Application of Aralar myocardial overexpression virus in preparation of medicine for treating heart failure
By specifically overexpressing the Aralar protein in cardiomyocytes using the recombinant adeno-associated virus type 9 (AAV9) vector, the problem of insufficient regulation of mitochondrial function in cardiomyocytes was solved, achieving effective treatment for heart failure and providing a novel gene therapy agent.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2026-03-02
- Publication Date
- 2026-05-22
Smart Images
Figure CN122070940A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the fields of biomedical and cardiovascular disease treatment technology, and in particular to the application of Aralar myocardial overexpression virus in the preparation of drugs for treating heart failure, specifically the use of Aralar to improve mitochondrial function by upregulating Aralar expression to treat heart failure. Background Technology
[0002] Heart failure is a chronic, progressive syndrome caused by structural or functional abnormalities of the heart, characterized by high hospitalization and mortality rates. While existing drug treatments can improve patient prognosis to some extent, they still suffer from insufficient efficacy and difficulty in effectively reversing disease progression. Research has identified mitochondrial energy metabolism disorders, increased oxidative stress, and kinetic imbalance as key mechanisms in the development of heart failure. Aralar, a protein closely coupled with mitochondrial metabolism, has attracted attention for its association with heart failure; however, its feasibility as a target in the treatment of heart failure still lacks systematic experimental evidence and a clear application plan.
[0003] In the development of heart failure treatment drugs based on the Aralar target, there is currently no viral vector application scheme that can achieve cardiac-targeted delivery and specifically overexpress Aralar protein in cardiomyocytes. It is impossible to improve cardiomyocyte mitochondrial function by targeting and regulating the expression of Aralar protein, thereby achieving effective treatment of heart failure. This has become a technical problem that urgently needs to be solved in the development of heart failure treatment drugs using the Aralar target. Summary of the Invention
[0004] To address the lack of existing viral vector applications that enable cardiac-targeted delivery and specific overexpression of Aralar protein in cardiomyocytes, thus preventing the effective treatment of heart failure by targeting and regulating Aralar protein expression to improve cardiomyocyte mitochondrial function, this application provides the use of Aralar myocardial overexpression viruses in the preparation of drugs for treating heart failure.
[0005] The application of an Aralar myocardial overexpression virus in the preparation of a drug for treating heart failure, as provided in this application, adopts the following technical solution: Firstly, this application provides the application of Aralar myocardial overexpression virus in the preparation of drugs for treating heart failure, using the following technical solution: The application of Aralar myocardial overexpression virus in the preparation of drugs for treating heart failure, wherein the Aralar myocardial overexpression virus is a recombinant adeno-associated virus type 9 (AAV9) vector, characterized in that: the AAV9 vector carries a myocardial-specific promoter and a nucleic acid molecule encoding the Aralar protein, and has cardiac-targeted delivery characteristics, for the specific overexpression of the Aralar protein in cardiomyocytes, wherein the Aralar protein treats heart failure by improving the mitochondrial function of cardiomyocytes.
[0006] By adopting the above technical solution, the AAV9 vector with cardiac-targeted delivery characteristics is selected as the basic vector. It is recombined with a myocardial-specific promoter and a nucleic acid molecule encoding the Aralar protein to achieve targeted delivery of the vector to cardiac tissue. At the same time, the Aralar protein is specifically overexpressed only in cardiomyocytes, avoiding ineffective intervention caused by expression in non-target tissues. As a mitochondrial-associated carrier protein, the Aralar protein can directly act on the mitochondria of cardiomyocytes to achieve functional regulation. The results were verified by a mouse model of aortic arch constriction and heart failure and a model of isoproterenol-induced cardiomyocyte hypertrophy. This recombinant virus can block the pathological progression of heart failure by regulating mitochondrial function, creating a new viral therapy for heart failure that targets cardiomyocytes.
[0007] Preferably, the myocardial-specific promoter is the cardiac troponin T promoter cTnT, and the AAV9 vector carries an expression cassette encoding the Aralar protein, forming a recombinant adeno-associated virus AAV9-cTnT-Aralar that has cardiac-targeted delivery characteristics and can specifically overexpress the Aralar protein in cardiomyocytes.
[0008] By employing the above-mentioned technical solution, cTnT is used as a myocardial-specific promoter and recombined with the AAV9 vector. Leveraging the transcriptional specificity of cTnT on cardiomyocytes, the nucleic acid molecule encoding Aralar protein is precisely driven to complete transcription and expression in cardiomyocytes. This, combined with the cardiac-targeted delivery characteristics of the AAV9 vector, forms a dual myocardial-specific regulation, significantly improving the expression efficiency and specificity of Aralar protein in cardiomyocytes and reducing non-specific expression in other tissues. This recombinant virus can effectively upregulate the expression levels of Aralar RNA and protein in myocardial tissue in a heart failure model, ensuring the targeted intervention effect of the virus on myocardial tissue.
[0009] Preferably, the Aralar is the Aralar protein encoded by the SLC25A12 gene, or a functional fragment or amino acid sequence variant derived from the protein and having mitochondrial functional regulatory activity.
[0010] By adopting the above technical solution, the Aralar protein encoded by the SLC25A12 gene is selected. This protein is a natural mitochondrial carrier protein that is closely associated with mitochondrial metabolism in cardiomyocytes and is a core protein for regulating mitochondrial function. Its functional fragments or amino acid sequence variants retain the core activity of mitochondrial function regulation and can exert the same mitochondrial regulatory effect as the natural Aralar protein. Using this type of protein as a core intervention factor, the core mechanism of mitochondrial dysfunction in the development of heart failure can be directly targeted, making the intervention of viral vectors more targeted and improving the treatment effectiveness of heart failure.
[0011] Preferably, the heart failure includes heart failure induced by aortic arch constriction or isoproterenol stimulation, as well as heart failure caused by stress overload, heart failure associated with myocardial hypertrophy, or heart failure accompanied by mitochondrial dysfunction.
[0012] By adopting the above-mentioned technical solutions, adaptive interventions can be carried out for heart failure caused by different factors. The above-mentioned types of heart failure all share the common core pathological mechanism of mitochondrial energy metabolism disorders, increased oxidative stress, or dynamic imbalance. This recombinant virus can improve the mitochondrial function of cardiomyocytes from the root by overexpressing Aralar protein, thereby adapting to the treatment needs of different types of heart failure, breaking through the limitations of treating heart failure caused by a single factor, and broadening the application scope of this recombinant virus in the field of heart failure treatment.
[0013] Preferably, the improvement of cardiomyocyte mitochondrial function includes at least one of maintaining or increasing mitochondrial membrane potential, inhibiting mitochondrial oxidative stress levels, and increasing the expression level of mitochondrial DNA transcription factor TFAM, wherein TFAM is related to the maintenance of mitochondrial DNA.
[0014] By employing the above-mentioned technical solutions, Aralar protein can regulate the mitochondrial function of cardiomyocytes from multiple dimensions. By maintaining or increasing the mitochondrial membrane potential, it can prevent mitochondrial functional impairment caused by abnormally low membrane potential. At the same time, it can inhibit the level of mitochondrial oxidative stress, reduce the production of oxidative stress products such as Mitosox, and increase the expression level of TFAM. With the help of TFAM to maintain mitochondrial DNA, it can ensure the stability and normal expression of mitochondrial DNA. It can repair damaged mitochondrial function from multiple levels of mitochondrial structure, oxidative stress, and genetic material, and block the mitochondrial-related pathological pathways in the development of heart failure.
[0015] Preferably, the improvement of cardiomyocyte mitochondrial function includes maintaining or promoting mitochondrial dynamic homeostasis, which involves the expression of mitochondrial fusion-related proteins MFN1 and MFN2 and the expression of mitochondrial division-related protein Drp1.
[0016] By employing the above-mentioned technical approach, the Aralar protein directly regulates the expression levels of mitochondrial fusion and division-related proteins. By upregulating the expression of mitochondrial fusion-related proteins MFN1 and MFN2, the mitochondrial fusion process is promoted, while the expression of mitochondrial division-related protein Drp1 is downregulated, inhibiting excessive mitochondrial division. This maintains and promotes the dynamic balance of cardiomyocyte mitochondria, improves the abnormal short, thick, and rod-shaped morphology of mitochondria in heart failure, restores the normal slender shape of mitochondria, and restores the normal structure and function of mitochondria. In this way, it alleviates the hypertrophic phenotype of cardiomyocytes caused by mitochondrial dynamic imbalance, and achieves effective treatment for heart failure.
[0017] Secondly, this application provides a gene therapy agent for treating heart failure, employing the following technical solution: A gene therapy formulation for treating heart failure comprises an effective amount of AAV9-cTnT-Aralar, wherein AAV9-cTnT-Aralar is a recombinant adeno-associated virus used to specifically overexpress the Aralar protein in cardiomyocytes.
[0018] By employing the above-mentioned technical solution, an effective amount of AAV9-cTnT-Aralar was used as the core active ingredient to prepare a gene therapy formulation. The dosage of the active ingredient was precisely controlled to ensure that the formulation could effectively overexpress the Aralar protein in cardiomyocytes after application. Utilizing the cardiac-targeted delivery and cardiomyocyte-specific expression characteristics of this recombinant virus, the formulation could directly act on the lesion site of the myocardial tissue and exert its therapeutic effect by regulating the mitochondrial function of cardiomyocytes. This formulation can effectively alleviate the symptoms of cardiac enlargement and improve cardiac function in mice with heart failure. At the same time, it can improve the isoproterenol-induced cardiomyocyte hypertrophy phenotype, providing a novel gene therapy formulation for heart failure.
[0019] Preferably, the gene therapy agent is administered via myocardial-targeted delivery.
[0020] By adopting the above technical solution, myocardial targeted delivery is used as the administration method of this gene therapy agent, which is compatible with the cardiac targeted delivery characteristics of AAV9-cTnT-Aralar. This further improves the enrichment efficiency of the agent in myocardial tissue, allowing more recombinant viruses to enter myocardial cells and exert their effects. It also reduces the distribution of the agent in non-target tissues such as the liver and kidneys, reduces the potential impact of non-target tissue expression, and ensures the effective expression level of Aralar protein in myocardial cells, thus giving full play to its core role in improving mitochondrial function and treating heart failure.
[0021] Preferably, the gene therapy formulation further comprises at least one of a pharmaceutically acceptable carrier, diluent, excipient, buffer system, or adjuvant.
[0022] By adopting the above technical solutions and adding pharmaceutically acceptable excipients such as carriers and diluents to the formulation, the overall physicochemical properties of the formulation can be adjusted according to the preparation requirements, thereby improving the stability of AAV9-cTnT-Aralar in the formulation and enhancing its bioavailability. This allows the excipients to work synergistically with the core active ingredient, ensuring that the formulation meets industry standards for pharmaceutical preparation and clinical application requirements. It also guarantees the effectiveness and safety of the formulation during storage, transportation, and clinical use, providing a feasible technical foundation for the industrialization and clinical translation of this gene therapy formulation.
[0023] Preferably, the gene therapy preparation is an injectable formulation.
[0024] By adopting the above technical solution, the gene therapy agent is prepared into an injectable dosage form, which enables rapid drug delivery and in vivo absorption, allowing AAV9-cTnT-Aralar to quickly reach myocardial tissue and exert a targeted intervention effect. At the same time, the injection dosage form is easy to administer and allows for flexible adjustment of the dosage and administration site according to the actual needs of clinical treatment, adapting to the treatment scenarios and physical conditions of different heart failure patients in clinical practice, thereby improving the convenience and practical applicability of the gene therapy agent in clinical applications.
[0025] In summary, this application has the following beneficial effects: 1. Since this application uses a recombinant adeno-associated virus type 9 (AAV9) vector as the basic vector, which carries a myocardial-specific promoter and a nucleic acid molecule encoding the Aralar protein, forming AAV9-cTnT-Aralar with cardiac-targeted delivery characteristics, it can specifically overexpress the Aralar protein in cardiomyocytes. Moreover, it has been verified by in vitro and in vivo heart failure models that this virus can effectively treat heart failure by improving the mitochondrial function of cardiomyocytes, and can also provide potential treatment strategies for the development of heart failure drugs.
[0026] 2. In this application, the Aralar protein encoded by the SLC25A12 gene or its functional fragments or amino acid sequence variants with mitochondrial function regulatory activity are preferably used as the core active protein. This type of protein can specifically improve the mitochondrial function of cardiomyocytes, and can regulate mitochondrial membrane potential, inhibit oxidative stress and regulate TFAM expression, thereby achieving the treatment of heart failure from the mitochondrial function level.
[0027] 3. The method of this application, through AAV9-cTnT-Aralar-mediated specific overexpression of Aralar protein in cardiomyocytes, can maintain and promote the mitochondrial dynamics homeostasis of cardiomyocytes, and precisely regulate the expression of mitochondrial fusion-related proteins MFN1 and MFN2 and division-related protein Drp1. This effect can be adapted to the treatment of various types of heart failure, covering heart failure caused by stress overload, myocardial hypertrophy and heart failure accompanied by mitochondrial dysfunction.
[0028] 4. This application prepares a gene therapy formulation containing an effective amount of AAV9-cTnT-Aralar. This formulation can specifically overexpress Aralar protein in cardiomyocytes. It can also be combined with pharmaceutically acceptable carriers, diluents, excipients and other excipients to make the formulation meet the requirements for pharmaceutical application. This provides a novel gene therapy formulation for the clinical treatment of heart failure and has a formulation basis for practical application.
[0029] 5. The gene therapy formulation of this application adopts a myocardial targeted delivery method and is formulated as an injection. This delivery method is compatible with the cardiac targeted delivery characteristics of AAV9-cTnT-Aralar, which can improve the delivery efficiency of the formulation in cardiomyocytes, ensure that Aralar protein can be expressed efficiently and specifically in cardiomyocytes, and give full play to its role in improving mitochondrial function and treating heart failure. Attached Figure Description
[0030] Figure 1 This is a comparison chart of serum Aralar expression levels between heart failure patients and normal patients provided in this application; Figure 2 This is an M-mode echocardiogram of cardiac function-related indicators in mice from the Sham and TAC groups provided in this application; Figure 3 These are the pathological morphological images of Masson staining and H&E staining of myocardial tissue from mice in the Sham and TAC groups provided in this application. Figure 4 This is a diagram of the Real-time PCR results for detecting Aralarm RNA expression levels in the myocardial tissues of mice in the Sham and TAC groups, as provided in this application. Figure 5 This is a Western blotting result of the detection of Aralar protein expression levels in myocardial tissues of mice in the Sham and TAC groups provided in this application; Figure 6 This is an M-mode echocardiogram of cardiac function-related indicators in mice in the TAC+AAV9-cTnT-NC group and the TAC+AAV9-cTnT-Aralar group provided in this application; Figure 7These are the pathological morphological images of Masson staining and H&E staining of myocardial tissue from mice in the TAC+AAV9-cTnT-NC group and the TAC+AAV9-cTnT-Aralar group provided in this application. Figure 8 This is a Real-time PCR result of the detection of Aralarm RNA expression levels in myocardial tissue of mice in the TAC+AAV9-cTnT-NC group and the TAC+AAV9-cTnT-Aralar group provided in this application; Figure 9 This is a Western blotting result of the detection of Aralar protein expression levels in myocardial tissue of mice in the TAC+AAV9-cTnT-NC group and the TAC+AAV9-cTnT-Aralar group provided in this application; Figure 10 This is a Real-time PCR result of the detection of Aralarm RNA expression levels in the ISO+AAV9-cTnT-NC group and the ISO+AAV9-cTnT-Aralar group of myocardial mast cell models provided in this application; Figure 11 This is a Western blotting result of the detection of Aralar protein expression levels in the ISO+AAV9-cTnT-NC group and the ISO+AAV9-cTnT-Aralar group of myocardial mast cell models provided in this application; Figure 12 These are confocal micrographs of mitochondrial membrane potential levels in the ISO+AAV9-cTnT-NC and ISO+AAV9-cTnT-Aralar groups of NRCM provided in this application. Figure 13 These are confocal microscope images of mitochondrial morphology observation in NRCM of the ISO+AAV9-cTnT-NC group and the ISO+AAV9-cTnT-Aralar group provided in this application; Figure 14 These are confocal micrographs of mitochondrial oxidative stress levels in the ISO+AAV9-cTnT-NC and ISO+AAV9-cTnT-Aralar NRCMs provided in this application. Figure 15 This is a Western blotting result of the detection of mitochondrial-related protein expression levels in the ISO+AAV9-cTnT-NC group and the ISO+AAV9-cTnT-Aralar group of NRCM provided in this application. Detailed Implementation
[0031] The following detailed description, using specific in vivo and in vitro experimental examples, further illustrates the application of AAV9-cTnT-Aralar in the preparation of drugs for treating heart failure. Animal models of heart failure and cardiomyocyte mast cell models were used to verify the effect of AAV9-cTnT-Aralar in targeting Aralar overexpression in cardiomyocytes, thus clarifying its application value in treating heart failure. Unless otherwise specified, all experimental methods used are conventional methods. All materials, reagents, methods, and instruments used, unless otherwise specified, are conventional materials, reagents, methods, and instruments in the art, which can be obtained commercially or prepared according to literature methods by those skilled in the art.
[0032] Technical concept: In the clinical treatment of heart failure, existing drugs are unable to effectively reverse the progression of the disease. The core reason is that they have not achieved precise targeted intervention on the key mechanism of heart failure development, namely the abnormal mitochondrial function of cardiomyocytes. At the same time, for the Aralar target, which is closely related to mitochondrial metabolism, there is a lack of viral vector application schemes that can achieve cardiac-targeted delivery and specific overexpression of this protein in cardiomyocytes. Therefore, it is impossible to improve myocardial mitochondrial function by regulating the expression of this target protein, and thus achieve targeted treatment of heart failure.
[0033] This technical solution uses recombinant adeno-associated virus type 9 as the base vector, integrating a myocardial-specific promoter and a nucleic acid molecule encoding the Aralar protein into the vector to construct AAV9-cTnT-Aralar with cardiac-targeted delivery characteristics, achieving specific overexpression of the Aralar protein in cardiomyocytes. Simultaneously, by regulating the expression of mitochondrial membrane potential, oxidative stress levels, and mitochondrial dynamics-related proteins in cardiomyocytes through this protein, it improves myocardial mitochondrial function from multiple dimensions. Furthermore, it prepares an injectable gene therapy formulation containing this recombinant virus, combined with pharmaceutically acceptable excipients, and uses a myocardial-targeted delivery method, thereby solving the technical problems of insufficient targeting and inability to precisely regulate the Aralar target to improve mitochondrial function in existing technologies.
[0034] I. Experimental Materials and Sources All reagents, instruments, and consumables used in this experiment were commercially available and qualified products. Specific categories, sources, and reagent preparation methods are as follows: 1. The main reagents and their manufacturers are shown in Table 1.
[0035] Table 1: Main reagents used in this experiment and their manufacturers Serial Number Experimental materials source 1 DNAMarker Jereh Company 2 DMEM high glucose medium Gibco 3 DMEM / F12 medium Gibco 4 fetal bovine serum Gibco 5 pancreatic enzymes Gibco 6 Penicillin-Streptomycin Mixture PS Gibco 7 methanol Sinopharm Chemical Reagent Co., Ltd. 8 Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. 9 chloroform Sinopharm Chemical Reagent Co., Ltd. 10 Isopropanol Sinopharm Chemical Reagent Co., Ltd. 11 periodic acid chev staining solution Beyotime 12 Benzylsulfonamide (PMSF) Beyotime 13 Tween-20 Beyotime 14 Tween-80 Beyotime 15 Two-color SDS-PAGE protein loading buffer (5X) Beyotime 16 Mitochondrial permeability transition pore (MPTP) assay kit Beyotime 17 skim milk powder Beyotime 18 BSA Beyotime 19 Hoechst33342 Beyotime 20 DAPI staining solution Beyotime 21 4% paraformaldehyde fixative Beijing Lanjieke Technology Co., Ltd. 22 MitoTrackerRedCMXRos dye Thermo Fisher Scientific (USA) 23 MitoSOX dyes Thermo Fisher Scientific (USA) 24 JC-1 dye (mitochondrial membrane potential probe) Thermo Fisher Scientific (USA) 25 Dihydroethidine (hydroethidine) Thermo Fisher Scientific (USA) 26 GAPDH antibody Proteintech 27 Drp1 antibody Proteintech 28 TFAM antibody Proteintech 29 Mfn1 antibody Proteintech 30 Mfn2 antibody Proteintech 31 VDAC antibody Proteintech 32 Small molecule compound ZINC1488036 Selleck 33 Goat anti-rabbit secondary antibody CellSignailing Technology (USA) 34 Goat anti-mouse secondary antibody CellSignailing Technology (USA) 35 Protein Extraction Lysis Buffer RIPA Beyotime 36 BCA Protein Concentration Assay Kit Shanghai Yamei 37 SDS-PAGE gel preparation kit Shanghai Yamei 38 100X Phosphatase Inhibitor Mixture Shanghai Yamei 39 PVDF membrane Millipore (USA) 40 ECL chemiluminescence reagent kit Millipore (USA) 41 Tris(hydroxymethyl)aminomethane Sigma Corporation (USA) 42 Sodium dodecyl sulfate Sigma Corporation (USA) 43 glycine Sigma Corporation (USA) 44 Triton X-100 Sigma Corporation (USA) 45 DMSO (dimethyl sulfoxide) Sigma Corporation (USA) 46 PBS powder Saville Company 47 HiScriptIVRTSuperMixforqPCR (+gDNAwiper) Novizan Biopharmaceuticals 2. The main instruments and their manufacturers are shown in Table 2.
[0036] Table 2: Main Instruments Used in This Experiment and Their Manufacturers Serial Number instrument source 1 PCR instrument AppliedBiosystems (USA) 2 Real-time quantitative PCR instrument BIO-RAD (USA) 3 DNA electrophoresis instrument BIO-RAD (USA) 4 Gel Imaging BIO-RAD (USA) 5 Horizontal shaking table Hualida Experimental Equipment Company 6 rocker bed Hualida Experimental Equipment Company 7 Cell incubator Thermo Fisher Scientific (USA) 8 NanodropOne Micro-Vitamin UV Spectrophotometer Thermo Fisher Scientific (USA) 9 Refrigerators with temperatures of 4℃ / -20℃ Samsung Corporation (South Korea) 10 Ultra-low temperature freezer SANYO Corporation (Japan) 11 ice maker SANYO Corporation (Japan) 12 Different sizes of pipettes and pipette tips Eppendorf (Germany) 13 Low-temperature high-speed centrifuge Eppendorf (Germany) 14 Electrophoresis and transfer apparatus BIO-RAD (USA) 15 ELISA reader BIO-TEK (USA) 16 constant temperature incubator Shanghai Hengyi Scientific Instruments Co., Ltd. 17 constant temperature water bath Shanghai Hengyi Scientific Instruments Co., Ltd. 18 Forced air drying oven Shanghai Hengyi Scientific Instruments Co., Ltd. 19 High temperature and high pressure sterilizer TOMY Corporation (USA) 20 Desktop handheld centrifuge TOMY Corporation (USA) 21 Chemiluminescent gel imaging Shanghai Tianneng 22 Inverted fluorescence microscope Leica (Germany) 23 Pure water system Millipore (USA) 24 Cell counter Invitrogen 25 Upright fluorescence microscope Olympus (Japan) 3. The experimental consumables and their manufacturers are shown in Table 1.
[0037] Table 3: List of experimental consumables and their manufacturers for this experiment Serial Number Consumable Name Production Company 1 Various models of gun heads KIRGEN 2 Various types of pipettes Corning 3 Various models of multi-well cell culture plates Corning 4 Various types of cell culture dishes Corning 5 cryopreservation tubes Corning 6 Centrifuge tubes of various models NEST 7 Clear 8-tube PCR Shanghai Sangon Biotech 8 96-cell PCR plate Shanghai Sangon Biotech 4. Preparation methods of main reagents The culture medium, virus stock solution, and electrophoresis-related buffers used in the experiment were all prepared according to the following methods and stored under the corresponding conditions for later use: 4.1 Complete culture medium: Add 1% penicillin antibiotics to DMEM high glucose medium containing 10% FBS, mix thoroughly, and store at 4°C.
[0038] 4.2 AAV9-cTnT-Aralar stock solution: Take AAV9-cTnT-Aralar powder (1E13vg / ml), dissolve it in 1ml DMEM medium, and store it at -20℃ in the dark after it is fully dissolved.
[0039] 4.3 10% SDS-PAGE separating gel: Prepare different volumes of 10% SDS-PAGE separating gel according to the volume ratios shown in the table below, and use after thoroughly mixing all components; Table 4: Preparation ratio of 10% SDS-PAGE separating gel, unit: mL.
[0040] Element 5 10 15 20 30 50 distilled water 1.0 2.0 3.0 4.0 6.0 10.0 30% Acr-Bis(29:1) 2.0 4.0 6.0 8.0 12.0 20.0 Tris-HCl (PH8.8) 1.9 3.8 5.7 7.6 11.4 19.1 10% SDS 0.05 0.1 0.15 0.2 0.3 0.5 10% APS 0.05 0.1 0.15 0.2 0.3 0.5 TEMED 0.002 0.004 0.006 0.008 0.012 0.02 4.4 5% PAGE Stacking Gel: Prepare different volumes of 5% SDS-PAGE stacking gel according to the volume ratios shown in the table below, and use after thoroughly mixing all components; Table 5: Preparation ratio of 5% PAGE stacking gel, unit: mL.
[0041] Element 2 3 4 6 8 10 Double distilled water 1.4 2.1 2.7 3.5 4.1 5.5 30% Acr-Bis(29:1) 0.33 0.5 0.67 0.83 1.0 1.3 Tris-HCl (PH6.8) 0.25 0.38 0.5 0.62 0.75 1.0 10% SDS 0.02 0.03 0.04 0.05 0.06 0.08 10% APS 0.02 0.03 0.04 0.05 0.06 0.08 TEMED 0.002 0.003 0.004 0.005 0.006 0.008 4.5 10× Electrophoresis Buffer: Weigh 30.2g Tris, 188g glycine, and 10g SDS, add double-distilled water to a final volume of 1L, and dissolve thoroughly before use.
[0042] 4.6 10× Transfer Buffer: Weigh 30.2g Tris and 188g glycine, add double-distilled water to a final volume of 1L, dissolve thoroughly and set aside.
[0043] 4.7 1×TBST buffer: Dissolve one packet of TBS powder in 1L of double-distilled water, then add 2ml of Tween-20 and mix thoroughly before use.
[0044] 4.8 Blocking solution: Take 50ml of 1×TBST buffer, add 2.5g of skim milk powder, dissolve thoroughly and set aside.
[0045] 4.9 Antibody working solution: Use dedicated primary and secondary antibody diluents to prepare the corresponding antibody working solution according to the concentration ratio indicated in the instructions for each antibody.
[0046] II. Experimental Procedure This experiment consists of four parts: design and preparation of Aralar myocardial overexpression virus, detection of heart failure patient samples, in vivo experiments in a mouse model of aortic arch constriction and heart failure, and in vitro experiments on isoproterenol (ISO)-induced hypertrophy of neonatal rat cardiomyocytes (NRCM). The efficacy of AAV9-cTnT-Aralar was verified through a combination of in vivo and in vitro experiments. The specific experimental steps are as follows: Example 1: Design and preparation of Aralar myocardial overexpression virus. This experiment was conducted in collaboration with Shanghai Jiman Biotechnology Co., Ltd., to design and customize a recombinant adeno-associated virus (AAV) targeting cardiomyocytes and overexpressing Aralar. The virus model is AAV9-cTnT-Aralar, and the vector number is 80528 (GPAAV-GalluscTNT-eGFP-Mouse_Slc25a12(Aralar)-WPRE). Its coding sequence is: SEQ ID NO:1 Example 2: Detection of Aralar protein expression level in myocardial tissue of patients with heart failure. Myocardial tissue samples were collected from patients with heart failure and normal individuals. The expression level of Aralar protein in the samples was detected by Western blotting. The trend of Aralar protein expression changes in patients with heart failure was compared and analyzed.
[0047] Example 3: Construction and in vivo detection of a mouse model of heart failure with aortic arch coarctation (TAC). Using C57BL / 6J mice as experimental subjects, a mouse model of heart failure with aortic arch coarctation was constructed. Three groups were set up: Sham sham operation group, TAC model group, and TAC+AAV9-cTnT-Aralar treatment group. The effects of AAV9-cTnT-Aralar on the heart failure model mice were verified by nucleic acid detection, protein detection, ultrasound detection, tissue staining and other methods.
[0048] 1. Preparation of a mouse model of heart failure due to aortic arch constriction Experimental animals: Female C57BL / 6J mice, weighing 20-25g, were selected and kept in a standard feeding environment with a 12-hour light / dark cycle for 1 week to adapt to the environment before the experiment. Anesthesia: Mice were induced with general anesthesia using isoflurane, and the anesthetic concentration was maintained at 1.5%-2%. Surgical procedure: Under aseptic conditions, a midline thoracic incision was made in the mouse to expose the aortic arch. 7-0 silk suture was wrapped around the aortic arch and ligated to narrow the aortic arch to about 50% of its diameter, creating a continuous blood flow load. Postoperative recovery: After the surgery, allow the mice to recover at room temperature for 1-2 hours, closely observe their vital signs, and return them to their cages for routine feeding once they have recovered. Grouping: Mice were randomly divided into three groups: the Sham sham surgery group (opening the chest but not ligating the aortic arch); the TAC model group (ligating the aortic arch to establish a heart failure model); and the TAC+AAV9-cTnT-Aralar treatment group (establishing a heart failure model followed by AAV9-cTnT-Aralar viral intervention).
[0049] 2. Animal tissue sampling, RNA extraction, reverse transcription of cDNA, and Real-time PCR detection. This step is used to detect the expression level of Aralarm RNA in mouse myocardial tissue. The specific operation is as follows: 2.1 Tissue sampling: Mice were anesthetized with a mask from a Reward anesthesia machine to maintain stable breathing. The chest cavity was cut open and the perfusion needle was inserted into the left apex of the heart. The heart was perfused with physiological saline. The perfusion was completed when the liver turned white and the mesentery became transparent. The chest cavity was cut open again to expose the heart. A portion of the heart tissue was taken and placed in an RNAaseFreeEP tube containing 200 μL of Trizol and placed in an ice box for later use.
[0050] 2.2 RNA extraction: (1) Homogenize the frozen heart tissue sample with a homogenizer, once every 30 seconds, for a total of 4 times, until no tissue fragments are visible to the naked eye; (2) Add 800 μL of Trizol to the homogenized sample and let it stand at room temperature for 5 min; (3) Add 200 μL of chloroform, shake rapidly and vigorously for 1 min, and let stand at room temperature for 5 min; (4) Centrifuge at 4℃ and 12000rpm for 15min; Gently aspirate the clear liquid from the top layer into a new RNAaseFreeEP tube, add an equal volume of isopropanol, gently invert the tube until no visible filaments are visible, and let stand at room temperature for 10 minutes. Centrifuge at 4℃ and 12000rpm for 15min, then discard the supernatant; Add 1 ml of anhydrous ethanol and mix gently. Centrifuge at 4°C and 12,000 rpm for 15 min and discard the supernatant. Invert the EP tube onto filter paper and let it air dry at room temperature until a translucent precipitate appears at the bottom of the tube. Add 20 μL of RNAase-free H2O to each tube, incubate in a 60°C water bath for 10 min to promote dissolution, and then place in an ice box to detect RNA concentration using an OD analyzer.
[0051] 2.3. Total RNA reverse transcription into cDNA: (1) DNA impurity removal: Prepare a 10 μL reaction system containing 2 μL of 5×g DNAwiperMix and 1 μg of RNA. Adjust the amount according to the RNA concentration. Add RNase-Free ddH2O to make up to 10 μL and react at 42℃ for 2 min. (2) cDNA reverse transcription: Prepare a 20 μL reaction system containing Oligo(dT)20VN1 μL, Randomhexamers1 μL, HiscriptⅢEnzymeMix2 μL, 5×EvoM-MLVRTReactionMixVer.24 μL, add the above DNA-removed RNA sample, and add RNase-FreeddH2O to 20 μL. React at 37℃ for 15 min and at 85℃ for 5 s. (3) Sample preservation: Dilute the cDNA obtained by reverse transcription with triple-distilled water 8 times and store it in a -20℃ refrigerator for later use.
[0052] 2.4 Primer Design and Synthesis: Primers were designed based on mouse Aralarm RNA sequences from the NCBI database. Primer specificity was verified using BLAST to predict amplification product size. Melting curves and agarose gel electrophoresis results were used to further verify primer specificity. Primers were synthesized by OBiO (Shanghai Heyuan). The specific sequences are shown in the table below. Table 6: Primer Sequence Listing
[0053] 2.5 Real-time PCR experiment: (1) Prepare the Real-time PCR reaction system (10 μL) according to the table below: Reagent Name Dosage (μL) 2×ChamqUniversalSYBRqPCRMasterMix 5 ForwardPrimer (10μM) 0.2 ReversePrimer (10μM) 0.2 cDNA 2 ddH2O 2.6 (2) PCR reaction using a Life Technology instrument, under the following conditions:
[0054] 3. Animal tissue sampling, protein extraction, and Western blotting detection This step is used to detect the expression levels of Aralar and mitochondrial-related proteins in mouse myocardial tissue. The specific procedure is as follows: 3.1 Protein Extraction: After anesthetizing mice, the heart was washed with cold PBS using the cardiac perfusion method to remove blood. The heart tissue was then quickly removed, cut in ice-cold PBS, and placed in lysis buffer containing protease inhibitors (PMSF). The tissue was then homogenized using a tissue homogenizer and centrifuged at 4°C and 12000×g for 10 minutes. The supernatant was collected as the protein sample from mouse myocardial tissue. The protein extraction method for neonatal rat myocardial cells was the same as above. After cell intervention, the cells were washed with PBS, lysed with lysis buffer containing PMSF, and centrifuged at 4°C and 12000×g for 10 minutes. The supernatant was collected as the cell protein sample. 3.2 Protein quantification: The concentration of extracted protein samples was quantified using the BCA (Bicinchoninic Acid Assay), and the protein sample concentration was adjusted to be consistent based on the quantification results; 3.3 Western blotting detection: Protein samples were subjected to 12% SDS-PAGE polyacrylamide gel electrophoresis at a constant voltage of 80V. Proteins were transferred from the gel to a PVDF membrane using a wet transfer method at a constant current of 200mA for 2 hours. After transfer, the PVDF membrane was placed in 5% skim milk + TBS-T buffer and blocked at room temperature for 1 hour. After blocking, the membrane was incubated with primary antibody overnight and washed three times with TBST buffer for 10 minutes each time. Secondary antibody was added and incubated at room temperature for 1 hour, followed by washing three times with TBST buffer for 10 minutes each time. Enhanced chemiluminescence (ECL) reagent was added for color development, and images were captured using a Bio-Rad ChemiDoc chemiluminescence imaging system to analyze protein expression levels.
[0055] 4. Ultrasound detection of cardiac function in mice Left ventricular function parameters in mice were detected using high-resolution small animal echocardiography equipment. The specific procedures are as follows: 4.1 Anesthesia: Mice were anesthetized with isoflurane mask gas to control the depth of anesthesia and avoid interference with the test results due to mouse movement; 4.2 Sample preparation: After anesthesia, the mice were placed on a heated bed and fixed in a supine position. Ultrasound coupling agent was applied to the chest of the mice to ensure good contact between the ultrasound probe and the skin. 4.3 Indicator Detection: Two-dimensional imaging was performed using the Vevo2100 small animal cardiac ultrasound device. M-mode scanning was used to record mouse cardiac function indicators, including left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic wall thickness (LVWTd), and left ventricular end-systolic wall thickness (LVWTs).
[0056] 5. Masson staining detection of mouse heart tissue This step is used to detect the degree of fibrosis in mouse myocardial tissue. The specific procedure is as follows: 5.1 Sample collection: After mice were anesthetized with isoflurane, a midline thoracic incision was made to expose and remove the heart. The heart was rinsed with cold PBS (pH 7.4) to remove any blood residue. 5.2 Tissue processing: The heart tissue undergoes a series of pathological slide preparation processes, including dewaxing, hydration, staining, differentiation, collagen staining, nuclear staining, dehydration, and mounting. 5.3 Microscopic observation: The prepared sections were placed under an Olympus BX51 optical microscope for observation, and images were taken and the myocardial fibrosis was analyzed.
[0057] 6. H&E staining detection of mouse heart tissue This step is used to detect morphological changes in mouse myocardial tissue. The specific procedure is as follows: 6.1 Sample collection: The sample collection method is the same as that for Masson staining. After anesthetizing the mice, the heart was collected and rinsed with cold PBS at pH 7.4 to remove blood residue. 6.2 Tissue processing: The heart tissue was dewaxed and hydrated, stained with hematoxylin, differentiated and blued, eosin stained, dehydrated and cleared, and mounted for pathological preparation. 6.3 Microscopic observation: The prepared sections were placed under an Olympus BX51 optical microscope for observation, and images were taken and the morphology, arrangement and nuclear changes of cardiomyocytes were analyzed.
[0058] Example 4: Construction of an isoproterenol (ISO)-induced neonatal rat cardiomyocyte (NRCM) hypertrophy model and detection of mitochondrial function An ISO-stimulated hypertrophic model of neonatal rat cardiomyocytes was constructed, and three groups were set up: a blank control group, an ISO model group, and an ISO+AAV9-cTnT-Aralar treatment group. The mitochondrial morphology, oxidative stress level, and membrane potential changes of cardiomyocytes were detected by mitochondrial probe staining to verify the ameliorative effect of AAV9-cTnT-Aralar on cardiomyocyte mitochondrial function.
[0059] 1. Construction of NRCM cardiomyocyte hypertrophy model 1.1 Heart sampling: After anesthetizing the suckling rats with isoflurane, a midline thoracic incision was made to expose and remove the heart. The heart was rinsed with cold PBS (pH 7.4) to remove any blood residue. 1.2 Digestion of cardiomyocytes: Cut the heart tissue into small pieces and place them in a digestion solution containing 0.1% collagenase I and 0.1% trypsin. Digest at 37°C with shaking for 30-40 minutes, gently shaking once every 10 minutes to ensure complete tissue dissociation. After digestion, add DMEM medium containing 10% fetal bovine serum to terminate the digestion. 1.3 Cell filtration and collection: The digested cell suspension was filtered through a 70μm cell sieve to remove undigested tissue fragments; the filtered cell suspension was centrifuged at 1000rpm for 5 minutes and the cell pellet was collected. 1.4 Cell Seeding and Culture: Resuspend the cell pellet in DMEM medium containing 10% FBS and 1% penicillin-streptomycin mixture, and seed the cells into 6-well or 24-well plates at a concentration of 100,000-200,000 cells / well. Incubate the plates in a constant temperature incubator at 37°C and 5% CO2. 1.5 Model Construction: After cell adhesion, 100 μmol / L of ISO drug was added for intervention, and the cells were cultured for a specified time to construct a cardiomyocyte hypertrophy model.
[0060] 2. MitoTracker probe detection of cardiomyocytes 2.1 Sample processing: Cardiac cells were extracted and seeded into plates for 48 hours, then washed once with PBS to remove residual culture medium; 2.2 Staining treatment: Add 100µL of 100nM MitoTrackerRed solution to each well, gently shake the culture plate to ensure full contact between the dye and the cells, and incubate at 4℃ and 5%CO2 for 30 minutes; after incubation, wash the cells 3 times with PBS, add Hoechst dye, incubate at 37℃ and 5%CO2 for 10 minutes, and then wash 3 times with PBS. Microscopic observation: The cell plate was placed under an Olympus IX83 confocal microscope to observe and photograph the mitochondrial morphology of cardiomyocytes at an excitation wavelength of 577 nm and an emission wavelength of 590 nm, and to analyze the division and fusion status of mitochondria.
[0061] 3. MitoSOX probe detection of cardiomyocytes 3.1 Sample processing: After seeding cardiomyocytes into plates for 48 hours, wash once with PBS to remove residual culture medium; 3.2 Staining treatment: Add an appropriate amount of 5µM MitoSOX working solution to each well and incubate at 37℃ and 5%CO2 for 30 minutes; after incubation, wash the cells 3 times with PBS, add Hoechst dye and incubate at 37℃ and 5%CO2 for 10 minutes, and then wash 3 times with PBS. 3.3 Microscopic observation: The cell plate was placed under an Olympus IX83 confocal microscope and the cells were observed and photographed at an excitation wavelength of 510 nm and an emission wavelength of 580 nm. The distribution and intensity of red fluorescence were analyzed to reflect the level of mitochondrial oxidative stress.
[0062] 4. JC-1 probe detection of cardiomyocytes 4.1 Sample processing: After seeding cardiomyocytes into plates for 48 hours, wash once with PBS to remove residual culture medium; 4.2 Staining treatment: Add an appropriate amount of 2µM JC-1 dye solution to each well and incubate in a 37℃ incubator for 30 minutes; after incubation, wash the cells 3 times with PBS, add Hoechst dye and incubate in a 37℃, 5% CO2 incubator for 10 minutes, and then wash 3 times with PBS. 4.3 Microscopic observation: The cell plate was placed under an Olympus IX83 confocal microscope. Red fluorescence (high membrane potential) was detected at an excitation wavelength of 490 nm / emission wavelength of 590 nm, and green fluorescence (low membrane potential) was detected at an excitation wavelength of 490 nm / emission wavelength of 530 nm. The distribution of red and green fluorescence in the cells was observed and photographed, and the red-green fluorescence ratio was calculated to reflect the changes in mitochondrial membrane potential.
[0063] III. Experimental Conclusions This experiment used samples from patients with heart failure, a mouse model of heart failure with aortic arch constriction, and an isoproterenol-induced cardiomyocyte hypertrophy model to conduct relevant tests. The results of each test were combined with corresponding statistical analysis methods for judgment. The specific results are as follows: like Figure 1 As shown, compared with normal patients, serum Aralar expression levels were significantly lower in patients with heart failure (p < 0.001). Compared with the control group, data are expressed as mean ± SEM. One-way ANOVA was used, and statistical significance was assessed using LSD (n = 60).
[0064] like Figure 2 As shown, in the M-mode echocardiography of mice, Figure AC shows that compared with the Sham group, the EF and FS of the TAC group mice decreased, and Figure DE shows that compared with the Sham group mice, the HW / BW and HW / TL increased (***p<0.001). Compared with the control group, the data are expressed as mean ± SEM. One-way ANOVA was used, and then LSD was used to assess statistical significance (n=6).
[0065] like Figure 3 As shown, Figure 3 A and Figure 3 B showed that, compared with the Sham group mice, the TAC group mice had more obvious Masson staining of myocardial fibrosis; Figure 3 C and Figure 3 D shows that, compared with the Sham group mice, H&E staining showed increased cell volume, irregular cell morphology, disordered arrangement, and nuclear condensation and necrosis. The scale bar is 50 μm and the magnification is ×20.
[0066] like Figure 4 As shown, Real-time PCR results indicated that Aralarm RNA expression was significantly reduced in mice after heart failure modeling. Nine mice were included in each group. Real-time PCR results were analyzed using a t-test (Student's T-test). Compared with the solvent group at the corresponding time points, ***P<0.001.
[0067] like Figure 5 As shown in the Westerblotting results, compared with the Sham group, the protein expression of Aralar in the TAC group was significantly reduced in heart failure (***P<0.001, n=3).
[0068] like Figure 6As shown, in the M-mode echocardiography of mice, Figure AC shows that compared with the TAC+AAV9-cTnT-NC group, the EF and FS of the TAC+AAV9-cTnT-Aralar group were decreased. Figure DE shows that compared with the TAC+AAV9-cTnT-NC group, the HW / BW and HW / TL of the TAC+AAV9-cTnT-Aralar group were reduced (p < 0.001). Compared with the control group, the data are expressed as mean ± SEM. One-way ANOVA was used, and then LSD was used to assess statistical significance (n = 6).
[0069] like Figure 7 As shown, Figure 7 A and Figure 7 B showed that compared with the TAC+AAV9-cTnT-NC group, the Masson staining of myocardial fibrosis in the TAC+AAV9-cTnT-Aralar group was significantly improved; Figure 7 C and Figure 7 As shown in Figure 1, compared with the TAC+AAV9-cTnT-NC group, the TAC+AAV9-cTnT-Aralar group showed reduced cell volume, decreased cell morphology, and reduced nuclear pyknosis and necrosis after H&E staining. (Scale bar: 50 μm, magnification × 20).
[0070] like Figure 8 As shown in the figure, Real-time PCR results indicated that, compared with the TAC+AAV9-cTnT-NC group, the expression level of Aralarm RNA in the TAC+AAV9-cTnT-Aralar group was significantly increased in heart failure. Nine mice were used in each group. Real-time PCR results were analyzed using a t-test (Student's T-test). Comparison with the solvent group at the corresponding time points showed no significant difference (***P<0.001).
[0071] like Figure 9 As shown in the Westerblotting results, compared with the TAC+AAV9-cTnT-NC group, the protein expression of Aralar in the TAC+AAV9-cTnT-Aralar group was significantly increased in heart failure (***P<0.001, n=3).
[0072] like Figure 10As shown in the figure, Real-time PCR results indicated that, compared with the ISO+AAV9-cTnT-NC group, the expression level of Aralarm RNA in the ISO+AAV9-cTnT-Aralar group was significantly increased in the myocardial mast cell model. Nine replicates were performed for each group, and Real-time PCR results were analyzed using a t-test (Student's T-test). Comparison with the solvent group at the corresponding time points showed no significant difference (***P<0.001).
[0073] like Figure 11 As shown in the Westerblotting results, compared with the ISO+AAV9-cTnT-NC group, the expression level of Aralar protein in the ISO+AAV9-cTnT-Aralar group was significantly increased in the myocardial mast cell model (***P<0.001, n=3).
[0074] like Figure 12 As shown, compared with the ISO+AAV9-cTnT-NC group, the ISO+AAV9-cTnT-Aralar group showed an increase in mitochondrial membrane potential, a decrease in green monomers, and an increase in red aggregates in the NRCM (representative confocal microscopy images are shown, scale bar: 50 μm; the data presented are representative of three independent experiments).
[0075] like Figure 13 As shown, compared with the ISO+AAV9-cTnT-NC group, the mitochondria in the NRCM of the ISO+AAV9-cTnT-Aralar group showed an increase in elongated mitochondria and a decrease in short, thick, or rod-shaped mitochondria, suggesting increased fusion and decreased division. Representative confocal microscopy images are shown, scale bar: 50 μm. The data presented are representative of three independent experiments.
[0076] like Figure 14 As shown, compared with the ISO+AAV9-cTnT-NC group, the red fluorescence of mitochondrial oxidative stress product Mitosox in the NRCM of the ISO+AAV9-cTnT-Aralar group was significantly reduced (representative confocal microscopy images are shown, scale bar: 50 μm, and the data presented are representative of three independent experiments).
[0077] like Figure 15 As shown, Figure 15 In AB, Westerblotting results showed that compared with the ISO+AAV9-cTnT-NC group, the expression of mitochondrial protein MFN1 in the NRCM of the ISO+AAV9-cTnT-Aralar group was increased, ***P<0.001, n=3; Figure 15In CD, Westerblotting results showed that compared with the ISO+AAV9-cTnT-NC group, the expression of mitochondrial protein MFN1 in the NRCM of the ISO+AAV9-cTnT-Aralar group was increased, ***P<0.001, n=3; Figure 15 In EF, Westerblotting results showed that compared with the ISO+AAV9-cTnT-NC group, the expression of mitochondrial protein DRP1 in the NRCM of the ISO+AAV9-cTnT-Aralar group was reduced, ***P<0.001, n=3; Figure 15 In AB, Western blotting results showed that compared with the ISO+AAV9-cTnT-NC group, the expression of mitochondrial protein TFAM in the NRCM of the ISO+AAV9-cTnT-Aralar group was increased, ***P<0.001, n=3.
[0078] In summary, this application demonstrated that in the heart of a mouse with heart failure, targeting cardiomyocytes with AAV9-cTnT-Aralar and overexpressing Aralar significantly reduced cardiac enlargement and improved cardiac function. In a cardiomyocyte hypertrophy model, AAV9-cTnT-Aralar intervention significantly altered mitochondrial dynamics, reducing mitochondrial fission and increasing fusion, with corresponding changes in the expression of mitochondrial fusion-related proteins Mfn1 and Mfn2, and the fission protein Drp1. Further examination of mitochondrial function showed that increased Aralar expression increased mitochondrial membrane potential, decreased reactive oxygen species (ROS) generation, and increased mitochondrial DNA expression. In conclusion, AAV9-cTnT-Aralar can improve mitochondrial dynamics, leading to increased mitochondrial DNA production, reduced ROS generation, and thus improving the course of heart failure.
[0079] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. The application of Aralar myocardial overexpression virus in the preparation of drugs for treating heart failure, wherein the Aralar myocardial overexpression virus is a recombinant adeno-associated virus type 9 (AAV9) vector, characterized in that: The AAV9 vector carries a myocardial-specific promoter and a nucleic acid molecule encoding the Aralar protein, and has cardiac-targeted delivery characteristics, for the specific overexpression of the Aralar protein in cardiomyocytes, which treats heart failure by improving mitochondrial function in cardiomyocytes.
2. The application of the Aralar myocardial overexpression virus according to claim 1 in the preparation of a drug for treating heart failure, characterized in that: The myocardial-specific promoter is the cardiac troponin T promoter cTnT, and the AAV9 vector carries an expression cassette encoding the Aralar protein, forming a recombinant adeno-associated virus AAV9-cTnT-Aralar that has cardiac-targeted delivery characteristics and can specifically overexpress the Aralar protein in cardiomyocytes.
3. The application of the Aralar myocardial overexpression virus according to claim 1 in the preparation of a drug for treating heart failure, characterized in that: The Aralar is the Aralar protein encoded by the SLC25A12 gene, or a functional fragment or amino acid sequence variant derived from this protein that has mitochondrial regulatory activity.
4. The application of the Aralar myocardial overexpression virus according to claim 1 in the preparation of a drug for treating heart failure, characterized in that: The heart failure includes heart failure induced by aortic arch constriction or isoproterenol stimulation, as well as heart failure caused by stress overload, heart failure associated with myocardial hypertrophy, or heart failure with mitochondrial dysfunction.
5. The application of the Aralar myocardial overexpression virus according to claim 1 in the preparation of a drug for treating heart failure, characterized in that: The improvement of cardiomyocyte mitochondrial function includes at least one of maintaining or increasing mitochondrial membrane potential, inhibiting mitochondrial oxidative stress levels, and increasing the expression level of mitochondrial DNA transcription factor TFAM, wherein TFAM is related to the maintenance of mitochondrial DNA.
6. The application of the Aralar myocardial overexpression virus according to claim 1 in the preparation of a drug for treating heart failure, characterized in that: The improvement of cardiomyocyte mitochondrial function includes maintaining or promoting mitochondrial dynamic homeostasis, which involves the expression of mitochondrial fusion-related proteins MFN1 and MFN2 and the expression of mitochondrial division-related protein Drp1.
7. A gene therapy agent for treating heart failure, characterized in that: It contains an effective amount of AAV9-cTnT-Aralar, wherein AAV9-cTnT-Aralar is a recombinant adeno-associated virus used to specifically overexpress the Aralar protein in cardiomyocytes.
8. A gene therapy agent for treating heart failure according to claim 7, characterized in that: The gene therapy agent is administered via myocardial-targeted delivery.
9. A gene therapy agent for treating heart failure according to claim 7, characterized in that: The gene therapy formulation further comprises at least one of a pharmaceutically acceptable carrier, diluent, excipient, buffer system, or adjuvant.
10. A gene therapy agent for treating heart failure according to claim 7, characterized in that: The gene therapy preparation is an injectable formulation.