recombinant adeno-associated virus AAV-Kir.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0061] Example 1

[0062] 1. Construction and histological examination of PD model mice.

[0063] The principle behind the 6-OHDA injection model into the ventral tegmental area (MFB) of the midbrain is primarily based on the selective toxicity of 6-hydroxydopamine (6-OHDA) to dopaminergic neurons. The MFB is a crucial neural pathway connecting the midbrain and striatum, with dopaminergic axons mainly originating from the substantia nigra and the ventral tegmental area (VTA). 6-OHDA is a compound structurally similar to dopamine and can be efficiently taken up into cells by the dopamine transporter (DAT). This allows 6-OHDA to enter dopaminergic neurons and cause cell damage.

[0064] First, using the classic mouse PD model, 150 nmol of 6-OHDA (10 mg / ml) was injected unilaterally into the medial forebrain bundle (MFB) of mice. After approximately 7 days, the mice exhibited significant somatic motor dysfunction, manifested as bradykinesia and somatic deviation. Histological examination revealed a large number of dead dopaminergic neurons in the substantia nigra of the mouse brain.

[0065] Results: A large number of dopaminergic neurons died on the modeling side in mice, resulting in significant somatic motor dysfunction.

[0066] 2. Construction of Kir2.1 viral expression vector

[0067] The ef1α promoter and Kir2.1 gene sequence were ligated into the adeno-associated virus vector AAV9 (purchased from Brinkes, catalog number BC-3673) to construct the viral vector AAV-ef1α-Kir2.1-GFP. Adeno-associated virus was then packaged according to existing techniques.

[0068] ef1α promoter sequence:

[0069] Human Kir2.1 sequence:

[0070] Human Kir4.1 sequence:

[0071] Human Kir3.2 sequence:

[0072] We injected 60 nl of AAV-ef1α-Kir2.1 / Kir3.2 / Kir4.1-GFP (titer 1*10^12 vg / ml) into the subthalamic nucleus on the ipsilateral side of the model mice. Three weeks later, we performed histological examination on the sections to confirm the injection site and expression status.

[0073] 3. Behavioral testing

[0074] Since viral expression requires a three-week time window, we first injected 60 nmol of AAV-ef1α-Kir2.1 / Kir3.2 / Kir4.1-GFP (titer 1*10^12 vg / ml) or 60 nmol of blank control virus AAV-ef1α-GFP (titer 1*10^12 vg / ml) into the subthalamic nucleus. In the last week of the three-week time window, we injected 150 nmol of 6-OHDA at a concentration of 10 mg / ml into the medial anterior tract. We compared the motor function, anxiety, and depression levels of mice in the control group (injected with saline in the medial forebrain tract), the model group (injected with 150 nmol of 6-OHDA in the medial forebrain tract and 60 nmol of control virus AAV-ef1α-GFP (titer 1*10^12 vg / ml) in the subthalamic nucleus), and the rescue group (injected with 6-OHDA in the medial forebrain tract and AAV-ef1α-Kir2.1-GFP (titer 1*10^12 vg / ml) in the subthalamic nucleus).

[0075] Results: Overexpression of Kir2.1 and Kir4.1 in the subthalamic nucleus of mice significantly increased the distance and speed of movement, and the mice did not exhibit a depressive-like phenotype; however, overexpression of Kir3.2 did not rescue the motor phenotype of the mice (Figure 6).

[0076] In the open field behavioral experiment, the total distance and average speed of the mice in the model group were significantly lower than those in the control group, and their immobility time was significantly prolonged. Furthermore, compared to the control group, the mice in the model group almost completely lost their counter-clockwise rotational behavior and were unable to stand on their hind limbs for sniffing. These behavioral indicators confirm the successful establishment of the Parkinson's disease model. Notably, the rescue group mice showed significant improvement in all of the above symptoms.

[0077] In the rotarod test, the mice in the model group were unable to walk normally, and their dwell time was significantly shorter than that of the control and rescue groups. Furthermore, in the tail suspension test, the mice in the model group showed no obvious depressive tendency, and their struggling intensity was lower than that of the control and rescue groups.

[0078] 4. Electrophysiological recording

[0079] 1) Brain slice preparation

[0080] We used C57BL / 6J mice, approximately 8 weeks old, after the above treatment for our experiments. The heart was perfused with pre-oxygenated 4°C slicing solution (95% O2 and 5% CO2) to replace systemic blood. The mice were then rapidly decapitated and the brain tissue was dissected. The brain tissue was prepared in pre-oxygenated 4°C slicing solution with the following formulation (unit: millimoles): 213 sucrose, 2.5 KCl, 2 MgSO4, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, 25 glucose (osmolarity 315 mOsm, pH 7.4). The tissue was sliced ​​into 250–350 μm thick slices using a vibratory microtome (Leica VT1200S). The slices were then transferred to artificial cerebrospinal fluid in a 35°C constant-temperature water bath and incubated for 35 minutes, after which they were transferred to room temperature until electrophysiological recording was performed. During electrophysiological recording, brain slices were placed in a recording chamber with artificial cerebrospinal fluid circulation and perfusion, maintained at a temperature of 34–35°C, with a perfusion rate of approximately 1.2 ml / min. Cells on the brain slices were observed on a monitor using an upright infrared differential interference contrast microscope (BX51WI or BX61WI, Olympus). The patch-clamp electrode resistance used for recording cell bodies was 5–8 MΩ.

[0081] 2) Action potential recording

[0082] Positive pressure was applied to the patch-clamp electrode, which had been infused with intracellular fluid, and it was maneuvered close to the fluorescently positive target cell. After the positive pressure was removed, the membrane was successfully sealed and ruptured. A series resistance below 15 MΩ was considered a satisfactory record. Action potentials were recorded in current-clamp mode. A series of depolarizing currents (e.g., 50–500 pA, 500 ms) were applied to induce action potentials in neurons, and a hyperpolarizing current (e.g., -50 pA, 500 ms) was applied to calculate the cell membrane input impedance. Signals were acquired at a sampling rate of 25 kHz and filtered at 10 kHz. Data recording was performed using a data acquisition system (pCLAMP 10.4 software). All recordings were performed at near-physiological temperatures (34–35 °C), maintained by a temperature controller (TC-324B, Warner Instruments).

[0083] Data were analyzed using Clampfit and MATLAB to quantify the characteristic parameters of action potentials, including amplitude, threshold, half-width, and frequency. Only cells with stable resting membrane potentials (≤-60mV) and access resistances below 15MΩ were included for data analysis. Discharge frequency-input current curves were plotted using the analyzed data. The results are shown in Figures 4 and 5.

[0084] Results Analysis: In mice with unilateral modeling, injection of AAV-ef1α-Kir2.1 / Kir3.2 / Kir4.1-GFP into the ipsilateral subthalamic nucleus resulted in tonic firing rather than burst firing. In the firing frequency-input current curves, neurons overexpressing Kir2.1 exhibited depolarization and inactivation under stimulation greater than 200 pA, with action potential frequency decreasing with increasing current. In the 6-OHDA model group, the frequency increased linearly with increasing current, indicating increased excitability. The action potential half-width at half-maximum (FWHM) and resting membrane potential (RMP) were significantly increased in the Kir2.1 overexpression group, indicating decreased neuronal excitability. In the Kir4.1 overexpression group, we observed that neuronal action potentials failed to fire normally. This is presumably due to enhanced potassium conductance mediated by the Kir4.1 channel, leading to increased potassium ion outflow and further reduction in membrane resistance. However, in the examples, Kir3.2 overexpression did not significantly alter neuronal excitability (Figures 7 and 8).

[0085] Since the Kir3.2 channel, together with the GABAb receptor, mediates the inhibitory potassium current generated by the activation of the GABAb receptor in neurons, thereby regulating the excitability of neurons, in this embodiment, we further compared the responses of subthalamic nucleus neurons in the control group and the model group to GABAb receptor agonists and antagonists to elucidate the specific role played by the Kir3.2 channel.

[0086] The experimental results showed that in the control group mice, blocking GABAb receptors significantly increased neuronal membrane resistance; however, in the model group mice, blocking GABAb receptors did not cause a significant change in membrane resistance. This indicates that the GABAb-Kir3.2 coupling pathway was impaired or downregulated in the model group mice, resulting in a significant reduction in the ability of GABAb receptors to regulate membrane resistance (Figures 9 and 10).

[0087] In this embodiment, a comparison of blood biochemical parameters between the Kir2.1 overexpression group and the model group showed no significant differences in alanine aminotransferase, alkaline phospholipase, aspartate aminotransferase, blood glucose levels, cholinesterase, and creatine kinase between the two groups; however, blood urea nitrogen levels were significantly increased, total protein levels were significantly decreased, and triglyceride levels also showed a significant decreasing trend. These results indicate that Kir2.1 overexpression did not cause systemic liver or muscle damage, but it led to specific metabolic changes characterized by abnormal protein and lipid metabolism, suggesting that Kir2.1 overexpression may produce systemic effects by affecting energy balance or nitrogen metabolism pathways (Figure 11).

[0088] Although significant changes in certain blood biochemical parameters were observed in Kir2.1-overexpressing mice in this experiment, such as increased blood urea nitrogen levels, decreased total protein levels, and a decreasing trend in triglycerides, these changes were not accompanied by significant increases in classic liver or muscle damage markers (such as alanine aminotransferase [ALT], aspartate aminotransferase [AST], creatine kinase [CK], cholinesterase [CHE], and alkaline phosphatase [ALP]). This indicates that although Kir2.1 overexpression causes metabolic changes, these changes do not lead to significant damage to systemic organs. Therefore, we can speculate that the biochemical changes caused by Kir2.1 overexpression are more due to the regulation of energy and nitrogen metabolism pathways than to systemic toxicity or tissue damage.

[0089] In terms of metabolism, the increase in blood urea nitrogen (BUN) may be related to the regulatory role of Kir2.1 in intracellular potassium ion balance. Abnormalities in potassium ion channel function often affect intracellular and extracellular ion balance and energy metabolism, which may in turn affect the metabolic function of the liver and kidneys, leading to elevated BUN. Furthermore, the decrease in total protein may reflect an imbalance between protein synthesis and degradation, or metabolic disorders caused by abnormal nitrogen metabolism, while the decrease in triglyceride levels may be related to the regulation of lipid metabolism. Although these biochemical changes show signs of metabolic disturbance, their degree is relatively mild and does not reach the level of significant organ dysfunction.

[0090] Importantly, although Kir2.1 overexpression led to specific metabolic alterations, these alterations did not affect the physiological functions of mice, nor did they cause systemic toxicity. In particular, no significant abnormalities were observed in common liver and muscle injury markers (such as ALT, AST, and CK), further indicating that the effects of Kir2.1 overexpression on these organs are manageable. This provides strong support for Kir2.1 as a potential therapeutic approach, especially in cardiovascular or neurological diseases, where it may exert therapeutic effects by regulating potassium ion channels.

[0091] Future research could further reduce the metabolic side effects of Kir2.1 by optimizing its expression levels and adjusting the dosage of its overexpression. More precise dosage control could potentially minimize its impact on systemic metabolism while maintaining the therapeutic efficacy of Kir2.1.

[0092] Neurons in the subthalamic nucleus are mainly divided into tonic firing neurons and burst firing neurons, which differ significantly in firing patterns, functions, and ion channel characteristics. Tonic firing neurons are characterized by the continuous and regular firing of action potentials, primarily in a stable, low-frequency, single firing pattern, which helps maintain the basal activity level of the subthalamic nucleus. This firing pattern plays a fundamental role in maintaining motor coordination in the basal ganglia pathway, helping to transmit a stable flow of low-frequency information, thereby achieving smooth motor control.

[0093] In contrast, burst-fire neurons exhibit rapid, multiple bursts of action potentials within a short period, followed by a quiet period. This explosive burst-fire pattern is typically used in response to specific synaptic inputs or physiological states, playing a crucial role in motor initiation, rapid adjustment, and feedback regulation.

[0094] From the perspective of ion channel characteristics, the sustained firing of tonic discharge neurons mainly depends on the homeostatic activity of sodium and potassium channels, while burst discharge neurons are closely related to the transient activation of calcium channels. The involvement of calcium channels gives burst discharges higher transient excitability, enabling them to transmit sudden motor commands, especially exerting a strong influence on postsynaptic neurons during rapid motor adjustments or specific behavioral demands. However, in pathological states (such as Parkinson's disease), the frequency and synchronicity of burst discharges increase significantly, potentially triggering abnormal synchronous activity, leading to motor disorders such as pathological tremors and rigidity. Therefore, these two firing patterns and neuron types exhibit different functional roles under normal motor regulation and pathological conditions, respectively.

[0095] Abnormal synchronized firing of neurons in the subthalamic nucleus, particularly enhanced burst firing, is closely related to the symptoms of Parkinson's disease. High-frequency burst firing causes excessive inhibition of downstream nuclei by the subthalamic nucleus, leading to impaired motor initiation and regulation. This abnormal firing activity manifests as symptoms such as bradykinesia, rigidity, and postural instability. Furthermore, abnormal burst firing is associated with the development of tremor because it can lead to abnormal rhythmic activity that synchronizes throughout the basal ganglia and the corticobasal ganglia pathway, thus producing the typical tremor symptoms of Parkinson's disease.

[0096] In our technique, overexpression of the Kir 2.1 potassium channel significantly affected neuronal firing patterns, thereby modulating abnormal firing in PD pathology. Potassium channels play a crucial role in regulating neuronal excitability, action potential formation, and frequency control. Overexpression of potassium channels typically enhances potassium ion efflux, thereby reducing neuronal excitability, decreasing aberrant burst firing, and mimicking the "desynchronization" effect of DBS, alleviating aberrant synchronization activity in the basal ganglia circuit, thus improving the PD phenotype.

[0097] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0098] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0099] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0100] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A recombinant adeno-associated virus (AAV-Kir), wherein the recombinant adeno-associated virus is an adeno-associated virus recombinantly containing the Kir gene on an expression vector.

2. The recombinant adeno-associated virus AAV-Kir as described in claim 1, characterized in that, The serotype of the recombinant adeno-associated virus is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13; And / or, the Kir gene includes any one or more of the Kir2.1, Kir3.2, or Kir4.1 genes.

3. The recombinant adeno-associated virus AAV-Kir as described in claim 1, characterized in that, The recombinant adeno-associated virus expression vector contains a promoter located upstream of the Kir gene.

4. The recombinant adeno-associated virus AAV-Kir as described in claim 3, characterized in that, The promoter is the ef1α promoter.

5. The recombinant adeno-associated virus AAV-Kir as described in claim 4, characterized in that, The nucleotide sequence of the ef1α promoter is shown in SEQ ID NO.

1.

6. The recombinant adeno-associated virus AAV-Kir as described in claim 2, characterized in that, The nucleotide sequence of the Kir2.1 gene is shown in SEQ ID NO.2; And / or, the nucleotide sequence of the Kir4.1 gene is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the Kir3.2 gene is shown in SEQ ID NO.

4.

7. A pharmaceutical composition comprising the recombinant adeno-associated virus AAV-Kir as described in any one of claims 1 to 6, and a pharmaceutically acceptable carrier or excipient.

8. Use of the recombinant adeno-associated virus AAV-Kir as described in any one of claims 1 to 6 or the pharmaceutical composition as described in claim 7 in the preparation of a product having one or more of the following functions: 1) Prevention and / or treatment of Parkinson's disease; 2) Reduce the excitability of neurons in the subthalamic nucleus; 3) Improve athletic ability; 4) Increase the distance and speed of movement.

9. The use as described in claim 8, characterized in that, The product will not cause anxiety or depression in individuals with Parkinson's disease.

10. The use as described in claim 8, characterized in that, The product is a drug administered via the subthalamus.

11. The use as described in claim 8, characterized in that, The product is an injectable drug administered via the subthalamic nucleus.

12. A method of treating Parkinson's disease, the method comprising administering to an individual with Parkinson's disease a preventative and / or therapeutically effective amount of the recombinant adeno-associated virus AAV-Kir as described in any one of claims 1 to 6, the pharmaceutical composition as described in claim 7, or the product used as described in any one of claims 8 to 11.

13. The method as described in claim 12, characterized in that, The recombinant adeno-associated virus (AAV-Kir), pharmaceutical composition, or product is administered to the subthalamus.

14. The method as described in claim 12, characterized in that, The recombinant adeno-associated virus (AAV-Kir), pharmaceutical composition, or product is administered to the subthalamic nucleus.

15. The method as described in claim 12, characterized in that, The method involves administering the recombinant adeno-associated virus AAV-Kir, a pharmaceutical composition, or a product to the subthalamus of an individual with Parkinson's disease using a microinjector. And / or, the method causes the subthalamus of the Parkinson's disease individual to overexpress the Kir gene.

16. The method as described in claim 12, characterized in that, The method involves administering the recombinant adeno-associated virus AAV-Kir, a pharmaceutical composition, or a product into the subthalamic nucleus of an individual with Parkinson's disease using a microinjector. And / or, the method causes the subthalamic nucleus of the Parkinson's disease individual to overexpress the Kir gene.

17. A nucleic acid construct containing a gene sequence for packaging the recombinant adeno-associated virus AAV-Kir as described in any one of claims 1 to 6.

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