Application of Adtrp virus in preparation of medicine for treating heart failure
Gene therapy based on the myoAAV1A-Adtrp viral vector reversed the symptoms of heart failure, solving the problem that existing treatments cannot cure heart failure and achieving a significant reduction in cardiac dysfunction and cardiomyopathy.
Patent Information
- Application Number
- CN202510738932.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-12-12
AI Technical Summary
Existing treatments for heart failure can only slow disease progression, not cure it, and there is a lack of effective new molecular mechanisms and treatments.
To develop a gene therapy based on the myoAAV1A-Adtrp viral vector, an Adtrp-deficient mouse model was established through CRISPR-Cas9 genome editing, and gene therapy was performed using the myoAAV1A-Adtrp viral vector to reverse the symptoms of heart failure.
It significantly reduces cardiac dysfunction, reverses the ratio of cardiomyocyte cross-sectional area to heart weight, reduces the percentage of ventricular cavity retention area, increases left ventricular wall thickness, reduces myocardial fibrosis, and blocks cardiac hypertrophy and heart failure.
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Abstract
Description
Technical Field
[0001] This invention relates to coronary heart disease susceptibility genes. ADTRP (Encoding an androgen-dependent TFPI regulatory protein) has a novel physiological function, and its role has been established. Adtrp As a novel candidate target for developing precision treatments for heart failure, a myoAAV1A-based approach was developed. Adtrp Viral vectors are being used in gene therapy to treat heart failure, using myoAAV1A- Adtrp Gene therapy can significantly reduce cardiac dysfunction and heart failure in CAD and TAC models of heart failure, providing a new treatment strategy for heart failure. Background Technology
[0002] Heart failure is a common and complex clinical condition characterized by shortness of breath, fatigue, and edema, caused by structural or functional abnormalities of the heart that prevent it from meeting the body's metabolic needs, leading to insufficient blood circulation. It is the end-stage of various cardiovascular diseases, including coronary artery disease (CAD), hypertension, valvular heart disease, and idiopathic dilated cardiomyopathy. Globally, heart failure is a significant public health problem, with the number of patients increasing every year.
[0003] Epidemiological data indicate that there are approximately 56.2 million people with heart failure worldwide. Among all causes of heart failure, cardiac arteriosclerosis (CAD) and its complications, myocardial infarction (MI) and hypertensive heart disease, are the most common factors, accounting for 26.5% and 26.2%, respectively. The 5-year mortality rate is close to 50%. Treatment options include pharmacological interventions such as diuretics, beta-blockers, and SGLT2 inhibitors; device therapies such as cardiac resynchronization therapy (CRT) and implantable cardioverter defibrillators (ICDs); and surgical options such as heart transplantation. However, current treatments can only slow the progression of heart failure, not cure the disease. Therefore, identifying new molecular mechanisms underlying the pathogenesis of heart failure and developing new treatments are of great significance.
[0004] Coronary heart disease susceptibility genes ADTRP Encoding androgen-dependent TFPI regulatory protein, ADTRP The protein is homologous to androgen-inducible protein 1 (AIG1) and is expressed on the cell surface of endothelial cells (ECs). It plays a role in regulating the expression of TFPI, an important natural inhibitor of coagulation and thrombosis. Previous studies in this invention have reported... ADTRP Androgens regulate endothelial cell cycle progression, proliferation, and apoptosis by modulating gene expression. ADTRP The thyroxine-responsive element (TGTTCT) in the promoter binds, directly activating the enzyme in a dose- and time-dependent manner. ADTRPTranscription, thereby inhibiting two endothelial cell functions involved in key processes of atherosclerosis development and progression: monocyte adhesion to endothelial cells and monocyte migration across the endothelial EC layer. ADTRP Also through positive regulation PIK3R3 Expression and AKT activation regulate the levels of other CAD-related genes, including MIA3 (TANGO1), COL7A1 (code) Collagen VII )and APOB ,and ADTRP - MIA3 - CollagenVII / APOB The pathway is related to monocyte adhesion in endothelial cells and monocyte transport across the EC layer, as previous studies in this invention have shown. ADTRP It has the effect of reducing the risk of coronary heart disease, myocardial infarction, and heart failure, and protecting the heart. This invention is the first to propose the development of a myoAAV1A- Adtrp Viral vectors are being used in gene therapy to treat heart failure, using myoAAV1A- Adtrp Gene therapy can significantly reduce cardiac dysfunction and heart failure in CAD and TAC models of heart failure, providing a new treatment strategy for heart failure and laying the foundation for future clinical applications in heart failure treatment. Summary of the Invention
[0005] This invention discloses a method based on myoAAV1A- Adtrp Viral vectors are used in gene therapy for the treatment of heart failure. This model employs a left anterior descending coronary artery (LAD) MI model and another heart failure TAC (aortic arch coarctation) model, both of which ultimately lead to heart failure. This invention establishes a viral vector model using CRISPR-Cas9 genome editing. Adtrp Knockout (KO) lines of deletion mice, effective against different genotypes ( Adtrp KO ( Adtrp - / - , Adtrp + / - ) and wild type ( Adtrp + / + Both models of ischemic heart failure based on myocardial infarction (MI) and non-ischemic heart failure based on cardiac arrest (TAC) were constructed, with Sham as the control. Echocardiography and histological methods were used to observe cardiac structure and function. Western blotting was used to identify... ADTRP Molecular mechanisms of KO-related heart failure. Adtrp The lack of cardiac function is impaired, and LAD ligation or TAC further exacerbates this effect. Echocardiography shows that, compared with WT mice, MI and TAC models... Adtrp + / - mice and Adtrp - / -Both LVEF% and LVFS% were significantly decreased in mice, indicating that Adtrp Causes cardiac dysfunction and heart failure. Adtrp - / - The mice's cardiac function was further impaired, with further reductions in LVEF% and LVFS%. There was no difference in the heart-to-body weight ratio among the three different genotypes in the sham-operated group, but compared to the WT mice in the MI group, Adtrp + / - mice and Adtrp - / - The heart-to-body weight ratio of the mice was significantly reduced; compared with the WT mice in the TAC group, Adtrp KO ( Adtrp + / - Adtrp - / - The heart-to-body weight ratio of mice was significantly increased; these results indicate that... Adtrp Knockout (KO) can lead to heart failure. Details are as follows: A type based on Adtrp The use of viruses in the preparation of drugs for treating heart failure, as described above. Adtrp The viral sequence is shown in SEQ ID NO:1.
[0006] The expression Adtrp The virus includes myoAAV1A- expressed in the body. Adtrp Viral vector.
[0007] The myoAAV1A- Adtrp Use of viral vectors in the preparation of drugs for treating ischemic heart failure based on left anterior descending coronary artery ligation MI.
[0008] The myoAAV1A- Adtrp In the use of viral vectors in the preparation of drugs for the treatment of ischemic heart failure based on MI, significant increases in LVEF% and LVFS% and a significant increase in the heart-to-body weight ratio have been achieved; or an increase in the cross-sectional area of cardiomyocytes and the heart-to-weight ratio, a decrease in the percentage of ventricular cavity slough area, an increase in left ventricular wall thickness, and a decrease in myocardial fibrosis have been achieved.
[0009] In some preferred cases, reversing the LVEF% and LVFS% in wild-type mice significantly reduced the reversal. Adtrp + / - mice and Adtrp - / - A significant decrease in the heart-to-body weight ratio in mice.
[0010] In some preferred cases, reversing wild-type MI mice and Adtrp + / - mice and Adtrp - / -Applications include: reduction in the cross-sectional area of mouse cardiomyocytes to the ratio of heart weight, increase in the percentage of ventricular cavity retention area, decrease in left ventricular wall thickness, and increase in myocardial fibrosis.
[0011] The myoAAV1A- Adtrp The viral vector inhibited the expression of Apob, collagen VII, Lepr, and endothelial cell inflammatory markers Icam-1, Vcam-1, E-selectin, and P-selectin, while increasing the expression of p65 and the endothelial angiogenesis marker CD31.
[0012] The myoAAV1A- Adtrp Use of viral vectors in the preparation of drugs for treating non-ischemic heart failure based on aortic transconstriction surgery (TAC).
[0013] The myoAAV1A- Adtrp The use of viral vectors in the preparation of drugs for treating TAC-based non-ischemic heart failure enables the blocking of cardiac hypertrophy and heart failure, and the reversal of increased cardiomyocyte cross-sectional area, percentage of ventricular cavity stasis area, left ventricular wall thickness, and myocardial fibrosis.
[0014] In some preferred cases, cardiac hypertrophy and heart failure were blocked. Reversal of LVEF% and LVFS% in wild-type mice was significant, reversing... Adtrp + / - mice and Adtrp - / - A significant increase in the heart-to-body weight ratio in mice. This treatment blocked cardiac hypertrophy and heart failure, and reversed increased cardiomyocyte cross-sectional area, percentage of ventricular cavity stasis area, left ventricular wall thickness, and myocardial fibrosis. The myoAAV1A- Adtrp In the use of viral vectors for the treatment of TAC-based non-ischemic heart failure, the pcAAV- Adtrp Gene therapy inhibited the expression of Apob, collagen VII, Lepr, and endothelial cell inflammatory markers Icam-1, Vcam-1, E-selectin, and P-selectin, while significantly increasing the expression of p65 and the vascular marker α-SMA.
[0015] A pharmaceutical composition for treating heart failure, the pharmaceutical composition comprising the aforementioned... Adtrp Virus, or the aforementioned myoAAV1A- Adtrp Viral vector.
[0016] myoAAV1A- Adtrp The viral vector was administered via intravenous injection for treatment.
[0017] This invention constructs a system driven by the heart-specific promoter cTNT. Adtrp The expressed adeno-associated virus vector, myoAAV1A- Adtrp The invention also includes a control vector, myoAAV1A-GdGreen. This invention detects myoAAV1A- Adtrp Efficacy of gene therapy in two different heart failure models (MI, TAC). Tail vein injection of myoAAV1A- Adtrp The vector completely reversed these effects, while the control vector myoAAV1A-GdGreen did not. Adtrp Gene therapy for heterozygotes in MI models Adtrp + / - Mice and homozygotes Adtrp - / - The rescue effect on heart failure in mice was similar. H&E and Masson staining showed that myoAAV1A- Adtrp Gene therapy also reversed the effects of gene therapy on wild-type MI model mice and Adtrp + / - and Adtrp - / - Decreased cross-sectional area and heart weight ratio of mouse cardiomyocytes, increased percentage of ventricular cavity retention area, decreased left ventricular wall thickness, and increased myocardial fibrosis; myoAAV1A- Adtrp Gene therapy also blocked cardiac hypertrophy and heart failure in the TAC model. In wild-type mice, TAC reduced LVEF% and LVFS%, and increased cardiomyocyte cross-sectional area, heart weight / body weight ratio, percentage of ventricular cavity retention area, left ventricular wall thickness, and myocardial fibrosis. However, injection of myoAAV1A via the tail vein... Adtrp With the vector (2.5E+11 vg per mouse), these effects were completely reversed, while those with the control vector myoAAV1A-GdGreen (2.5E+11 vg per mouse) were not. In TAC heterozygotes... Adtrp + / - Mice and homozygous Adtrp - / - Similar rescue results were obtained in mice. Attached Figure Description
[0018] Figure 1 Constructing using CRISPR-Cas9 gene editing technology Adtrp - / - KO mice. Figure 1 Development of AC gene editing technology Adtrp - / - A schematic diagram of the strategy for knocking out mice. Figure 1 medium D wild type Adtrp + / + mice and Adtrp - / - Illustration of PCR amplification primer sequences and gene identification in KO mice. Figure 1 Real-time RT-PCR analysis in China showed Adtrp In heterozygotes Adtrp + / - homozygote Adtrp - / - Reduced mRNA expression levels in KO mice. Figure 1 Mid-F Western blotting showed Adtrp - / - In KO mice Adtrp Protein deficiency, Adtrp + / - In KO mice Adtrp Protein expression is decreased. *** P <0.001 (n=12 / group).
[0019] Figure 2 MI model Adtrp KO ( Adtrp + / - Adtrp - / - It can lead to cardiac dysfunction and heart failure. Figure 2 China A Adtrp + / + , Adtrp + / - and Adtrp - / - Representative echocardiographic measurements of cardiac function parameters were taken from the MI model group and the Sham (sham surgery control group). Figure 2 China BC Adtrp + / + , Adtrp + / - and Adtrp - / - Statistical analysis of LVEF% (left ventricular ejection fraction) and LVFS% (left ventricular fractional shortening) in the MI or Sham groups, Sham group Adtrp KO ( Adtrp + / - Adtrp - / - The LVEF% and LVFS% of mice were significantly reduced, and the MI group showed a further reduction. Figure 2 DMI and Sham group Adtrp + / - and Adtrp - / - Heart weight to body weight ratio in mice. MI group Adtrp + / - and Adtrp- / - The heart-to-body weight ratio decreased in mice. P <0.05; **, P <0.01; ***, P <0.001 (n=12 / group).
[0020] Figure 3 TAC model Adtrp KO ( Adtrp + / - Adtrp - / - It can lead to cardiac dysfunction and heart failure. Figure 3 China A Adtrp + / + , Adtrp + / - and Adtrp - / - Representative echocardiographic measurements of cardiac function parameters were performed in the TAC model group and the Sham group. Figure 3 China BC Adtrp + / + , Adtrp + / - and Adtrp - / - Statistical analysis of LVEF% (left ventricular ejection fraction) and LVFS% (left ventricular fractional shortening) in the TAC or Sham groups, Sham group Adtrp KO ( Adtrp + / - Adtrp - / - The LVEF% and LVFS% of mice were significantly reduced, and the TAC group showed a further reduction. Figure 3 D TAC and Sham Group Adtrp + / - and Adtrp - / - Heart weight to body weight ratio in mice. TAC group. Adtrp + / - and Adtrp - / - Increased heart-to-body weight ratio in mice. Normal range, not significant; ** P <0.01; ***, P <0.001 (n=12 / group).
[0021] Figure 4 : Adtrp Cardiac function testing for heart failure caused by gene therapy MI. Figure 4 pcAAV injection in China Adtrp Compared with control pcAABV-GdGreen, Adtrp + / + , Adtrp + / - and Adtrp - / - Representative echocardiograms of mice in the MI or Sham groups. Figure 4 BC pcAAV- Adtrp Gene therapy reversed the effects of myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - Decreased LVEF% (B) and LVFS% (C) in mice. NS, not significant; ***, P <0.001 (n=12 / group).
[0022] Figure 5 : Adtrp Morphological testing of heart failure caused by gene therapy for myocardial infarction (MI). Figure 5 AC, EF H&E staining detection and analysis of pcAAV- Adtrp Gene therapy reversed MI Adtrp + / + , Adtrp + / - and Adtrp - / - The mouse cardiomyocyte cross-sectional area (C) decreased, the ventricular cavity retention area (E) increased, and the left ventricular wall thickness (F) decreased. Figure 5 pcAAV-1 injection in D Adtrp Gene therapy reversed the effects of myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - Decreased heart-to-body weight ratio (D) in mice. Figure 5 GK Masson staining analysis of pcAAV- Adtrp Gene therapy reversed the effects of myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - In mice, left ventricular wall thickness was reduced, myocardial fibrosis was increased, the area of ventricular cavity retention was increased, and the cross-sectional area of cardiomyocytes was reduced. P <0.01; ***, P <0.001 (n=12 / group).
[0023] Figure 6 : Adtrp The molecular mechanism of gene therapy for MI. In a MI-induced heart failure model, AdtrpGene therapy upregulated Adtrp and p65, and downregulated Apob, collagen VII, Lepr, Icam-1, Vcam-1, E-selectin, and P-selectin. Figure 6 AH Western blot analysis showed pcAAV- Adtrp Gene therapy significantly increased Adtrp expression (AB) and inhibited post-myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - Expression of Apob, collagen VII and Lepr(CH) in mice. Figure 6 Chinese IP Western blot analysis showed that pcAAV- Adtrp Gene therapy can significantly inhibit post-myocardial infarction Adtrp + / + , Adtrp + / - and Adtrp - / - Expression of endothelial cell inflammatory markers Icam-1, Vcam-1, E-selectin and P-selectin in mice. Figure 6 QR Western blot analysis showed pcAAV- Adtrp Gene therapy significantly increases the risk of myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - p65 expression levels in mice. P <0.05; **, P <0.01; ***, P <0.001 (n=9 / group).
[0024] Figure 7 : Adtrp Molecular mechanisms of gene therapy for MI (immunohistochemical detection). Figure 7 AF pcAAV- Adtrp Immunostaining analysis of Adtrp, Apob, collagen VII, Lepr, and CD31 after gene therapy. P <0.001 (n=9 / group).
[0025] Figure 8 : Adtrp Cardiac function testing for TAC-induced cardiac hypertrophy and heart failure induced by gene therapy. Figure 8 China A Adtrp+ / + , Adtrp + / - and Adtrp - / - Mice in the TAC or Sham groups were injected with pcAAV- Adtrp Representative echocardiograms compared to control pcAABV-GdGreen. Figure 8 BC pcAAV- Adtrp Gene therapy reversed TAC Adtrp + / + , Adtrp + / - and Adtrp - / - Decreased LVEF% (B) and LVFS% (C) in mice. NS, not significant; ***, P <0.001 (n=12 / group).
[0026] Figure 9 : Adtrp Morphological detection of TAC-induced cardiac hypertrophy and heart failure. Figure 9 AC, E-FH&E staining analysis of pcAAV- Adtrp Gene therapy reversed TAC Adtrp + / + , Adtrp + / - and Adtrp - / - The cross-sectional area (C) of mouse cardiomyocytes, the area of ventricular cavity retention (E), and the thickness (F) of the left ventricular wall increased. Figure 9 pcAAV-1 injection in D Adtrp Gene therapy reversed the effects of myocardial infarction. Adtrp + / + , Adtrp + / - and Adtrp - / - Increased heart-to-body weight ratio (D) in mice. Figure 9 JN Masson staining analysis of pcAAV- Adtrp Gene therapy reversed TAC Adtrp + / + , Adtrp + / - and Adtrp - / - Increased myocardial fibrosis (I), increased left ventricular wall thickness (J), and increased cardiomyocyte cross-sectional area (K) in mice. NS, not significant; **, P <0.01; ***, P <0.001 (n=12 / group).
[0027] Figure 10 : Adtrp The molecular mechanism of gene therapy for TAC. In a TAC-induced heart failure model, Adtrp Gene therapy upregulated Adtrp and p65, and downregulated Apob, collagen VII, Lepr, Icam-1, Vcam-1, E-selectin, and P-selectin. Figure 10 AH Western blot analysis showed that pcAAV- Adtrp Gene therapy significantly increased TAC Adtrp + / + , Adtrp + / - and Adtrp - / - In mice, the expression of Adtrp (AB) inhibited the expression of Apob, collagen VII, and Lepr (CH). Figure 10 Chinese IP Western blot analysis showed that pcAAV- Adtrp Gene therapy can significantly inhibit TAC Adtrp + / + , Adtrp + / - and Adtrp - / - Expression of inflammatory markers Icam-1, Vcam-1, E-selectin and P-selectin in mouse endothelial cells. Figure 10 QR Western blot analysis showed that pcAAV- Adtrp Gene therapy significantly improved TAC Adtrp + / + , Adtrp + / - and Adtrp - / - p65 expression in mice. P <0.05; **, P <0.01; ***, P <0.001 (n=9 / group).
[0028] Figure 11 : Adtrp Molecular mechanisms of gene therapy TAC (immunohistochemical detection). Figure 11 AF pcAAV- Adtrp Immunostaining analysis of Adtrp, Apob, collagen VII, Lepr, and α-SMA after gene therapy. P <0.001 (n=9 / group).
[0029] Figure 12 : ADTRPMechanism diagram of the action of the NF-κB p65 pathway on cardiac microenvironment endothelial cells. Coronary artery disease susceptibility genes. ADTRP Knockout of pcAAV-cTNT- leads to heart failure and insufficiency by affecting the function of endothelial cells in the cardiac microenvironment. Adtrp Viral vector gene therapy alleviates ischemic heart failure in a MI (myocardial infarction) model and pressure overload-induced heart failure in a TAC (transcatheter aortic arch stenosis) model by reducing cardiac fibrosis and mitigating adverse remodeling. Mechanistically, ADTRP This study activates NF-κB p65 signaling, downregulates Leptin receptor (LepR), ApoB, and Collagen VII, and inhibits endothelial cell inflammation (reducing ICAM-1, VAM-1, P-Selection, and E-Selectin). This provides a novel therapeutic strategy for the treatment of heart failure. Detailed Implementation
[0030] Example 1 Build Adtrp Whole gene knockout (KO) mice and heart-specific knockout CKO mice Heterozygotes of wild-type (WT) C57BL / 6J mice and exon 2-4 deletions Adtrp + / - The mice were obtained from Cyagen (Suzhou, China). (Created) Adtrp Strategies for knocking out mice, such as Figure 1 As shown. PCR method was used to... Adtrp Genotyping and identification were performed on KO mice using the following primers: PCR primer 1 Forward: 5'-TTTACCGAGATGGCATTTGTAAC-3' (SEQ ID NO:2); Reverse: 5'-GAAACAGA CAGTGTCAAGCCAC-3' (product size: 553bp) (SEQ ID NO:3); PCR primer 2 Forward: 5'-GGTCATCATCATTGCTTTCTAAC-3' (SEQ ID NO:4); Reverse: 5'-GAAACAGACAGTGTCAAGCCAC-3' (product size: 583bp) (SEQ ID NO:5). Wild-type Adtrp + / + A band is shown at 583 bp, indicating a heterozygote. Adtrp + / - Two bands are shown at 553 bp and 583 bp, indicating homozygotes. Adtrp - / - A stripe is displayed at 553 bp.
[0031] Example 2 Constructing a MI-based model of ischemic heart failure C57BL6J mice (10-12 weeks old) were used to establish a mouse model of myocardial infarction by ligating the left anterior descending coronary artery (LAD). The mice were divided into two groups: a myocardial infarction (MI) model group and a normal control group (Sham). Mice in the Sham group underwent the same surgery, but without LAD ligation. The mice were then divided into... Adtrp - / - , Adtrp + / - Three genotype groups (WT, 12 mice per group) were used. The specific procedure was as follows: Mice of various genotypes were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Hair was removed from the lower jaw to the upper abdomen. The mice were fixed on a surgical board and placed on a heated plate. A PVC cannula for connecting to a small animal ventilator was inserted into the mouse's trachea and connected to the ventilator. After turning on the ventilator, the second intercostal space in the thoracic cavity was cut, the heart was expelled from the thoracic cavity, the pericardium was cut open, and the left atrial appendage and coronary artery were located. A suture needle with sutures was held in place using a micro-needle holder and passed through the myocardium from right to left, ligating the left anterior descending coronary artery (LAD). The heart was then placed back into the thoracic cavity. The thoracic cavity was combed to ensure organ repositioning. The thoracic cavity walls were closed layer by layer and sutured. The mice were removed from the ventilator and placed in a 37°C incubator overnight. Sham mice underwent the same procedures as the surgical mice, but without LAD ligation. Cardiac function was assessed preoperatively and postoperatively by echocardiography. Echocardiography and TTC staining confirmed myocardial infarction. Twenty-eight days postoperatively, cardiac function was reassessed, and the mice were euthanized. Heart tissue was collected for histological analysis (H&E and Masson staining) to assess infarct area and ventricular remodeling. Western blotting and immunohistochemistry were used to detect the expression levels of Adtrp, Apob, collagen VII, Lepr, Icam-1, Vcam-1, E-selectin, P-selectin, and p65. Example 3 Constructing a TAC-based model of non-ischemic heart failure A TAC model was established in C57BL6J mice (10-12 weeks old) to induce myocardial hypertrophy and heart failure. Mice were divided into a Sham group and a TAC group, each containing three different strains. Adtrp Genotypic composition: Adtrp KO ( Adtrp - / - and Adtrp + / - ) and wild type ( Adtrp + / +Twelve mice of each genotype were used. The surgical procedure was as follows: Mice of each genotype were divided into two groups and anesthetized with sodium pentobarbital (50 mg / kg) via intraperitoneal injection. Endotracheal intubation was performed, and the skin was disinfected with alcohol. A transverse incision was made parallel to the midpoint of the mouse's forearm, with the incision position ideally allowing clear visualization of the second rib. The subcutaneous fascia at the incision site was then clamped and cut. A traction suture was placed, the sternum was cut, and the thoracic cavity was opened. After opening the thoracic cavity, the thymus was clearly visible. The thymus was bluntly dissected to expose the aortic arch and its branches. A small incision was made in the soft tissue between the aortic arch, brachiocephalic trunk, and left common carotid artery, and a suture was threaded through. The aortic arch was ligated with 7-0 silk suture. The chest was closed, air was expelled, the skin was sutured, the wound was sutured layer by layer, disinfected, the tube removed, and the mice were resuscitated with a heating pad. The sham-operated group (Sham) mice underwent the same procedure as the surgical group, but without the TAC procedure. Postoperatively, cardiac function was assessed using a small animal ultrasound system. Doppler ultrasound was performed 28 days postoperatively, and cardiac tissue was collected for histological analysis. H&E and Masson staining were used to assess ventricular cavity retention, myocardial fibrosis, and ventricular remodeling. Western blotting and immunohistochemistry were used to detect the expression levels of Adtrp, Apob, collagen VII, Lepr, Icam-1, Vcam-1, E-selectin, P-selectin, and p65.
[0032] Example 4 Echocardiographic analysis Echocardiography was performed on mice using a Mindray Resona R9 Doppler color ultrasound system. The linear array probe (L20-5U) operated at a frequency of 14.5 MHz, a scanning speed of 200 cm / s, and an image depth of 2.5 cm. Anesthesia was administered as described above, with the animals tilted 30° to the left. Two-dimensional ultrasound imaging was used to obtain parasternal left ventricular short-axis views. Left ventricular ejection fraction (EF%) and short-axis segmentation shortening (FS%) were measured using M-mode echocardiography. Preoperatively, postoperatively, and 4 weeks after treatment, pcAAV-cTNT- Adtrp Assess cardiac function.
[0033] Example 5 Hematoxylin and eosin staining (H&E staining) In MI and TAC or AAV-cTNT- Adtrp Four weeks after treatment, the mice were anesthetized and euthanized. Hearts were prepared by cryosectioning and paraffin sectioning, dewaxed in xylene, hydrated with graded ethanol, and rinsed with water. Sections were stained with hematoxylin, differentiated, stained with eosin, dehydrated, mounted with neutral resin, examined under a microscope, and images were acquired and analyzed.
[0034] Example 6 Masson staining Heart sections were harvested, fixed, and embedded in paraffin. Sections were dewaxed, rehydrated, and stained overnight with Masson A solution. After incubation in Masson B and C solutions, sections were differentiated using 1% hydrochloric acid-ethanol and stained with Masson D, E, and F solutions. After rinsing and differentiation with 1% glacial acetic acid, sections were dehydrated in anhydrous ethanol, cleared with xylene, mounted with neutral resin, examined under a microscope, and images were acquired and analyzed.
[0035] Example 7 Immunohistochemical analysis Mice were euthanized, and cardiac tissue was fixed as described above. The tissue was dehydrated, embedded in paraffin, and sectioned. Sections were dewaxed, rehydrated, and antigen recovered in a microwave oven using citrate antigen retrieval buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and non-specific binding was blocked with 3% bovine serum albumin. Sections were treated overnight at 4°C with primary antibodies: anti-Adtrp (1:50; Sigma), anti-Apob (1:100; PTGLAB), anti-collagen VII (1:50; Abcam), anti-Lepr (1:100; PTGLAB), anti-CD31 (1:100; PTGLAB), and anti-α-SMA (1:100; PTGLAB). After incubation with secondary antibodies at room temperature, DAB chromogenic agent was used for staining. Hematoxylin was used to counterstain cell nuclei, allowing differentiation, followed by a blue staining. Finally, the sections were dehydrated, mounted with neutral resin, and imaged using an Olympus BX53F optical microscope (Olympus, USA).
[0036] Example 8 Real-time quantitative RT-PCR analysis Total RNA was isolated from mouse tissues using TRIzol (Takara). Contaminating DNA was removed by incubation with DNase I, and the RNA was converted to cDNA using M-MLV reverse transcriptase (Promega, USA) and random primers. Real-time RT-PCR was performed using a FastStart SYBR Green Master (Roche Applied Sciences, USA) Step one Plus Real-time PCR system (Thermo Fisher, USA). Each qPCR reaction required a 10 µL volume containing 5 µL of SYBR Green I Master Mix (Roche), 200 nM forward and reverse primers, and 1 µL of cDNA template. Quantitative PCR amplification primers were used, employing 2... ⁻ΔΔCt The method determined the relative gene expression of GAPDH.
[0037] Example 9 Western blot analysis Proteins were extracted from mouse tissues using RIPA lysis buffer and analyzed by Western blot to detect the protein expression levels of Adtrp, Apob, collagen VII, Lepr, Icam-1, Vcam-1, E-selectin, P-selectin, and p65 in mouse tissues. Primary antibodies included anti-Adtrp (Sigma, USA), anti-Apob, anti-collagen VII (Abcam, UK), anti-Lepr, anti-β-actin, anti-α-Tubulin, anti-GAPDH, anti-NF-κB p65, anti-Icam-1, anti-Vcam-1, anti-E-selectin, and anti-P-selectin (PTGLAB Group, Inc., China). Secondary antibodies included horseradish peroxidase-labeled anti-mouse IgG and anti-rabbit IgG (Thermo Fisher).
[0038] Example 10 Injection of adeno-associated virus vector pcAAV-cTNT- Adtrp Gene therapy for myocardial infarction (MI) and TAC-induced heart failure in mice Two weeks after establishing the MI and TAC models, mice were injected via tail vein with the adeno-associated virus vector pcAAV-cTNT- Adtrp (OBIO Technology, China) The dosage was 2.5E + 11 vg per mouse. The control group received an equal amount of pcAAV-cTNT-GdGreen vector. Cardiac function was assessed by Doppler ultrasound 4 weeks after treatment. Mice were then anesthetized, euthanized, and TTC staining was performed to assess improvement in myocardial ischemia (MI model group). HE, Masson staining, and immunofluorescence analysis were used to assess ventricular cavity preservation and myocardial necrosis. Immunohistochemistry and Western blotting were used to detect the expression levels of Adtrp, Apob, collagen VII, and Lepr.
[0039] Based on the content of Examples 1-10 above, the following conclusions are drawn: (1) Adtrp The lack of [certain nutrients] leads to heart failure, and CAD / MI further exacerbates cardiac dysfunction. In this study, the invention established [a system / mechanism] through CRISPR-Cas9 genome editing. Adtrp The knockout (KO) mouse line, analyzed by Western blot, Adtrp + / - Adtrp protein expression in mice is reduced by approximately 50%. Adtrp - / -The disappearance of Adtrp protein expression in mice confirms... Adtrp KO mice were successfully constructed. Ischemic heart failure models based on myocardial infarction (MI) and non-ischemic heart failure models based on cardiac arrest (TAC) were constructed. Doppler echocardiography was used to confirm successful modeling on day 2 after modeling. Cardiac function parameters were assessed by echocardiography on day 28 after modeling. The models of this invention conform to clinical characteristics. Compared with WT mice, the MI and TAC models showed... Adtrp + / - mice and Adtrp - / - Both LVEF% and LVFS% were significantly reduced in mice. There was no difference in the heart-to-body weight ratio among the three different genotypes in the sham-operated group. (MI model) Adtrp + / - mice and Adtrp - / - Compared with WT mice with MI, the heart-to-body weight ratio of the mice was significantly reduced in the TAC model. Adtrp + / - mice and Adtrp - / - Compared with WT mice in the TAC model, the heart-to-body weight ratio was significantly increased in the MI model; the MI model showed decreased cardiomyocyte cross-sectional area, increased percentage of ventricular cavity retention area, decreased left ventricular wall thickness, and increased myocardial fibrosis; the TAC model showed myocardial hypertrophy, with increased cardiomyocyte cross-sectional area, increased percentage of ventricular cavity retention area, increased left ventricular wall thickness, and increased degree of myocardial fibrosis; indicating that... Adtrp The absence of these factors leads to cardiac dysfunction and heart failure; MI and TAC further exacerbate cardiac dysfunction. See the relevant steps in Examples 1, 2, 3, 4, 5, 6, and 8.
[0040] (2) Adeno-associated virus vector myoAAV1A- Adtrp Effectively blocks the progression of heart failure. This invention discovers... Adtrp KO mice exhibited impaired cardiac contractility and heart failure. This invention hypothesizes that through mice... Adtrp Overexpression of gene therapy can alleviate heart failure. This invention constructs a gene therapy driven by the heart-specific promoter cTNT. Adtrp The expressed adeno-associated virus vector myoAAV1A- Adtrp And a control vector myoAAV1A-GdGreen. This invention detects myoAAV1A- Adtrp The efficacy of gene therapy in two different heart failure models: a myocardial ischemic heart failure model based on myocardial infarction (MI) and a non-ischemic heart failure model based on transcatheter arterial therapy (TAC). In both the MI and TAC models, WT mice showed significantly reduced left ventricular ejection fraction (LVEF%) and left ventricular susceptibility (LVFS%). However, tail vein injection of myoAAV1A- AdtrpThe viral vector completely reversed these effects, while the control vector myoAAV1A-GdGreen did not. Adtrp Gene therapy for heterozygotes of MI Adtrp + / - Mice and homozygotes Adtrp - / - The rescue effect on heart failure in mice was similar. H&E and Masson staining showed that myoAAV1A- Adtrp Gene therapy also reversed the effects in wild-type MI mice and Adtrp + / - mice and Adtrp - / - Decreased cross-sectional area of mouse cardiomyocytes and heart weight / body weight ratio, increased percentage of ventricular cavity retention area, decreased left ventricular wall thickness, and increased myocardial fibrosis. myoAAV1A- Adtrp Gene therapy also blocked cardiac hypertrophy and heart failure in the TAC model; reduced LVEF% and LVFS% in TAC wild-type mice; and increased cardiomyocyte cross-sectional area, heart / body weight ratio, percentage of ventricular cavity stasis area, left ventricular wall thickness, and degree of myocardial fibrosis. These phenotypic defects were addressed by tail vein injection of myoAAV1A- Adtrp The vector (2.5E+11 vg per mouse) was completely reversed, while the control vector myoAAV1A-GdGreen (2.5E+11 vg per mouse) was not reversed. In TAC heterozygotes... Adtrp + / - Mice and homozygous Adtrp - / - Similar rescue results were obtained in mice. See the relevant steps in Examples 1, 2, 3, 4, 5, 6, 8, and 10.
[0041] (3) MyoAAV1A- Adtrp The molecular mechanism of gene therapy. This study used Western blot and immunohistochemical analysis to reveal the molecular mechanism of MyoAAV1A- Adtrp Molecular mechanisms of gene therapy. Western blot analysis showed that MI induces... Adtrp Upregulated expression of the gene therapy vector myoAAV1A- Adtrp Further significant increase Adtrp The expression of [the gene] was present in the control vector myoAAV1A-GdGreen, but not in the wild-type [vector]. Adtrp + / + Mouse, heterozygote Adtrp + / - Mice and homozygotes Adtrp - / - Similar results were obtained in mice. These data indicate that myoAAV1A-Adtrp The carrier successfully increased Adtrp The expression. Adtrp Three downstream genes Apob , Col7a1 and Lepr The MI group significantly upregulated the expression levels of these proteins, and also significantly upregulated the expression levels of cell adhesion molecules Icam-1, Vcam-1, E-selectin, and P-selectin, while significantly reducing the expression level of p65. Meanwhile, the gene therapy vector myoAAV1A- Adtrp This effect was reversed, while the control vector myoAAV1A-GdGreen did not. The data indicate that myoAAV1A- Adtrp Gene therapy successfully suppressed ischemic heart failure by reducing the expression of Apob, Col7a1, Lepr, Icam-1, Vcam-1, E-selectin, and P-selectin, and increasing p65 expression. Similar gene therapy experiments were conducted in TAC-induced cardiac hypertrophy and heart failure models. Western blot analysis showed that compared with the control vector myoAAV1A-GdGreen, myoAAV1A-GdGreen significantly reduced the expression of p65. Adtrp Gene therapy has increased significantly. Adtrp The expression was significantly upregulated. Adtrp The expression levels of downstream genes Apob, Col7a1, and Lepr, as well as cell adhesion molecules ICAM-1, VCAM-1, E-selectin, and P-selectin, were significantly reduced, and the expression level of p65 was significantly decreased. The gene therapy vector myoAAV1A- Adtrp This effect was reversed, while the control vector myoAAV1A-GdGreen did not. Data indicate that myoAAV1A- Adtrp The vector successfully inhibited TAC-induced heart failure by reducing the expression of Apob, Col7a1, Lepr, Icam-1, Vcam-1, E-selectin, and P-selectin, and increasing the expression of p65. See the relevant steps in Examples 1, 2, 3, 4, 7, 8, 9, and 10.
[0042] (4) ADTRP Mechanism of action on cardiac microenvironment endothelial cells via the NF-κB p65 pathway. This invention identifies a susceptibility gene for coronary artery disease. ADTRP Knockout leads to cardiac dysfunction and heart failure by affecting the function of endothelial cells in the cardiac microenvironment, and can be further aggravated by non-cardiac ischemia (MI) or pressure overload (TAC). pcAAV-cTNT- AdtrpViral vector gene therapy alleviates ischemic heart failure in a MI (myocardial infarction) model and pressure overload-induced heart failure in a TAC (transcatheter aortic arch stenosis) model by reducing cardiac fibrosis and mitigating adverse remodeling. Mechanistically, ADTRP Activation of NF-κB p65 signaling, downregulation of Leptin receptor (LepR), ApoB and Collagen VII, and inhibition of endothelial cell inflammation (reducing ICAM-1, VAM-1, P-Selection and E-Selectin) alleviate cardiac dysfunction and heart failure in MI myocardial infarction mice and TAC mice. Adtrp Further translation of gene therapy may provide new avenues for precision treatment of heart failure. This invention provides a novel treatment strategy for heart failure. See the relevant steps in Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
[0043] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method based on Adtrp The use of viruses in the preparation of drugs for treating heart failure, as described above. Adtrp The viral sequence is shown in SEQ ID NO:
1.
2. The use according to claim 1, characterized in that, The expression Adtrp The virus includes myoAAV1A- expressed in the body. Adtrp Viral vector.
3. The use according to claim 2, characterized in that, The myoAAV1A- Adtrp Use of viral vectors in the preparation of drugs for treating ischemic heart failure based on left anterior descending coronary artery ligation MI.
4. The use according to claim 3, characterized in that, The myoAAV1A- Adtrp In the use of viral vectors in the preparation of drugs for the treatment of ischemic heart failure based on MI, significant increases in LVEF% and LVFS% and a significant increase in the heart-to-body weight ratio have been achieved; or an increase in the cross-sectional area of cardiomyocytes and the heart-to-weight ratio, a decrease in the percentage of ventricular cavity slough area, an increase in left ventricular wall thickness, and a decrease in myocardial fibrosis have been achieved.
5. The use according to claim 3, characterized in that, The myoAAV1A- Adtrp The viral vector inhibited the expression of Apob, collagen VII, Lepr, and endothelial cell inflammatory markers Icam-1, Vcam-1, E-selectin, and P-selectin, while increasing the expression of p65 and the endothelial angiogenesis marker CD31.
6. The use according to claim 2, characterized in that, The myoAAV1A- Adtrp Use of viral vectors in the preparation of drugs for treating non-ischemic heart failure based on aortic transconstriction surgery (TAC).
7. The use according to claim 6, characterized in that, The myoAAV1A- Adtrp The use of viral vectors in the preparation of drugs for treating TAC-based non-ischemic heart failure aims to block cardiac hypertrophy and heart failure, and reverse increased cardiomyocyte cross-sectional area, percentage of ventricular cavity stasis area, left ventricular wall thickness, and myocardial fibrosis.
8. The use according to claim 6, characterized in that, The myoAAV1A- Adtrp In the use of viral vectors for the treatment of TAC-based non-ischemic heart failure, the pcAAV- Adtrp Gene therapy inhibited the expression of Apob, collagen VII, Lepr, and endothelial cell inflammatory markers Icam-1, Vcam-1, E-selectin, and P-selectin, while significantly increasing the expression of p65 and the vascular marker α-SMA.
9. A pharmaceutical composition for treating heart failure, characterized in that, The pharmaceutical composition comprises that described in claim 1. Adtrp Virus, or myoAAV1A as described in any one of claims 2-8 Adtrp Viral vector.
10. The pharmaceutical composition for treating heart failure according to claim 9, characterized in that, myoAAV1A- Adtrp The viral vector was administered via intravenous injection for treatment.