Gene medicine for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation as well as preparation method and application of gene medicine

Abstract

The invention relates to the technical field of gene engineering, in particular to a gene medicine for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation as well as a preparation method and application of the gene medicine. Effective components of the gene drug provided by the invention comprise shuttle plasmids; the shuttle plasmid comprises an AAV9 vector and a PNPLA2 gene. The PNPLA2 gene with normal function is delivered through the AAV9 carrier, lipid metabolism disorder caused by mutation is repaired, fat deposition in cardiac muscle is reduced, and then the pathological change of ACM is improved. By recovering the function of the PNPLA2 gene, the process of myocardial fibrosis can be effectively inhibited, and the cardiac function can be improved. The cardiac muscle specific promoter cTnT is adopted to ensure that the PNPLA2 gene is only expressed in cardiac muscle, so that the targeting property of gene therapy is improved, and side effects caused by systemic expression are avoided. The strategy can effectively reduce the side effects of gene therapy and ensure the accuracy and safety of therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and in particular to a gene medicine for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation, and a preparation method and application thereof. Background Art

[0002] Arrhythmogenic cardiomyopathy (ACM) is a cardiomyopathy caused by genetic factors, characterized by the gradual replacement of myocardial tissue with fat and fibrosis, leading to ventricular arrhythmias and progressive heart function decline. ACM usually affects the right ventricle as the main site of involvement, but can also affect the left ventricle. The disease is mainly inherited in families, and common pathogenic gene mutations include PKP2, DSG2, DSP, CTNNA3, etc. In recent years, more and more studies have found that abnormal lipid metabolism, especially PNPLA2 gene mutations, is also one of the key pathogenic factors of ACM.

[0003] The protein encoded by the PNPLA2 (patatin-like phospholipase domain-containing protein 2) gene is mainly responsible for catalyzing the hydrolysis of triglycerides and is a very important enzyme in lipid metabolism. Mutations in PNPLA2 can lead to lipid metabolism disorders, which in turn cause fat deposition in cardiomyocytes and myocardial fibrosis. This pathological change significantly increases the risk of sudden death in patients. In particular, the PNPLA2 c.G245A (p.G82D) mutation has been confirmed by many studies to be closely related to the occurrence of arrhythmogenic cardiomyopathy (ACM). According to studies, this mutation causes the loss of PNPLA2 enzyme activity, leading to fat deposition in the myocardium and triggering pathological changes in the heart, which seriously affects heart function. The clinical manifestations of ACM include symptoms such as arrhythmias, myocardial hypertrophy, right ventricular enlargement, and heart failure, and often occur in young patients, usually around the age of 40, greatly affecting the patient's quality of life and prognosis.

[0004] At present, the treatment methods for ACM are limited, and the existing treatment methods mainly focus on symptom control, drug therapy and heart transplantation. In terms of symptom control, antiarrhythmic drugs are mainly used to relieve arrhythmias and improve heart function. However, these drugs can only relieve symptoms and cannot cure the disease. For heart transplantation, it is the only effective treatment for patients with end-stage ACM. Although heart transplantation can significantly improve the quality of life of patients, due to the shortage of donor hearts, many patients cannot receive transplantation in time, and heart transplantation itself is also accompanied by problems such as immune rejection. The long-term survival rate and prognosis of patients are not ideal. Most of the current research focuses on the treatment of ACM caused by structural protein mutations, and not enough attention has been paid to the cause of lipid metabolism disorders. In particular, there is currently no effective gene therapy program for ACM caused by PNPLA2 gene mutations. Most of the existing research is to simply repair structural protein genes, rather than lipid metabolism treatment.

[0005] Therefore, there is currently a lack of a treatment option that can fundamentally cure ACM, especially ACM caused by PNPLA2 gene mutations, and there is an urgent need to develop new treatment strategies to address the problems of lipid metabolism disorders and myocardial fat infiltration. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a gene drug for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation, and its preparation method and application. The present invention is based on the AAV9 vector and adopts the gene therapy strategy of the myocardial specific promoter cTnT, which restores the function of the PNPLA2 gene by repairing lipid metabolism disorders, thereby improving the pathological changes of ACM.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a gene drug for treating arrhythmogenic cardiomyopathy caused by PNPLA2 mutation. The effective component of the gene drug includes a shuttle plasmid; the shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, and the PNPLA2 gene is located downstream of a cTnT promoter on the AAV9 vector.

[0009] Preferably, the nucleotide sequence of the PNPLA2 gene is as shown in SEQ ID NO.1.

[0010] Preferably, the AAV9 vector is a pHBAAV vector.

[0011] Preferably, the gene drug is a viral particle packaged by a lentivirus.

[0012] Preferably, the titer of the virus particles in the gene drug is 1×10 12 ~1×1013 vg / mL.

[0013] The present invention provides a method for preparing the gene medicine described in the above technical solution, comprising the following steps:

[0014] The shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid were mixed to obtain a three-plasmid system;

[0015] The three-plasmid system is packaged and purified in HEK293T cells to obtain the gene medicine.

[0016] Preferably, the mass ratio of the shuttle plasmid, the pAAV-RC plasmid and the pHelper plasmid is 1:1:2.

[0017] Preferably, the virus packaging reagent includes liposomes; the mass volume ratio of the three-plasmid system and the liposomes is 40 μg:120 μL.

[0018] The present invention provides the use of the gene drug described in the above technical solution or the gene drug prepared by the preparation method described in the above technical solution in the preparation of a product for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy.

[0019] Preferably, the PNPLA2 mutation is PNPLA2 c.245G>A (p.G82D).

[0020] Beneficial effects:

[0021] The present invention provides a gene drug for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation, wherein the active ingredient of the gene drug includes a shuttle plasmid; the shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, and the PNPLA2 gene is located downstream of the cTnT promoter on the AAV9 vector. The present invention delivers a functional PNPLA2 gene through an AAV9 vector to repair lipid metabolism disorders caused by mutations, reduce fat deposition in the myocardium, and thus improve the pathological changes of ACM. By restoring the function of the PNPLA2 gene, the process of myocardial fibrosis can be effectively inhibited and cardiac function can be improved. The present invention uses a myocardial-specific promoter cTnT to ensure that the PNPLA2 gene is expressed only in the myocardium, thereby improving the targeting of gene therapy and avoiding side effects caused by systemic expression. This strategy can effectively reduce the side effects of gene therapy and ensure the accuracy and safety of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is the pHBAAV-TNT-3flag-P2A-EGFP vector map.

[0024] Figure 2 The sequencing results of PNPLA2 c.G245A / c.G245A mutant mice and wild-type mice; the upper part is the sequencing result of wild-type mice, and the lower part is the sequencing result of PNPLA2 c.G245A / c.G245A mutant mice.

[0025] Figure 3 The electrophoresis results of PNPLA2 c.G245A / c.G245A mutant mice and wild-type mice.

[0026] Figure 4 Figure 1. PNPLA2 c.G245A / c.G245A mutation reproduces ACM phenotype in mice. a. Sanger sequencing detected the sequence of PNPLA2 gene in Pnpla2 c.G245A / c.G245A knock-in mice and wild-type (WT) control mice. b. Kaplan-Meier survival analysis comparing the survival of wild-type (WT, n=10) and Pnpla2c.G245A / c.G245A (Hom, n=13) mice. c. Representative echocardiographic images showing long-axis and short-axis views of WT and Hom mice. d. M-mode echocardiogram of WT and Hom mice. e. Quantitative analysis of ejection fraction (EF), fractional shortening (FS), end-diastolic volume (EDV), and end-systolic volume (ESV) comparing WT and Hom mice. f. Representative electrocardiogram (ECG) waveforms from WT (n=6) and Hom (n=5) mice. g. Representative images of the hearts of WT and Hom mice. h. Ratio of heart weight to body weight of WT (n=6) and Hom (n=5) mice. i H&E staining shows the histological features of right ventricular (RV) tissues of WT and Hom mice. The scale bar on the left is 500 μm, and the scale bar on the right is 25 μm. j. Masson trichrome staining shows fibrosis in the right ventricular tissues of WT and Hom mice. The scale bar on the left is 500 μm, and the scale bar on the right is 25 μm. k. Quantitative analysis of the changes in Col1a1 expression in the right ventricle of WT (n=6) and Hom (n=5) mice. l. Oil red O staining shows lipid accumulation in WT and Hom mice. The scale bar for each grid is 20 μm. m. Transmission electron microscopy (TEM) images show the ultrastructure of heart tissues of WT and Hom mice. The scale bar is 1 μm.

[0027] Figure 5Experimental results of early PNPLA2 supplementation to prevent the ACM phenotype of PNPLA2 mutant mice. a. Schematic diagram of AAV9 vector. b. Schedule of AAV9 vector delivery by intrathoracic injection on postnatal day 5. c. Echocardiogram showing cardiac function of untreated (Hom), AAV9-PNPLA2 treated (Treated), and wild-type (WT) mice. d. Comparison of ejection fraction (EF) and fractional shortening (FS) in WT (n=6), Hom (n=5), and Treated (n=6) mice. e. CK-MB levels in WT (n=6), Hom (n=5), and Treated (n=6) mice. f. Gross images of the heart in WT, untreated, and treated mice. g. Ratio of heart weight to body weight in WT (n=6), Hom (n=5), and Treated (n=6) mice. h. H&E staining of myocardial tissue in WT, untreated, and treated mice, with the left scale being 500 μm and the right scale being 25 μm. i. Masson trichrome staining of right ventricular tissues of WT, untreated and treated mice, with a scale of 500 μm on the left and 25 μm on the right. j. Quantitative analysis of fibrosis in the right ventricle of WT, untreated and treated mice. k. Transmission electron microscopy (TEM) images of heart tissues of WT, Hom and Treated mice, with a scale of 1 μm. l. Immunofluorescence of GFP-tagged PNPLA2 expression in the hearts of AAV9-treated mice, with a scale of 20 μm per grid.

[0028] Figure 6The experimental results of late-stage AAV9-PNPLA2 supplementation therapy to reverse the progression of ACM in PNPLA2 mutant mice. a. Schematic diagram of AAV9 vector delivery to 6-week-old PNPLA2 c.G245A / c.G245A mice by intrathoracic injection. b. Gross images of the hearts of WT, untreated (Hom) and treated (Treated) mice. c. Echocardiographic quantitative analysis of ejection fraction (EF) of WT (n=6), untreated (Hom, n=5) and AAV9-PNPLA2 treated (Treated, n=6) mice. d. Quantitative analysis of EF and FS of WT (n=6), Hom (n=5) and Treated (n=6) mice. e. CK-MB levels of WT (n=6), Hom (n=5) and Treated (n=6) mice. f. Ratio of heart weight to body weight of WT (n=6), Hom (n=5) and Treated (n=6) mice. g. H&E staining of myocardial tissue of WT, Hom and Treated mice, the left bar is 500μm, and the right enlarged bar is 25μm. h. Masson trichrome staining of ventricular tissue of WT, Hom and Treated mice, the left bar is 500μm, and the right enlarged bar is 25μm. i. Quantitative analysis of right ventricular fibrosis in WT, Hom and Treated mice. j. Transmission electron microscopy (TEM) images of cardiac ultrastructure of WT, Hom and Treated mice, the scale bar is 1μm. k. Immunofluorescence of GFP-tagged PNPLA2 expression in the hearts of AAV9-treated mice, each grid scale bar is 20μm.

[0029] Figure 7The experimental results show that AAV9-PNPLA2 treatment significantly prolongs the survival of PNPLA2 mutant mice. a. Schematic diagram of 6-week-old Pnpla2 c.G245A / c.G245A mice treated with AAV9-PNPLA2 vector. b. Kaplan-Meier survival analysis comparing the survival of WT, Hom and Treated mice. c. Echocardiographic quantitative analysis of ejection fraction (EF) and fractional shortening (FS) in WT (n=6, 32 weeks old), Hom (n=5, 12 weeks old) and Treated (n=3, 32 weeks old) mice at 12 and 32 weeks. d. Serum CK-MB levels in WT (n=6), Hom (n=5) and Treated (n=6) mice. e. Gross images of the heart in WT, Hom and Treated mice. f. Ratio of heart weight to body weight in WT (n=6), Hom (n=5) and Treated (n=6) mice. g. H&E staining of myocardial tissues of WT, Hom, and Treated mice. h. Masson trichrome staining of ventricular tissues of WT, Hom, and Treated mice. i. Quantitative analysis of myocardial fibrosis in WT (n=6), Hom (n=5), and Treated (n=6) mice. j. Transmission electron microscopy (TEM) images of the ultrastructure of the heart of WT, Hom, and Treated mice, showing lipid deposition between cardiomyocytes, with a scale bar of 2 μm. k. Immunofluorescence of GFP-tagged PNPLA2 expression in the hearts of AAV9-treated mice, with a scale bar of 20 μm per grid.

[0030] Figure 8 and Fig. 9 The results of the experiment revealing the changes in lipid metabolism of cardiomyocytes after AAV9-PNPLA2 treatment were obtained from the genome sequencing of mononuclear mice. Figure 8 Middle a. Experimental workflow for cardiac tissue sampling and single nucleus RNA sequencing (snRNA-seq) for wild-type and treated mice, with representative unified manifold approximation and projection (UMAP) plots after data integration. Each nucleus is color-coded according to the different stages of sample origin and the associated cell type (from left to right, top to bottom). Figure 8 Middle b. The proportion of each type of cells in untreated (Hom), AAV9-PNPLA2-treated (Treated), and wild-type (WT) mice. Figure 8 Middle c. The signature plot shows the expression of classic marker genes used to define each cell type. Figure 8 Middle d. Violin plots show the distribution of lipid metabolism scores calculated based on the expression levels of Abhd5, Acat1, Cd36, Cpt1a, Cpt1b, Dgat1, Lpl, Pparg and Srebf1 in experimental groups and cardiomyocyte subpopulations. Figure 8 e. GO enrichment analysis showed differentially expressed genes between the untreated group (Hom) and the AAV9-PNPLA2 treated group (Treated). Fig. 9 Middle f. Reconstructed cell trajectory diagram showing the transition from stage 5 to stage 1 obtained by Monocle 2 trajectory analysis and associated with pseudotime. Fig. 9 Middle g. Heat map showing the dynamic expression of genes in cardiomyocytes. CM, cardiomyocytes; FB, fibroblasts; EC, endothelial cells; MC, macrophages; PC, pericytes; LEC, lymphatic endothelial cells; NC, neurons.

[0031] Fig.10 Experimental results for the safety evaluation of AAV9-PNPLA2 gene therapy. a. AAV9-GFP transduction rate in heart and liver tissues of Pnpla2c.G245A / c.G245A mice injected on day 5 after birth (group A) 12 weeks after injection, each scale bar is 20μm. b. AAV9-GFP transduction rate in heart and liver tissues of mice treated at 6 weeks of age (group B) 6 weeks after treatment, each scale bar is 20μm. c. AAV9-GFP transduction rate in heart and liver tissues of mice treated at 6 weeks of age and analyzed at 32 weeks (group C), each scale bar is 20μm. d Quantification of AAV9 transduction rate in heart and liver tissues of groups A, B and C. e. H&E staining of liver tissues of wild-type and AAV9-PNPLA2-treated Pnpla2c.G245A / c.G245A mice. f. Serum AST / ALT levels of wild-type (n=6, 32 weeks old), syngeneic (n=5, 12 weeks old) and treated (n=15) mice. DETAILED DESCRIPTION

[0032] The present invention provides a gene drug for treating arrhythmogenic cardiomyopathy caused by PNPLA2 mutation. The effective component of the gene drug includes a shuttle plasmid; the shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, and the PNPLA2 gene is located downstream of a cTnT promoter on the AAV9 vector.

[0033] As an embodiment, the nucleotide sequence of the PNPLA2 gene is shown as SEQ ID NO.1.

[0034] As an embodiment, the AAV9 vector is a pHBAAV vector.

[0035] As an embodiment, the gene medicine is a viral particle packaged by a lentivirus.

[0036] As an embodiment, the titer of the virus particles in the gene drug is 1×10 12 ~1×10 13 vg / mL.

[0037] The gene drug provided by the present invention is a gene therapy drug based on adeno-associated virus type 9 (AAV9) vector, which can treat arrhythmogenic cardiomyopathy (ACM) caused by PNPLA2 gene mutation. The core component of the gene therapy drug is the AAV9 vector, which carries the myocardial specific promoter cTnT and the human PNPLA2 gene. Through gene delivery of the AAV9 vector, the PNPLA2 gene can be specifically expressed in cardiac tissue, thereby correcting the lipid metabolism abnormalities caused by the PNPLA2 mutation and improving cardiac function.

[0038] AAV9 (adeno-associated virus type 9) is the core delivery vector of the present invention, which has high cardiac targeting and low immunogenicity and is an ideal gene delivery tool. The composition of AAV9 virus is as follows.

[0039] 1.1 Inverted terminal repeat sequence (ITR): used in the virus packaging and transduction process to ensure that the AAV9 virus can successfully enter the target cells and stably express the target gene.

[0040] 1.2 Myocardial-specific promoter cTnT: This promoter drives the specific expression of the PNPLA2 gene in the myocardium. The cTnT (cardiac troponin T) promoter is highly myocardial-selective and can ensure the expression of the PNPLA2 gene in cardiac tissue without causing unnecessary expression in other tissues.

[0041] 1.3 Human PNPLA2 gene: The human PNPLA2 gene is used to replace the PNPLA2 gene that has lost its function due to gene mutation in the patient's body, restore the lipid metabolism balance of the heart, and slow down or reverse the pathological changes in the heart caused by PNPLA2 deficiency.

[0042] The virus particle structure of the AAV9 vector includes VP1, VP2 and VP3 proteins, and the molecular ratio is as follows: VP1: VP2: VP3 = 1: 1: 10. These viral proteins together constitute the capsid of the AAV9 virus, which is responsible for the stability, targeting and binding of the virus to the host cells. The diameter of the AAV9 virus particle is about 20 to 26 nm, which is a relatively small virus particle. It is easy to reach the target tissue through the blood circulation and can effectively penetrate the cell membrane to enter the cell. The titer range of the AAV9 virus in the present invention is 1×10 12 ~1×10 13vg / mL. This titer ensures sufficient gene delivery capacity to achieve efficient PNPLA2 gene expression in cardiac tissue. The virus solution uses phosphate buffered saline (PBS) as a solvent, and glycerol is added as a stabilizer. The role of glycerol is to form a protective film to prevent the aggregation or structural damage of virus particles when stored at low temperatures, and to ensure the stability of virus particles during freezing and thawing.

[0043] The drug solution is a colorless and transparent liquid. The virus particles are evenly dispersed in PBS. The glycerol concentration is 5% (w / v) to maintain the stability of the virus particles. At the same time, the gene therapy drug can be stored for a long time under low temperature conditions to ensure that the activity and titer of the virus particles do not decrease. The titer decreases by less than 5% within 12 months of freezing at -80°C; the titer decreases by less than 10% within 7 days of storage at room temperature at 25°C.

[0044] The diameter of AAV9 virus particles is 20-26 nm. This physical size enables AAV9 to efficiently penetrate cell membranes, especially in cardiomyocytes. At the same time, through the use of glycerol protective agent, AAV9 vectors can be stored stably for a long time under low temperature conditions, reducing aggregation or structural damage caused by low temperature. The titer of AAV9 virus in the drug solution is 1×10 12 ~1×10 13 vg / mL, ensuring the high efficiency of gene delivery and meeting the amount of viral vector required for treatment. The ratio of VP1, VP2 and VP3 proteins in AAV9 virus particles is 1:1:10. This ratio ensures the structural stability and transduction efficiency of virus particles.

[0045] Compared with existing treatment options, the gene therapy drugs of the present invention, especially the treatment method based on AAV9 vector delivery of PNPLA2 gene, have significant advantages, including higher targeting, better therapeutic effect, higher safety and more persistent gene expression. The specific advantages are as follows:

[0046] 1. Myocardium-specific delivery and expression

[0047] The present invention uses the AAV9 vector, which is an adeno-associated virus serotype with myocardial superiority and can efficiently penetrate cardiac tissue. Compared with other AAV serotypes, AAV9 has stronger targeting in the myocardium, which can ensure that the delivered gene is mainly expressed in the heart, reduce gene expression in non-target organs, and thus reduce potential side effects and toxicity. By using the cTnT promoter (myocardial-specific promoter) to drive the expression of the human PNPLA2 gene, it is further ensured that the gene is efficiently expressed only in myocardial cells, significantly improving the targeting and specificity of the treatment. In the prior art, most promoters (such as the CMV promoter) have the risk of systemic expression, which may lead to side effects in non-target organs. The present invention solves this problem through the use of the myocardial-specific promoter cTnT, thereby improving the accuracy and safety of the treatment.

[0048] 2. Efficient gene delivery and stable expression

[0049] The AAV9 vector system of the present invention can achieve efficient gene delivery in cardiomyocytes and ensure long-term gene expression. Compared with traditional viral vectors or other non-viral vectors, AAV9 has lower immunogenicity and longer expression persistence, and can therefore provide a more stable therapeutic effect. In animal experiments, the use of AAV9 to deliver the PNPLA2 gene significantly restored the lipid metabolism balance of the myocardium and improved cardiac function, and this effect remained stable in long-term follow-up. The sustained effect of gene therapy is one of its greatest advantages, avoiding the problem of repeated administration of traditional drug treatments and being able to suppress the progression of the disease for a long time.

[0050] 3. Improve heart function and reverse pathological changes

[0051] The experimental results showed that AAV9-PNPLA2 gene therapy can significantly improve the cardiac function of PNPLA2 gene mutant mice, including improvements in ejection fraction (EF) and fractional shortening (FS). The heart weight / body weight ratio was restored to a level close to that of wild-type mice, and the cardiac structure and tissue morphology were significantly restored. Pathological and ultrastructural analysis showed that fat infiltration in the myocardial tissue of the treatment group was reduced and myocardial fibrosis was significantly reversed, all of which indicate that the present invention can effectively treat cardiomyopathy caused by PNPLA2 mutations. This effect is significantly better than traditional drug therapy and existing gene therapy programs, especially in reversing pathological changes and restoring cardiac function.

[0052] 4. Excellent safety

[0053] Compared with other high-dose gene delivery schemes, the AAV9 vector used in the present invention has low immunogenicity and low toxicity. No significant hepatotoxicity or immune response was observed during the treatment, and the myocardial-specific expression of the AAV9 vector effectively avoided toxicity to non-target organs. Liver function indicators such as AST / ALT levels did not show a significant increase after treatment, ensuring the safety of the treatment. This safety advantage stems from the unique properties of the AAV9 vector and the myocardial-specific expression of the cTnT promoter, which reduces adverse reactions and side effects of the drug.

[0054] Compared with the prior art, the gene therapy drug of the present invention has higher targeting, stronger therapeutic effect and higher safety. Its advantages mainly come from the use of AAV9 vector for efficient myocardial targeted gene delivery, the application of myocardial specific promoter cTnT to ensure efficient gene expression in myocardial cells, and the long-term stability and safety of genes during treatment. In addition, the recovery of cardiac function and reversal of pathological changes after treatment also show that this technical solution has significant advantages over existing treatment methods. Therefore, the present invention not only provides an innovative treatment method for ACM caused by PNPLA2 mutation, but also provides new ideas and solutions for gene therapy of cardiovascular diseases.

[0055] The present invention provides a method for preparing the gene medicine described in the above technical solution, comprising the following steps:

[0056] The shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid were mixed to obtain a three-plasmid system;

[0057] The three-plasmid system is packaged and purified in HEK293T cells to obtain the gene medicine.

[0058] As an embodiment, the mass ratio of the shuttle plasmid, the pAAV-RC plasmid and the pHelper plasmid is 1:1:2.

[0059] As an embodiment, the virus-packaging reagent includes liposomes; the mass volume ratio of the three-plasmid system and the liposomes is 40 μg:120 μL.

[0060] The present invention provides the use of the gene drug described in the above technical solution or the gene drug prepared by the preparation method described in the above technical solution in the preparation of a product for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy.

[0061] As one embodiment, the PNPLA2 mutation is PNPLA2 c.245G>A (p.G82D).

[0062] To further illustrate the present invention, a gene drug for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation, a preparation method thereof and an application thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] A gene therapy drug based on adeno-associated virus type 9 (AAV9) vector was commissioned to Hanheng Biotechnology (Shanghai) Co., Ltd. for preparation, including: constructing a shuttle plasmid based on AAV9 vector to deliver the PNPLA2 gene, and performing virus packaging, purification and quality control in HEK293T cells through an optimized three-plasmid system, ultimately forming a high-titer, myocardial-specific gene delivery drug.

[0065] 1) The shuttle plasmid (pHBAAV-TNT-3flag-P2A-EGFP) carries the human PNPLA2 gene (NM_020376) and enhances gene expression and mRNA stability in cardiomyocytes through the cTnT myocardial-specific promoter and the PolyA signal sequence. The construction method is as follows: the target gene is inserted between the Nhe I and Hind III restriction sites of the pHBAAV vector to obtain the shuttle plasmid (pHBAAV-TNT-3flag-P2A-EGFP). The vector map is shown in Figure 1 .

[0066] The nucleotide sequence of the target gene (human PNPLA2 gene) is shown in SEQ ID NO.1, and is as follows:

[0067]

[0068] 2) The other two plasmids are pAAV-RC plasmid (encoding AAV9 capsid protein and rep protein) and pHelper plasmid (providing E1A / E1B genes). AAV-293 cells were used as host cells and cultured in DMEM (high glucose) medium supplemented with 10% fetal bovine serum. When the AAV-293 cells were cultured to 80% to 90% confluence, co-transfection of the three plasmids was started. In the transfection system, the shuttle plasmid, pAAV-RC, and pHelper plasmid were configured in a mass ratio of 1:1:2. During the transfection process, the total mass of the three plasmids was mixed with liposomes (Lipofectamine TM ) were mixed at a mass volume ratio of 40 μg:120 μL to form a transfection complex, which was then added to the cell culture medium. The cells were cultured at 37°C for 6 hours and then replaced with fresh culture medium containing 10% FBS. 72 hours after transfection, cells and culture supernatant were collected for subsequent virus extraction.

[0069] 3) Virus extraction was performed using the freeze-thaw method. The collected cells were placed between liquid nitrogen and a 37°C water bath and repeatedly frozen and thawed three times, with an interval of 30 seconds each time, to lyse the cells and release viral particles. After freezing and thawing, the lysate was centrifuged at 4°C and 2000×g for 5 minutes to remove cell debris, and the supernatant was collected for subsequent treatment. The purification of viral particles was first precipitated using 8% PEG-8000 and 0.5M NaCl, mixed and allowed to stand at 4°C for 12 hours, and then the precipitate was collected by centrifugation at 12000×g for 30 minutes and resuspended in PBS. The nucleic acid residues and other impurities were further removed by the Biomiga AAV purification column to finally obtain a high-purity viral liquid.

[0070] 4) The purified virus solution was concentrated to 1 mL through an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. The virus titer range was 1×10 12 ~1×10 13 The quality control process includes using fluorescent quantitative PCR to detect virus titer, HeLa cells for sterility testing, and PCR to detect mycoplasma contamination to ensure that the quality of the virus sample meets the standards.

[0071] 5) Finally, the purified virus solution was diluted with PBS to the required concentration (1×10 12 ~1×10 13 vg / mL), and add 5% (w / v) glycerol or sucrose as a stabilizer to ensure the stability of the virus particles when stored at low temperatures. The prepared virus solution is divided into sterile EP tubes and stored at -80°C to ensure the stability of the virus particles under long-term storage conditions.

[0072] Example 2 AAV9 delivery of PNPLA2 gene therapy for PNPLA2 mutant mice

[0073] 1. Experimental Procedure

[0074] 1.1 Construction of mouse model

[0075] This experiment used a PNPLA2 c.G245A homozygous mutation (PNPLA2 c.G245A / c.G245A) mouse model, which was constructed by Saiye (Suzhou) Biotechnology Co., Ltd. using CRISPR / Cas9 technology. This mutation mimics the c.G245A mutation of the human PNPLA2 gene, which is known to be associated with ARVC. The construction method includes: the mouse strain is: C57BL / 6; the mouse Pnpla2 gene (GenBank accession number: NM_001163689.1; Ensembl: ENSMUSG00000025509), exon 2 is selected as the target, and the G82D (GGT>GAT) mutation site in the donor oligo is introduced into exon 2 by homologous directed replication.

[0076] Sequence analysis and BamH I restriction endonuclease analysis were performed on mice injected with mRNA. The results are shown in Figure 2 and Figure 3 .Depend on Figure 2 and Figure 3 It can be seen that the PNPLA2 c.G245A mutant mouse model was successfully constructed.

[0077] The presence of the PNPLA2 c.G245A / c.G245A mutation in Pnpla2c.G245A / c.G245A mice (Hom) and wild-type (WT) control mice was confirmed by Sanger sequencing ( Figure 4 Middle a, Figure 5 The survival of Pnpla2 c.G245A / c.G245A mice was significantly shortened, and all homozygous mice died before 14 weeks of age, indicating that this mutation had a severe impact on survival ( Figure 4 (b)

[0078] To explore the functional characteristics of Pnpla2 mutant mice, the present invention performed echocardiographic evaluation and observed that the left ventricular systolic function of 6-week-old Pnpla2 c.G245A / c.G245A mice was significantly impaired, as manifested by a significant decrease in ejection fraction (EF) and fractional shortening (FS). Figure 4 These mice also showed significant increases in end-diastolic volume (EDV) and end-systolic volume (ESV), suggesting severe systolic dysfunction and ventricular dilatation ( Figure 4In addition, frequent premature ventricular contractions (PVCs) were observed in Pnpla2c.G245A / c.G245A mice, suggesting electrophysiological abnormalities consistent with ACM ( Figure 4 (f)

[0079] The hearts of Pnpla2 c.G245A / c.G245A mice were significantly enlarged, with obvious fatty infiltration, and the heart-to-body weight ratio was significantly increased compared with that of WT mice ( Figure 4 g and h). Histological analysis showed extensive myocardial thickening and lipid accumulation in the right ventricular wall of Pnpla2 c.G245A / c.G245A mice ( Figure 4 Masson trichrome staining results showed that the right ventricle of Pnpla2c.G245A / c.G245A mice had obvious fibrosis, and the expression of fibrosis marker Col1a1 was increased ( Figure 4 j and k).

[0080] Ultrastructural examination of cardiac tissue by transmission electron microscopy (TEM) revealed significant abnormalities, including enlarged lipid droplets in the intermyofibril spaces and disorganized sarcomere structure ( Figure 4 These results indicate that the Pnpla2c.G245A / c.G245A mouse model has highly similar cardiac characteristics to ACM patients.

[0081] 1.2 Preparation and delivery of viral vectors

[0082] The mice were randomly divided into two groups: the early treatment group (treated on the 5th day after birth, Figure 5 Middle b), late treatment group (treated at 6 weeks of age, Figure 6 (a). Each group contained at least 5 mice.

[0083] The virus titer prepared in Example 1 was 1×10 12 vg / mL of virus solution was delivered by intrathoracic injection (IT), with each mouse in the early treatment group (5 days after birth) being injected with 20 μL of virus solution, and each mouse in the late treatment group (6 weeks old) being injected with 50 μL of virus solution.

[0084] 1.3 Cardiac function assessment

[0085] At 2, 4, and 8 weeks after treatment, cardiac function was evaluated by echocardiography, mainly measuring ejection fraction (EF) and fractional shortening (FS). At the same time, electrocardiogram (ECG) monitoring was performed to check for arrhythmias, including premature arrhythmias (PVCs) and ventricular tachycardia (VT).

[0086] 1.4 Pathological analysis

[0087] After treatment, the mouse hearts were subjected to histological analysis, and HE staining and Masson staining were used to evaluate the structural changes, fibrosis, and fat deposition of the myocardium. Immunohistochemistry (IHC) and Western blot were used to detect the changes in PNPLA2 protein expression and related lipid metabolism genes (such as ABHD5, LPL, PPARγ, etc.).

[0088] 2. Experimental results

[0089] 2.1 Experimental results of the early treatment group are shown in Figure 5 .

[0090] Pnpla2 c.G245A / c.G245A mice treated with AAV9-PNPLA2 showed significantly improved cardiac function, with ejection fraction (EF) and fractional shortening (FS) significantly higher than those of untreated mutant mice. After 2 weeks of treatment, the ejection fraction (EF) increased from about 40% before treatment to 60%, and the fractional shortening (FS) increased from 15% to 30%. After 4 weeks of treatment, EF was close to the level of WT mice (70%), and FS increased to 40%, indicating that cardiac contractile function was maintained ( Figure 5 Creatine kinase isoenzyme MB (CK-MB) levels are a marker of myocardial injury. Untreated Pnpla2c.G245A / c.G245A mice had CK-MB levels that were almost twice as high as those in wild-type mice, whereas AAV9-PNPLA2 treatment reduced CK-MB levels to wild-type levels, indicating that cardiac injury was alleviated ( Figure 5 e). Gross examination revealed that the heart morphology of AAV9-PNPLA2 treated Pnpla2 c.G245A / c.G245A mice was normal, with no fat deposition, similar to that of wild-type mice ( Figure 5 f). The heart weight to body weight ratio of the treated mice was also reduced, approaching that of the wild-type mice, indicating that heart enlargement was effectively alleviated ( Figure 5 Middle g). H&E staining after 12 weeks showed that the myocardial structure of mice in the treatment group was intact and no inflammatory infiltration was observed ( Figure 5 In contrast to the marked fibrosis in untreated mutant mice, the absence of right ventricular fibrosis in the treated group demonstrated the protective effect of gene supplementation therapy ( Figure 5 Transmission electron microscopy (TEM) analysis showed that the myometrial structure of the heart tissue of the treated mice was well arranged, the mitochondrial ultrastructure was intact, the intercellular connections were stable, and no abnormal lipid accumulation or organelle damage was observed ( Figure 5 Immunofluorescence experiments confirmed the expression of GFP-tagged PNPLA2 protein in the hearts of AAV9-treated mice, verifying the effective delivery and expression of exogenous proteins ( Figure 5 (middle l).

[0091] These results indicate that early AAV9-mediated PNPLA2 gene supplementation significantly prevented the occurrence of ACM in Pnpla2c.G245A / c.G245A mice and preserved cardiac structure and function.

[0092] 2.2 Experimental results of the late treatment group are shown in Figure 6 After treatment, the cardiac morphology of PNPLA2 c.G245A / c.G245A mice was similar to that of wild-type mice, with no obvious external fat accumulation ( Figure 6 (middle b). Echocardiography 6 weeks after treatment showed that the ejection fraction (EF) and fractional shortening (FS) of mice in the AAV9-PNPLA2 treatment group were significantly improved, with EF and FS increased to 50% and 20%, respectively, compared with untreated mice, although still lower than wild-type levels, indicating partial recovery ( Figure 6 (c, d). Treatment initiated after the onset of ACM stabilized cardiac function but failed to completely reverse the existing functional deficits. Serum CK-MB levels have dropped to wild-type levels, indicating reduced myocardial damage ( Figure 6 The heart weight to body weight ratio of the treated mice has dropped to a level comparable to that of the wild type, which is an improvement compared to the ACM mice ( Figure 6 Middle f). H&E staining showed that the myocardial structure of the treated mice was normal, with neatly arranged muscle fibers, indicating that the histology had returned to normal ( Figure 6 (g). Compared with untreated mice, the treated group showed reduced fibrosis, suggesting that fibrosis was reversed ( Figure 6 h, i). Transmission electron microscopy (TEM) analysis showed that the myometrial structure of the heart tissue of the treated mice was orderly, the mitochondria were intact, the intercellular connections were stable, and there was no abnormal lipid accumulation, reflecting the recovery of the ultrastructure ( Figure 6 Immunofluorescence staining confirmed the expression of GFP-tagged PNPLA2 in the hearts of AAV9-treated mice, verifying the effectiveness of gene delivery ( Figure 6 These results suggest that late-stage AAV9-PNPLA2 gene therapy can partially reverse ACM pathological changes and stabilize cardiac function, highlighting its potential benefit in the treatment of advanced ACM.

[0093] 3. Summary and clinical significance

[0094] The treatment of PNPLA2 gene delivered by AAV9 vector can effectively improve the cardiac function, pathological manifestations and rhythm stability of PNPLA2 c.G245A / c.G245A mutant mice. The effect of early treatment is better than that of late treatment. Early treatment can not only significantly restore cardiac function, reduce fibrosis and fat accumulation, but also significantly prolong the survival of mice. Although late treatment prolongs the survival period, the effect on improving cardiac function is limited, especially for the structural damage of the heart that has already occurred, the degree of recovery is poor. These experimental results provide important preclinical evidence for the treatment of ARVC based on AAV9 delivery of PNPLA2 gene, and also lay the foundation for the application of this strategy in clinical practice.

[0095] Example 3

[0096] To evaluate the long-term efficacy of AAV9-PNPLA2 treatment, we treated 6-week-old Pnpla2c.G245A / c.G245A mice and monitored their survival until 32 weeks ( Figure 7 Although treatment prolonged the survival of mice, echocardiography at 12 weeks showed that the ejection fraction (EF) and fractional shortening (FS) of treated mice were still significantly lower than those of wild-type mice and similar to those of untreated mutant mice ( Figure 7 This suggests that although late-stage gene therapy prolonged survival, it failed to completely prevent cardiac dysfunction. At 32 weeks, serum CK-MB levels in treated mice were lower than in untreated control mice, indicating reduced myocardial damage ( Figure 7 Gross examination revealed no obvious fat accumulation in the hearts of AAV9-PNPLA2-treated Pnpla2 c.G245A / c.G245A mice, and H&E staining confirmed minimal adipocyte infiltration in the myocardial tissue ( Figure 7 The heart weight to body weight ratio of the treated mice has dropped to the wild-type level, which is improved compared with the ACM mice ( Figure 7 Compared with untreated mutant mice, myocardial fibrosis in treated mice was reduced, but still higher than that in wild-type mice at 32 weeks of age ( Figure 7 h, i). Transmission electron microscopy (TEM) analysis showed that lipid deposition still existed between cardiomyocytes ( Figure 7 Immunofluorescence staining confirmed the sustained expression of GFP-tagged PNPLA2 in the hearts of AAV9-treated mice, verifying the presence of exogenous protein ( Figure 7 These results demonstrate that late-stage AAV9-PNPLA2 gene therapy significantly prolonged the survival of mutant mice despite the presence of cardiac dysfunction, suggesting that this treatment has the potential benefit of extending lifespan even in the presence of cardiac damage.

[0097] Example 4: Mononuclear mouse genome sequencing confirms that AAV9-PNPLA2 gene therapy regulates cardiomyocyte function by improving lipid metabolism

[0098] To further explore the effects of AAV9-PNPLA2 gene therapy on transcriptome levels, we collected heart tissues from wild mice, PNPLA2 c.G245A / c.G245A mice, and mice that received AAV9-PNPLA2 gene therapy for single-nucleus mouse genome sequencing. We placed five mouse heart tissues in a test tube for cell nuclei extraction and used 10x Genomics technology for sequencing, library construction, and data analysis ( Figure 8 A total of 6434 nuclei were analyzed, and cells could be labeled based on genes specifically expressed in each cell type. Based on specific marker genes, these cells could be divided into seven categories: cardiomyocytes (CM), fibroblasts (FB), endothelial cells (EC), macrophages (MC), pericytes (PC), lymphatic endothelial cells (LEC), and neurons (NC). Notably, cardiomyocytes increased significantly after gene therapy, highlighting the efficacy of AAV9-PNPLA2 treatment ( Figure 8 (middle ac).

[0099] Based on the expression levels of key genes in classical lipid metabolism (such as Abhd5, Acat1, Cd36, Cpt1a, Cpt1b, Dgat1, Lpl, Pparg, and Srebf1), the lipid metabolism score was significantly improved after gene therapy ( Figure 8 By comparing the differentially expressed genes (DEGs) related to lipid metabolism in cardiomyocytes before and after gene therapy, we found that the expression of genes related to lipid metabolism was significantly different. GO enrichment analysis further supported this finding ( Figure 8 e). Using the Monocle 2 analysis method, we observed that after treatment, stage 1 cells transformed into stage 5, which is characterized by improved lipid metabolism and consistent with cell fate determination. This transformation was accompanied by a significant upregulation of lipid metabolism-related genes (such as Cdk8 and Ppara), providing more evidence for enhanced lipid metabolism in cardiomyocytes after gene therapy ( Fig. 9 fg).

[0100] Example 5 Safety Assessment of AAV9-PNPLA2 Gene Therapy

[0101] As a potential side effect, AAV9 is mainly associated with hepatotoxicity. To evaluate the target specificity and long-term safety of AAV9-PNPLA2 therapy, we examined the expression of AAV9-GFP in the heart and liver tissues of treated Pnpla2c.G245A / c.G245A mice. In mice injected with AAV9 on postnatal day 5 (group A), the transduction rates of the heart and liver were 39% and 8%, respectively, at 12 weeks after injection (group A). Fig.10 In contrast, mice treated at 6 weeks of age (group B) had a transduction rate of 77% in the heart and 9% in the liver, respectively, 6 weeks after treatment ( Fig.10 Notably, mice injected with AAV9 at 6 weeks of age and analyzed at 32 weeks (group C) had reduced transduction rates in the heart and liver to 34% and 1%, respectively ( Fig.10 These findings suggest that AAV9 is gradually cleared from heart and liver tissues over time.

[0102] In addition, histopathological analysis of liver tissues from wild-type and AAV9-PNPLA2-treated Pnpla2c.G245A / c.G245A mice revealed no pathological abnormalities or tumor formation ( Fig.10 e). Serum aspartate aminotransferase (AST) / alanine aminotransferase (ALT) levels in all AAV9-PNPLA2-treated mice were comparable to those in the control group, indicating the absence of hepatotoxicity ( Fig.10 These results suggest that AAV9-PNPLA2 treatment did not adversely affect liver function in mice or induce hepatotoxicity, supporting its potential as a safe therapeutic option for clinical translational applications.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene drug for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation, characterized in that: The active ingredient of the gene medicine includes a shuttle plasmid; the shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, and the PNPLA2 gene is located downstream of the cTnT promoter on the AAV9 vector.

2. The gene medicine according to claim 1, characterized in that: The nucleotide sequence of the PNPLA2 gene is shown in SEQ ID NO.

1.

3. The gene medicine according to claim 1, characterized in that: The AAV9 vector is a pHBAAV vector.

4. The gene medicine according to any one of claims 1 to 3, characterized in that: The gene medicine is a virus particle packaged by a lentivirus.

5. The gene medicine according to claim 4, characterized in that: The titer of the viral particles in the gene drug is 1×10 12 ~1×10 13 vg / mL.

6. The method for preparing a gene drug according to any one of claims 1 to 5, characterized in that: The following steps are involved: The shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid were mixed to obtain a three-plasmid system; The three-plasmid system is packaged and purified in HEK293T cells to obtain the gene medicine.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the shuttle plasmid, the pAAV-RC plasmid and the pHelper plasmid is 1:1:

2.

8. The preparation method according to claim 6, characterized in that: The virus packaging reagent includes liposomes; the mass volume ratio of the three-plasmid system and the liposomes is 40 μg:120 μL.

9. Use of the gene drug according to any one of claims 1 to 5 or the gene drug prepared by the preparation method according to any one of claims 6 to 8 in the preparation of a product for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation.

10. The use according to claim 9, characterized in that: The PNPLA2 mutation is PNPLA2c.245G>A (p.G82D).

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