A gene drug for treating PNPLA2 mutation-induced arrhythmogenic cardiomyopathy and a preparation method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, in particular to a kind of gene medicine for treating PNPLA2 mutation arrhythmogenic cardiomyopathy and its preparation method and application.The effective component of gene medicine provided in the present application includes shuttle plasmid;The shuttle plasmid includes AAV9 carrier and PNPLA2 gene.The present application delivers normal PNPLA2 gene by AAV9 carrier, repairs the lipid metabolism disorder caused by mutation, reduces the fat deposition in myocardium, and then improves the pathological changes of ACM.By restoring the function of PNPLA2 gene, the process of myocardial fibrosis can be effectively inhibited, and the heart function can be improved.The present application uses myocardial specific promoter cTnT to ensure that PNPLA2 gene is only expressed in myocardium, so as to improve the targeting of gene therapy, and avoid the 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.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene drug for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy, its preparation method, and its application. Background Technology

[0002] Arrhythmogenic cardiomyopathy (ACM) is a genetically induced cardiomyopathy characterized by the gradual replacement of myocardial tissue by fat and fibrosis, leading to ventricular arrhythmias and progressive cardiac dysfunction. ACM typically affects the right ventricle, but can also impact the left ventricle. The disease is predominantly familial, and common pathogenic gene mutations include PKP2, DSG2, DSP, and CTNNA3. Recent studies have increasingly identified lipid metabolism abnormalities, particularly mutations in the PNPLA2 gene, as a key contributing factor to ACM.

[0003] The protein encoded by the PNPLA2 (patatin-like phospholipase domain-containing protein 2) gene is primarily responsible for catalyzing the hydrolysis of triglycerides and is a crucial enzyme in lipid metabolism. Mutations in PNPLA2 lead to lipid metabolism disorders, resulting in fat deposition in cardiomyocytes and myocardial fibrosis. These pathological changes significantly increase the risk of sudden death in patients. In particular, the PNPLA2 c.G245A (p.G82D) mutation has been confirmed by multiple studies to be closely related to the development of arrhythmogenic cardiomyopathy (ACM). Studies have shown that this mutation leads to the loss of PNPLA2 enzyme activity, resulting in fat deposition in the myocardium and triggering pathological changes in the heart, severely affecting cardiac function. Clinical manifestations of ACM include arrhythmias, myocardial hypertrophy, right ventricular enlargement, and heart failure. It often occurs in young patients, typically around age 40, significantly impacting patients' quality of life and prognosis.

[0004] Currently, treatment options for ACM are limited, with existing methods primarily focusing on symptom control, medication, and heart transplantation. Symptom control mainly relies on antiarrhythmic drugs to alleviate arrhythmias and improve cardiac function; however, these drugs only relieve symptoms and do not cure the disease. Heart transplantation is the only effective treatment for end-stage ACM patients. Although it significantly improves patients' quality of life, the shortage of donor hearts prevents many patients from receiving timely transplants, and heart transplantation itself involves issues such as immune rejection, resulting in less than ideal long-term survival and prognosis. Current research largely focuses on treating ACM caused by structural protein mutations, neglecting the etiology of lipid metabolism disorders. In particular, there are currently no effective gene therapy options for ACM caused by PNPLA2 gene mutations; existing research mostly focuses on repairing structural protein genes rather than addressing lipid metabolism issues.

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

[0006] To address the aforementioned problems, this invention provides a gene therapy drug for treating PNPLA2-mutant arrhythmic cardiomyopathy, its preparation method, and its application. Based on the AAV9 vector and employing a myocardial-specific promoter cTnT gene therapy strategy, this invention restores the function of the PNPLA2 gene by repairing lipid metabolism disorders, thereby improving the pathological changes in ACM.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a gene therapy for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy. The active ingredient of the gene therapy includes a shuttle plasmid. The shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector. The PNPLA2 gene is located downstream of the cTnT promoter on the AAV9 vector.

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

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

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

[0012] Preferably, the viral particle titer in the gene therapy drug is 1×10⁻⁶. 12 ~1×1013 vg / mL.

[0013] This invention provides a method for preparing the gene drug 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 was used for viral packaging and purification in HEK293T cells to obtain the gene drug.

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

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

[0018] This invention provides the application 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 products for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy.

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

[0020] Beneficial effects:

[0021] This invention provides a gene therapy for treating PNPLA2-mutant arrhythmic cardiomyopathy (ACM). The active ingredient of the gene therapy includes a shuttle plasmid. The shuttle plasmid comprises an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, wherein the PNPLA2 gene is located downstream of the cTnT promoter on the AAV9 vector. This invention delivers a functional PNPLA2 gene via the AAV9 vector, repairing lipid metabolism disorders caused by mutations, reducing intramyocardial fat deposition, and thus improving the pathological changes of ACM. By restoring the function of the PNPLA2 gene, the progression of myocardial fibrosis can be effectively inhibited, and cardiac function can be improved. This invention uses the myocardium-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 precision and safety of treatment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

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

[0024] Figure 2 Sequencing results for PNPLA2 c.G245A / c.G245A mutant mice and wild-type mice; the upper part shows the sequencing results for wild-type mice, and the lower part shows the sequencing results for PNPLA2 c.G245A / c.G245A mutant mice.

[0025] Figure 3 Electrophoresis results for PNPLA2 c.G245A / c.G245A mutant mice and wild-type mice.

[0026] Figure 4 The results of reproducing the ACM phenotype in mice using the PNPLA2 c.G245A / c.G245A mutation were presented. Specifically: a. Sanger sequencing was used to detect the PNPLA2 gene sequence in Pnpla2 c.G245A / c.G245A knock-in mice and wild-type (WT) control mice. b. Kaplan-Meier survival analysis was performed to compare the survival of wild-type (WT, n=10) and Pnpla2c.G245A / c.G245A (Hom, n=13) mice. c. Representative echocardiographic images showing the long and short axis views of WT and Hom mice. d. M-mode echocardiography of WT and Hom mice. e. Quantitative analysis comparing ejection fraction (EF), fractional shortening (FS), end-diastolic volume (EDV), and end-systolic volume (ESV) in WT and Hom mice. f. Representative electrocardiogram (ECG) waveforms from WT (n=6) and Hom (n=5) mice, respectively. g. Representative images of the hearts of WT and Hom mice. h. Heart weight to body weight ratio of WT (n=6) and Hom (n=5) mice. i. H&E staining showing the histological features of the right ventricle (RV) tissue of WT and Hom mice; left icon bar is 500 μm, right magnified icon bar is 25 μm. j. Masson trichrome staining showing fibrosis in the right ventricle tissue of WT and Hom mice; left icon bar is 500 μm, right magnified icon bar is 25 μm. k. Quantitative analysis of changes in Col1a1 expression in the right ventricle of WT (n=6) and Hom (n=5) mice. l. Oil Red O staining showing lipid accumulation in WT and Hom mice; each scale bar is 20 μm. m. Transmission electron microscopy (TEM) images showing the ultrastructure of the heart tissue of WT and Hom mice; scale bar is 1 μm.

[0027] Figure 5Results of experiments demonstrating early PNPLA2 supplementation to prevent the ACM phenotype in PNPLA2 mutant mice. The figures include: a. Schematic diagram of the AAV9 vector. b. Timeline of AAV9 vector delivery via intrathoracic injection on day 5 after birth. c. Echocardiography showing cardiac function in 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. Heart weight to body weight ratio 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; left icon bar is 500 μm, right magnified icon bar is 25 μm. i. Masson's trichrome staining of right ventricular tissue from WT, untreated, and treated mice. Left scale bar: 500 μm; right scale bar: 25 μm. j. Quantitative analysis of right ventricular fibrosis in WT, untreated, and treated mice. k. Transmission electron microscopy (TEM) images of heart tissue from WT, Hom, and treated mice. Scale bar: 1 μm. l. Immunofluorescence of GFP-labeled PNPLA2 expression in the hearts of AAV9-treated mice. Scale bar: 20 μm.

[0028] Figure 6Results of experiments reversing ACM progression in PNPLA2 mutant mice with supplemental AAV9-PNPLA2 therapy in late-stage mice. a. Schematic diagram of AAV9 vector delivery via intrapleural injection to 6-week-old PNPLA2 c.G245A / c.G245A mice. b. Gross images of the hearts of WT, untreated (Hom), and treated (Treated) mice. c. Echocardiographic quantitative analysis of ejection fraction (EF) in WT (n=6), untreated (Hom, n=5), and AAV9-PNPLA2-treated (Treated, n=6) mice. d. Quantitative analysis of EF and 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. Heart weight to body weight ratio in WT (n=6), Hom (n=5), and Treated (n=6) mice. g. H&E staining of myocardial tissue from WT, Hom, and Treated mice; left scale bar is 500 μm, right scale bar is 25 μm. h. Masson trichrome staining of ventricular tissue from WT, Hom, and Treated mice; left scale bar is 500 μm, right scale bar is 25 μm. i. Quantitative analysis of right ventricular fibrosis in WT, Hom, and Treated mice. j. Transmission electron microscopy (TEM) images of the ultrastructure of the heart from WT, Hom, and Treated mice; scale bar is 1 μm. k. Immunofluorescence of GFP-labeled PNPLA2 expression in the heart of AAV9-treated mice; scale bar is 20 μm per division.

[0029] Figure 7The experimental results show that AAV9-PNPLA2 treatment significantly prolonged the survival of PNPLA2 mutant mice. Specifically: a. Schematic diagram of 6-week-old Pnpla2 c.G245A / c.G245A mice treated with the AAV9-PNPLA2 vector. b. Kaplan-Meier survival analysis comparing the survival of WT, Hom, and Treated mice. c. Quantitative echocardiographic 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 cardiac images of WT, Hom, and Treated mice. f. Heart weight to body weight ratio in WT (n=6), Hom (n=5), and Treated (n=6) mice. g. H&E staining of myocardial tissue from WT, Hom, and Treated mice. h. Masson trichrome staining of ventricular tissue from 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 from WT, Hom, and Treated mice, showing lipid deposition between cardiomyocytes, scale bar at 2 μm. k. Immunofluorescence of GFP-labeled PNPLA2 expression in the heart of AAV9-treated mice, scale bar at 20 μm.

[0030] Figure 8 and Figure 9 Experimental results from mononuclear mouse genome sequencing revealing changes in lipid metabolism in cardiomyocytes after AAV9-PNPLA2 treatment. Among them, Figure 8 The experimental workflow for cardiac tissue sampling and single-nuclear RNA sequencing (snRNA-seq) is presented in a table, applicable to both wild-type and treatment mice, and a representative unified manifold approximation and projection (UMAP) plot of the integrated data is displayed. Each cell nucleus is color-coded (from left to right, top to bottom) according to the different stages of sample origin and the associated cell type. Figure 8 b. The proportions of various cell types in untreated (Hom), AAV9-PNPLA2-treated (Treated), and wild-type (WT) mice. Figure 8 c. Feature map shows the expression of classic marker genes used to define each cell type. Figure 8 The middle d. violin plot shows the distribution of lipid metabolism scores calculated based on the expression levels of Abhd5, Acat1, Cd36, Cpt1a, Cpt1b, Dgat1, Lpl, Pparg, and Srebf1 in the experimental group and cardiomyocyte subsets. Figure 8 e.GO enrichment analysis revealed differentially expressed genes between the untreated group (Hom) and the AAV9-PNPLA2 treated group (Treated). Figure 9 The reconstructed cell trajectory plot in f. shows the transition from stage 5 to stage 1 obtained through Monocle 2 trajectory analysis and is correlated with pseudo-time. Figure 9 The heatmap in the middle section shows the dynamic expression of genes in cardiomyocytes. CM, cardiomyocytes; FB, fibroblasts; EC, endothelial cells; MC, macrophages; PC, pericytes; LEC, lymphoendothelial cells; NC, nerve cells.

[0031] Figure 10 Results of the safety assessment of AAV9-PNPLA2 gene therapy. Specifically: a. AAV9-GFP transduction rate in heart and liver tissues of Pnpla2c.G245A / c.G245A mice (Group A) injected on day 5 after birth, 12 weeks post-injection, with each scale bar at 20 μm. b. AAV9-GFP transduction rate in heart and liver tissues of mice treated at 6 weeks of age (Group B), 6 weeks post-treatment, with each scale bar at 20 μm. c. AAV9-GFP transduction rate in heart and liver tissues of mice treated at 6 weeks of age and analyzed at 32 weeks of age (Group C), with each scale bar at 20 μm. d. Quantification of AAV9 transduction rate in heart and liver tissues of Groups A, B, and C. e. H&E staining of liver tissues from wild-type and AAV9-PNPLA2-treated Pnpla2c.G245A / c.G245A mice. f. Serum AST / ALT levels in wild-type (n=6, 32 weeks old), homologous (n=5, 12 weeks old) and treatment group (n=15) mice. Detailed Implementation

[0032] This invention provides a gene therapy for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy. The active ingredient of the gene therapy includes a shuttle plasmid. The shuttle plasmid includes an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector. The PNPLA2 gene is located downstream of the cTnT promoter on the AAV9 vector.

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

[0034] In one embodiment, the AAV9 carrier is a pHBAAV carrier.

[0035] In one implementation, the gene drug is a viral particle packaged with a lentivirus.

[0036] In one embodiment, the viral particle titer in the gene therapy drug is 1 × 10⁻⁶. 12 ~1×10 13 vg / mL.

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

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

[0039] 1.1 Inverted terminal repeat (ITR): Used in the packaging and transduction of the virus to ensure that the AAV9 virus can successfully enter the target cell 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, ensuring the expression of the PNPLA2 gene in cardiac tissue without triggering unwanted expression in other tissues.

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

[0042] The viral particle structure of the AAV9 vector includes VP1, VP2, and VP3 proteins in the following molecular ratio: VP1:VP2:VP3 = 1:1:10. These viral proteins together form the capsid of the AAV9 virus, responsible for viral stability, targeting, and binding to host cells. The AAV9 viral particles have a diameter of approximately 20–26 nm, classifying them as relatively small viral particles that easily reach target tissues via blood circulation and can effectively penetrate cell membranes to enter the cell. The AAV9 viral titer range in this invention is 1 × 10⁻⁶. 12 ~1×10 13vg / mL. This titer ensures sufficient gene delivery capacity for efficient PNPLA2 gene expression in cardiac tissue. The virus solution uses phosphate-buffered saline (PBS) as the solvent and glycerol as a stabilizer. Glycerol forms a protective film to prevent viral particle aggregation or structural damage during cryopreservation, ensuring the stability of the viral particles during freezing and thawing.

[0043] The drug solution is a colorless and transparent liquid. Viral particles are uniformly dispersed in PBS with a glycerol concentration of 5% (w / v) to maintain viral particle stability. Simultaneously, this gene therapy drug can be stored long-term under low-temperature conditions, ensuring that the activity and titer of the viral particles do not decrease. Titer decrease is less than 5% within 12 months of freezing at -80°C; titer decrease is less than 10% within 7 days of storage at room temperature (25°C).

[0044] The AAV9 virus particle diameter is 20–26 nm, a size that allows AAV9 to efficiently penetrate cell membranes, especially in cardiomyocytes. Furthermore, the use of glycerol as a preservative ensures long-term stable storage of the AAV9 vector at low temperatures, reducing aggregation or structural damage caused by cold. The titer of AAV9 virus in the drug solution is 1 × 10⁻⁶. 12 ~1×10 13 The concentration of viral vectors (vg / mL) ensures efficient gene delivery and meets the required viral vector volume for treatment. The ratio of VP1, VP2, and VP3 proteins in the AAV9 viral particle is 1:1:10. This ratio ensures the structural stability and transduction efficiency of the viral particle.

[0045] Compared to existing treatments, the gene therapy drugs of this invention, particularly the treatment method based on AAV9 vector delivery of the PNPLA2 gene, have significant advantages, including higher targeting, better therapeutic efficacy, higher safety, and more durable gene expression. Specific advantages are as follows:

[0046] 1. Myocardial-specific delivery and expression

[0047] This invention utilizes the AAV9 vector, an adeno-associated virus serotype with superior myocardial properties and the ability to efficiently penetrate cardiac tissue. Compared to other AAV serotypes, AAV9 exhibits stronger targeting in the myocardium, ensuring that the delivered gene is primarily expressed within the heart, reducing gene expression in non-target organs, thereby minimizing potential side effects and toxicity. By using the cTnT promoter (a myocardial-specific promoter) to drive the expression of the human PNPLA2 gene, the gene is further guaranteed to be efficiently expressed only in cardiomyocytes, significantly improving the targeting and specificity of the treatment. In existing technologies, most promoters (such as the CMV promoter) carry the risk of systemic expression, which may lead to side effects in non-target organs. This invention solves this problem through the application of the myocardial-specific cTnT promoter, thereby improving the precision and safety of the treatment.

[0048] 2. Efficient gene delivery and stable expression

[0049] The AAV9 vector system of this invention enables highly efficient gene delivery within cardiomyocytes and ensures long-term gene expression. Compared to traditional viral vectors or other non-viral vectors, AAV9 exhibits lower immunogenicity and longer expression persistence, thus providing a more stable therapeutic effect. In animal experiments, delivery of the PNPLA2 gene using AAV9 significantly restored myocardial lipid metabolism balance and improved cardiac function, and this effect remained stable over long-term follow-up. The sustained effect of gene therapy is one of its greatest advantages, avoiding the need for repeated drug administration required by traditional drug therapy and enabling long-term inhibition of disease progression.

[0050] 3. Improves cardiac function and reverses pathological changes

[0051] Experimental results show that AAV9-PNPLA2 gene therapy can significantly improve cardiac function in PNPLA2 gene mutant mice, including improvements in ejection fraction (EF) and fractional shortening (FS). The heart weight / body weight ratio was restored to near the level of wild-type mice, and cardiac structure and tissue morphology were significantly restored. Pathological and ultrastructural analyses showed reduced fatty infiltration and significant reversal of myocardial fibrosis in the treated group, all indicating that the present invention can effectively treat cardiomyopathy caused by PNPLA2 mutations. This effect is significantly superior to traditional drug therapy and existing gene therapy regimens, especially in reversing pathological changes and restoring cardiac function.

[0052] 4. Excellent safety

[0053] Compared to other high-dose gene delivery protocols, the AAV9 vector used in this invention exhibits low immunogenicity and low toxicity. No significant hepatotoxicity or immune responses were observed during treatment, and the myocardial-specific expression of the AAV9 vector effectively avoided non-target organ toxicity. Liver function indicators such as AST / ALT levels did not show significant increases after treatment, ensuring the safety of the therapy. This safety advantage stems from the unique properties of the AAV9 vector and the myocardial-specific expression of the cTnT promoter, reducing adverse drug reactions and side effects.

[0054] Compared to existing technologies, the gene therapy drug of this invention exhibits higher targeting specificity, stronger therapeutic efficacy, and greater safety. Its advantages primarily stem from the use of the AAV9 vector for highly efficient delivery of myocardial-targeted genes, the application of the myocardial-specific promoter cTnT to ensure efficient gene expression in cardiomyocytes, and the long-term stability and safety of the gene during treatment. Furthermore, the recovery of cardiac function and reversal of pathological changes after treatment demonstrate the significant advantages of this technology over existing treatment methods. Therefore, this invention not only provides an innovative treatment method for ACM caused by PNPLA2 mutations but also offers new ideas and solutions for gene therapy of cardiovascular diseases.

[0055] This invention provides a method for preparing the gene drug 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 was used for viral packaging and purification in HEK293T cells to obtain the gene drug.

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

[0059] In one embodiment, the reagent for virus packaging includes liposomes; the mass-to-volume ratio of the three plasmid system to the liposomes is 40 μg: 120 μL.

[0060] This invention provides the application 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 products for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy.

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

[0062] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a gene therapy for treating PNPLA2 mutation-induced arrhythmic cardiomyopathy, its preparation method, and its application, but these descriptions 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, prepared by Hanheng Biotechnology (Shanghai) Co., Ltd., includes: constructing a shuttle plasmid based on the AAV9 vector to deliver the PNPLA2 gene, and performing viral packaging, purification and quality control in HEK293T cells through an optimized three-plasmid system to finally form 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 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 below. Figure 1 .

[0066] The nucleotide sequence of the target gene (human PNPLA2 gene) is shown in SEQ ID NO.1, 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 with 10% fetal bovine serum. When the AAV-293 cells reached 80%–90% confluence, co-transfection with the three plasmids began. In the transfection system, the shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid were prepared at a mass ratio of 1:1:2. During transfection, the total mass of the three plasmids was mixed with liposomes (Lipofectamine). TM The transfection complex was formed by mixing the two components at a mass-to-volume ratio of 40 μg:120 μL and then added to the cell culture medium. The cells were cultured at 37°C for 6 hours, then the medium was replaced with fresh medium containing 10% FBS. 72 hours after transfection, the cells and culture supernatant were collected for subsequent virus extraction.

[0069] 3) Virus extraction was performed using a freeze-thaw method. Collected cells were subjected to three freeze-thaw cycles between liquid nitrogen and a 37°C water bath, with 30-second intervals between each thaw, to lyse the cells and release viral particles. After freeze-thaw, the lysate was centrifuged at 4°C, 2000×g for 5 minutes to remove cell debris, and the supernatant was collected for further processing. Virus particle purification began with precipitation using 8% PEG-8000 and 0.5M NaCl. After mixing, the mixture was incubated at 4°C for 12 hours, then centrifuged at 12000×g for 30 minutes to collect the precipitate and resuspend it in PBS. Further purification using a Biomiga AAV column removed residual nucleic acid and other impurities, ultimately yielding a high-purity viral solution.

[0070] 4) The purified virus solution was concentrated to 1 mL using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, resulting in a virus titer range of 1 × 10⁻⁶. 12 ~1×10 13 vg / mL. The quality control process includes using quantitative real-time PCR to detect viral titers, performing sterility tests on HeLa cells, and PCR testing for mycoplasma contamination to ensure that the quality of viral samples meets the standards.

[0071] 5) Finally, the purified virus solution was diluted with PBS to the required concentration (1×10⁻⁶). 12 ~1×10 13 The virus solution was prepared by adding 5% (w / v) glycerol or sucrose as a stabilizer to ensure the stability of the virus particles during low-temperature storage. The prepared virus solution was aliquoted 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 to PNPLA2 mutant mice

[0073] 1. Experimental Procedure

[0074] 1.1 Construction of the mouse model

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

[0076] Sequence analysis and BamHI restriction endonuclease analysis were performed on mice injected with mRNA. The results are shown in [Table / Reference]. Figure 2 and Figure 3 .Depend on Figure 2 and Figure 3 It can be seen that the PNPLA2 c.G245A mutant mouse model has been 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 verified by Sanger sequencing. Figure 4 a, Figure 5 (a) The lifespan of Pnpla2 c.G245A / c.G245A mice was significantly shortened, with all homozygous mice dying 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, echocardiographic evaluation was performed. It was observed that 6-week-old Pnpla2 c.G245A / c.G245A mice exhibited significantly impaired left ventricular systolic function, manifested as a significant reduction in ejection fraction (EF) and fractional shortening (FS). Figure 4 (c and d). These mice also showed a significant increase in end-diastolic volume (EDV) and end-systolic volume (ESV), suggesting severe systolic dysfunction and ventricular dilation. Figure 4Furthermore, frequent premature ventricular contractions (PVCs) were observed in Pnpla2c.G245A / c.G245A mice, suggesting an electrophysiological abnormality consistent with ACM. Figure 4 (f)

[0079] The hearts of Pnpla2 c.G245A / c.G245A mice were significantly enlarged, with marked fatty infiltration, and the heart-to-body weight ratio was significantly increased compared to 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 (g and h). Figure 4 Masson trichrome staining results showed that Pnpla2c.G245A / c.G245A mice had significant fibrosis in the right ventricle, and the expression of the fibrosis marker Col1a1 was elevated. Figure 4 (j and k).

[0080] Ultrastructural examination of cardiac tissue using transmission electron microscopy (TEM) revealed significant abnormalities, including enlarged lipid droplets in the myofibril spaces and disordered sarcomere structures. Figure 4 (l and m). These results indicate that the Pnpla2c.G245A / c.G245A mouse model is highly similar to ACM patients in terms of cardiac characteristics.

[0081] 1.2 Preparation and Delivery of Viral Vectors

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

[0083] The virus titer prepared in Example 1 was 1 × 10⁻⁶. 12 The viral solution was delivered via intrapleural injection (IT). Mice in the early treatment group (day 5 after birth) were injected with 20 μL of viral solution each, while mice in the late treatment group (6 weeks old) were injected with 50 μL of viral solution each.

[0084] 1.3 Cardiac Function Assessment

[0085] Cardiac function was assessed by echocardiography at 2, 4, and 8 weeks post-treatment, primarily measuring ejection fraction (EF) and fractional shortening (FS). Simultaneously, electrocardiogram (ECG) monitoring was performed to check for arrhythmias, including early ventricular arrhythmias (PVCs) and ventricular tachycardia (VT).

[0086] 1.4 Pathological Analysis

[0087] Histological analysis of mouse hearts was performed after treatment. HE staining and Masson staining were used to assess myocardial structural changes, fibrosis, and fat deposition. Immunohistochemistry (IHC) and Western blot were used to detect changes in PNPLA2 protein expression and related lipid metabolism genes (such as ABHD5, LPL, PPARγ, etc.).

[0088] 2. Experimental Results

[0089] 2.1 The experimental results of the early treatment group are shown in [the table below]. Figure 5 .

[0090] Pnpla2 c.G245A / c.G245A mice treated with AAV9-PNPLA2 showed significantly improved cardiac function, with significantly higher ejection fraction (EF) and fractional shortening (FS) than untreated mutant mice. After 2 weeks of treatment, the ejection fraction (EF) increased from approximately 40% to 60%, and the fractional shortening (FS) increased from 15% to 30%. At 4 weeks of treatment, EF approached the level of WT mice (70%), and FS increased to 40%, indicating that cardiac contractile function was maintained. Figure 5 (c, d). Creatine kinase isoenzyme MB (CK-MB) levels are a biomarker of myocardial injury. Untreated Pnpla2c.G245A / c.G245A mice had CK-MB levels almost twice that of wild-type mice, while AAV9-PNPLA2 treatment reduced CK-MB levels to wild-type levels, indicating a reduction in cardiac injury. Figure 5 (e). Gross examination revealed that the hearts of AAV9-PNPLA2-treated Pnpla2 c.G245A / c.G245A mice had normal morphology, no fat deposition, and were similar to those of wild-type mice. Figure 5 (f). The heart weight to body weight ratio in the treatment group mice was also reduced, approaching the value of wild-type mice, indicating that cardiac enlargement was effectively alleviated. Figure 5 (g). H&E staining after 12 weeks showed that the myocardial structure of the treated mice was intact, with no inflammatory infiltration observed. Figure 5 (h). Compared with the obvious fibrosis in untreated mutant mice, no right ventricular fibrosis was observed in the treatment group, demonstrating the protective effect of gene supplementation therapy. Figure 5 (i, j). Transmission electron microscopy (TEM) analysis showed that the myosarcomere structure in 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 assays confirmed the expression of GFP-labeled PNPLA2 protein in the hearts of AAV9-treated mice, validating the efficient delivery and expression of the exogenous protein. Figure 5 (l).

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

[0092] 2.2 The experimental results of the late-stage treatment group are shown in [the table below]. Figure 6 After treatment, the heart morphology of PNPLA2 c.G245A / c.G245A mice was similar to that of wild-type mice, with no obvious external fat accumulation. Figure 6 (b) Echocardiography at 6 weeks post-treatment showed significant improvement in ejection fraction (EF) and fractional shortening (FS) in mice treated with AAV9-PNPLA2, with EF and FS increasing to 50% and 20%, respectively. While still below wild-type levels compared to untreated mice, this indicated partial recovery. Figure 6 (c, d). Treatment initiated after the onset of ACM stabilized cardiac function but failed to completely reverse existing functional deficits. Serum CK-MB levels have decreased to wild-type levels, indicating reduced myocardial damage. Figure 6 (e). The heart weight to body weight ratio in the treatment group mice had decreased to a level comparable to that of wild-type mice, showing improvement compared to ACM mice. Figure 6 (f). H&E staining showed that the myocardial structure of the treated mice was normal, and the muscle fibers were neatly arranged, indicating that the histological structure had been restored to normal. Figure 6 (g). Compared with untreated mice, the treatment group showed reduced fibrosis, suggesting that fibrosis was reversed. Figure 6 (h, i). Transmission electron microscopy (TEM) analysis showed that the myosarcomere structure in the heart tissue of the treated mice was orderly, the mitochondria were intact, the intercellular connections were stable, and no abnormal lipid accumulation was observed, reflecting the restoration of ultrastructure. Figure 6 Immunofluorescence staining confirmed GFP-labeled PNPLA2 expression in the hearts of AAV9-treated mice, validating the effectiveness of gene delivery. Figure 6 These results indicate that advanced AAV9-PNPLA2 gene therapy can partially reverse pathological changes in ACM and stabilize cardiac function, highlighting its potential benefits in the treatment of advanced ACM.

[0093] 3. Summary and Clinical Significance

[0094] Treatment with AAV9 vector-delivered PNPLA2 gene effectively improved cardiac function, pathological manifestations, and cardiac rhythm stability in PNPLA2 c.G245A / c.G245A mutant mice. Early treatment was more effective than late treatment, significantly restoring cardiac function, reducing fibrosis and fat accumulation, and significantly prolonging survival. While late treatment prolonged survival, its effect on improving cardiac function was limited, especially in recovering from existing cardiac structural damage. These experimental results provide important preclinical evidence for AAV9-based PNPLA2 gene delivery therapy for ARVC and lay the foundation for its clinical application.

[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 up to 32 weeks. Figure 7 (a, b). Although treatment prolonged the survival of mice, echocardiography at 12 weeks showed that the ejection fraction (EF) and fractional shortening (FS) of the treated mice were still significantly lower than those of wild-type mice, and similar to those of untreated mutant mice. Figure 7 (c) This indicates that while late-stage gene therapy prolonged survival, it failed to completely prevent cardiac dysfunction. At week 32, serum CK-MB levels in the treatment group mice were lower than in the untreated control mice, indicating a reduction in myocardial damage. Figure 7 (d). Gross examination revealed no significant 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 (e, g). The heart weight to body weight ratio in the treatment group mice had decreased to wild-type levels, showing improvement compared to ACM mice. Figure 7 (f). Compared with untreated mutant mice, the treated group of mice showed reduced myocardial fibrosis, but it was still higher than that in 32-week-old wild-type mice. Figure 7 h, i). Transmission electron microscopy (TEM) analysis showed that lipid deposits were still present between cardiomyocytes (h, i). Figure 7 Immunofluorescence staining confirmed the sustained expression of GFP-labeled PNPLA2 in the hearts of AAV9-treated mice, verifying the presence of the exogenous protein. Figure 7 These results indicate that, despite cardiac dysfunction, late AAV9-PNPLA2 gene therapy significantly prolonged the survival of mutant mice, suggesting that this treatment has a potential life-extending benefit even in the presence of cardiac damage.

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

[0098] To further investigate the impact of AAV9-PNPLA2 gene therapy on the transcriptome, we collected heart tissues from wild-type mice, PNPLA2 c.G245A / c.G245A mice, and mice receiving AAV9-PNPLA2 gene therapy for single-nucleus mouse genome sequencing. We extracted cell nuclei from the heart tissues of the five mice in a test tube, sequenced them using 10x Genomics technology, constructed libraries, and performed data analysis. Figure 8 (a) A total of 6434 cell nuclei were analyzed, and cells were labeled based on genes specifically expressed in each cell type. Based on specific marker genes, these cells were classified into seven categories: cardiomyocytes (CM), fibroblasts (FB), endothelial cells (EC), macrophages (MC), pericytes (PC), lymphoendothelial cells (LEC), and nerve cells (NC). Notably, cardiomyocytes significantly increased after gene therapy, highlighting the efficacy of AAV9-PNPLA2 treatment. Figure 8 (c).

[0099] Based on the expression levels of key genes in classic lipid metabolism (such as Abhd5, Acat1, Cd36, Cpt1a, Cpt1b, Dgat1, Lpl, Pparg, and Srebf1), lipid metabolism scores were significantly improved after gene therapy. Figure 8 (d). By comparing the differential expression (DEGs) of lipid metabolism-related genes in cardiomyocytes before and after gene therapy, we found significant differences in the expression of these genes. GO enrichment analysis further supports this finding. Figure 8 (e). Using Monocle 2 analysis, we observed that after treatment, stage 1 cells transitioned to stage 5, a stage characterized by improved lipid metabolism consistent with cell fate determination. This transition was accompanied by a significant upregulation of lipid metabolism-related genes (such as Cdk8 and Ppara), providing further evidence for enhanced lipid metabolism in cardiomyocytes following gene therapy. Figure 9 (fg).

[0100] Example 5: Safety assessment of AAV9-PNPLA2 gene therapy

[0101] AAV9 is a potential side effect, primarily associated with hepatotoxicity. To evaluate the target specificity and long-term safety of AAV9-PNPLA2 treatment, 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 day 5 after birth (Group A), the transduction rates in the heart and liver were 39% and 8%, respectively, at 12 weeks post-injection. Figure 10 (a, d). In contrast, mice treated at 6 weeks of age (group B) showed cardiac and hepatic transduction rates of 77% and 9%, respectively, 6 weeks after treatment. Figure 10 (b, d). Notably, in mice injected with AAV9 at 6 weeks of age and analyzed at 32 weeks (group C), the transduction rates in the heart and liver were reduced to 34% and 1%, respectively. Figure 10 (c, d). These findings suggest that AAV9 is gradually cleared from heart and liver tissue over time.

[0102] Furthermore, histopathological analysis of liver tissues from wild-type and AAV9-PNPLA2-treated Pnpla2c.G245A / c.G245A mice revealed no pathological abnormalities or tumor formation. Figure 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 no hepatotoxicity. Figure 10 (f). These results suggest that AAV9-PNPLA2 treatment did not adversely affect liver function in mice or induce hepatotoxicity, supporting its potential as a safe treatment option for clinical translation.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a gene therapy drug in the preparation of a product for treating PNPLA2-mutant arrhythmic cardiomyopathy, characterized in that, The active ingredient of the gene therapy drug includes a shuttle plasmid; the shuttle plasmid comprises an AAV9 vector and a PNPLA2 gene inserted into the AAV9 vector, the PNPLA2 gene being located downstream of the cTnT promoter on the AAV9 vector; the nucleotide sequence of the PNPLA2 gene is shown in SEQ ID NO.1; the AAV9 vector is a pHBAAV vector; the gene therapy drug is a lentiviral packaged viral particle; the titer of the viral particle in the gene therapy drug is 1 × 10⁻⁶. 12 ~1×10 13 vg / mL.

2. The application according to claim 1, characterized in that, Includes the following steps: The shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid were mixed to obtain a three-plasmid system; The three-plasmid system was used for viral packaging and purification in HEK293T cells to obtain the gene drug.

3. The application according to claim 2, characterized in that, The mass ratio of the shuttle plasmid, pAAV-RC plasmid, and pHelper plasmid is 1:1:

2.

4. The application according to claim 2, characterized in that, The reagents used to package the virus include liposomes; the mass-to-volume ratio of the three plasmid systems to the liposomes is 40 μg: 120 μL.

5. The application according to claim 1, characterized in that, The PNPLA2 mutation is PNPLA2 c.245G>A (p.G82D).

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