Double-mutant inactivated telomerase, vector comprising same, and use thereof

Abstract

Provided is a double-mutation inactivated telomerase, the double-mutation inactivated telomerase comprising two mutation sites. A first mutation site is Y707, and a second mutation site is D868. Further provided are a vector comprising the aforementioned double-mutation inactivated telomerase and the use thereof in a drug for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renocardiac syndrome, and cardiorenal syndrome, and improving myocardial cell contractility.

Description

Double-mutated inactive telomerase, vectors containing it, and their uses

[0001] Citation of relevant applications

[0002] This application claims the benefit of Chinese Patent Application No. 2025100177762, filed on January 6, 2025 with the State Intellectual Property Office of the People's Republic of China, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the fields of biomedicine and genetic engineering, specifically to an inactivated telomerase, a vector containing the inactivated telomerase, and its uses. Background Technology

[0004] For decades, cardiovascular disease has been a leading cause of death globally. In 2019, cardiovascular disease accounted for 33% of all deaths worldwide. With an aging and growing population, cardiovascular disease is increasingly becoming a severe burden on global healthcare systems. Although significant progress has been made in the treatment of cardiovascular disease in recent years, the prevalence of heart failure (HF), a common cardiovascular disease, continues to rise, limiting overall survival extension. Currently, approximately 38 million people worldwide suffer from heart failure, resulting in more than 1 million hospitalizations annually in the United States and Europe. In China, the overall prevalence of HF among adults is 1.3%, and it is rising with an aging population. The current 5-year mortality rate for HF is approximately 40% to 70%, worse than the prognosis of many cancers. Heart failure is the end stage of various heart diseases and has become a significant public health issue affecting the health of the population.

[0005] Heart failure is a syndrome in which the heart is unable to effectively pump blood to meet the body's metabolic needs. Its causes include coronary artery disease, hypertension, and cardiomyopathy. Heart failure is a common problem in the elderly, with an incidence increasing with age. Its core pathological mechanisms are myocardial dysfunction and ventricular remodeling. Increased ventricular wall tension, activation of the neuroendocrine system, and cell death are key factors leading to heart failure. Clinically, patients often present with shortness of breath, fatigue, ankle edema, and weight gain. Based on the timing and rate of onset, heart failure can be classified as chronic heart failure (CHF) or acute heart failure (AHF). There are two forms of acute heart failure (AHF): one is a sudden worsening or acute exacerbation of the symptoms or signs of chronic heart failure, called "acute decompensated heart failure (ADHF)," which is one of the main forms of AHF, accounting for about 80% to 90%; the other is a first-time attack in patients with or without underlying heart disease, due to the aggravation of underlying heart disease or the occurrence of acute cardiac lesions, or due to non-cardiac factors, called "new-onset acute heart failure," accounting for about 10% to 20%.

[0006] Currently, treatment for heart failure includes pharmacological therapy (such as ACE inhibitors, beta-blockers, and diuretics), device therapy (such as pacemakers and implantable cardioverter-defibrillators), and surgical treatment (such as heart transplantation). Based on the left ventricular ejection fraction (LVEF) level measured by echocardiography at the patient's initial assessment, heart failure can be classified into three basic types: "heart failure with reduced ejection fraction (HFrEF)," "heart failure with mildly reduced ejection fraction (HFmrEF)," and "heart failure with preserved ejection fraction (HFpEF)." Specifically, in HFrEF, LVEF < 40%; in HFmrEF, LVEF is 40%-49%; and in HFpEF, LVEF ≥ 50%. HFpEF can be further divided into "heart failure with normal ejection fraction (HFnEF)" and "heart failure with supra-normal ejection fraction (HFsnEF)". In addition, there are several special types: "heart failure with improved ejection fraction (HFimpEF)", "heart failure with recovered ejection fraction (HFrecEF)", "heart failure with decreased ejection fraction (HFdecEF)", and "improved heart failure with preserved ejection fraction (HFpimpEF)".

[0007] The pathogenesis of heart failure (HF) involves diverse mechanisms and a complex molecular basis, including increased hemodynamic load, ischemia-related dysfunction, myocardial loss, excessive neurohumoral stimulation, abnormal calcium ion response, ventricular remodeling, myocardial fibrosis, and genetic mutations. Standard treatment for HF includes the administration of angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, beta-blockers, or calcium channel blockers to reduce the burden on cardiomyocytes and alleviate symptoms. However, these treatments have limited effect on improving overall survival. New treatment strategies are urgently needed to address this significant and growing public health problem.

[0008] In recent years, telomere / telomerase biology has gained increasing prominence in the pathogenesis of age-related cardiovascular diseases (CVD), such as atherosclerosis, hypertension, myocardial infarction (MI), and heart failure. Telomerase, a cellular reverse transcriptase (TERT; also known as TP2; TRT; EST2; TCS1), is considered an important upstream signal for the activation of the DNA damage response (DDR) caused by excessively short telomeres, which is believed to induce permanent cell cycle arrest in cardiomyocytes. This limitation may be related to the decreased proliferative potential of cardiac stem cells (CSCs) and cardiomyocytes. The link between cardiovascular disease and telomere shortening lays the foundation for developing therapeutic techniques aimed at lengthening telomeres and thereby restoring the proliferative capacity of the adult mammalian heart. This phenomenon offers a fascinating prospect for the treatment and prevention of cardiovascular disease (CVD). Further research into telomerase gene therapy in the field of cardiac regenerative medicine makes sense, as encouraging results have been shown in mouse models, demonstrating the beneficial effects of reactivating telomerase in the heart after myocardial infarction (Mohammed Abdel-Gabbar et al, Telomere-based treatment strategy of cardiovascular diseases: imagination comes to reality, Genome Instability & Disease (2024) 5:61-75).

[0009] Research by Yin Cai et al. indicates that in the hearts of aging Rap1- / - mice, telomere shortening is accompanied by extensive DNA damage, manifested as a decreased T / S ratio and an increase in nuclear γH2AX. Simultaneously, age-related phenotypes such as mitochondrial ultrastructural alterations, enhanced aging, myocardial hypertrophy, and dysfunction are also evident. Mechanistically, acetylated p53 and nuclear p53 are increased in the hearts of Rap1- / - mice, while PPARα is decreased. Importantly, p53 directly binds to the promoter of PPARα in the mouse heart and inhibits PPARα transcription. They suggest that Rap1 can link telomere biology with fatty acid metabolism and age-related cardiac lesions by regulating the p53 / PPARα signaling pathway, thus becoming a therapeutic target for preventing / alleviating cardiac aging (Deficiency of telomere-associated repressor activator protein 1 precipitates cardiac aging in mice via p53 / PPARα signaling, Theranostics 2021, Vol. 11, Issue 10). However, Yin Cai et al. did not disclose what specific disease they studied, especially heart disease, or the data from their research.

[0010] Currently, how to regulate telomerase-related signaling pathways to achieve the treatment of age-related cardiovascular diseases remains a research hotspot in this field. Summary of the Invention

[0011] In order to address the problems existing in the prior art, the present invention aims to provide a double-mutant inactive telomerase for preparing drugs for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome and / or improving the contractile ability of myocardial cells.

[0012] In one aspect, the present invention provides a double-mutant inactive telomerase having two mutation sites, wherein the first mutation site is Y707 and the second mutation site is D868.

[0013] On the other hand, the present invention provides a vector containing a double-mutant inactive telomerase, the vector containing the coding sequence of the double-mutant inactive telomerase, wherein the double-mutant inactive telomerase has two mutation sites, wherein the first mutation site is Y707 and the second mutation site is D868. Specifically, the first mutation is Y707L and the second mutation is D868V; or the first mutation site is Y707F and the second mutation site is D868A.

[0014] In another aspect, the present invention provides the use of double-mutated inactive telomerase or a carrier containing double-mutated inactive telomerase in the preparation of medicaments for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome and / or improving myocardial cell contractility.

[0015] Compared to wild-type human TERT, the double-mutant inactivated telomerase provided by this invention exhibits alterations in telomerase activity and nuclear localization properties. Due to the synergistic effect of the first mutation site Y707 and the second mutation site D868, the inactivated telomerase of this invention can repair telomeres and achieve a new telomere homeostasis. By reactivating the protective effect of telomeres, it improves the contractile capacity of cardiomyocytes and prevents further degeneration of cardiac function. According to embodiments of this invention, in various models such as the AngII heart failure mouse model, the DMD-induced heart failure mouse model, the TAC-induced heart failure mouse model, and the myocardial infarction mouse model, the inactivated telomerase of this invention demonstrates safety and can significantly improve the ejection fraction in disease model mice. It can be used to prepare drugs for the prevention or treatment of myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, and cardiorenal syndrome, and to improve the contractile capacity of cardiomyocytes. Attached Figure Description

[0016] Figure 1 illustrates the relationship between telomeres and the pathogenesis of heart failure, as well as the mechanism of action of gene therapy drugs in treating heart failure.

[0017] Figure 2 shows that JV001 can effectively improve AngII-induced cardiomyocyte functional impairment. a. JV001 increases the beating rate of cardiomyocytes. b. JV001 improves the contraction velocity of cardiomyocytes. c. JV001 stabilizes the contraction waveform of cardiomyocytes.

[0018] Figure 3 shows the screening of CI-TERT mutants in disease-related human induced pluripotent stem cells (DCM-hiPSC).

[0019] Figure 4 shows the improvement of left ventricular ejection fraction and left ventricular fractional shortening in Ang II mice by JV001. a. Left ventricular ejection fraction over time in different treatment groups. b. Left ventricular fractional shortening over time in different treatment groups. c. Heart weight / body weight ratio in different treatment groups. d. Heart weight / tibia length ratio in different treatment groups.

[0020] Figure 5 shows the distribution of JV001 in different organs of the Ang II mouse model.

[0021] Figure 6 shows the left ventricular ejection fraction (LVEF) values ​​in mice after administration of the Y707L+D868V mutant. a. Dynamic trends of LVEF in the control group, model group, and Y707L+D868V group at different time points after AngII injection; b. Comparison of specific LVEF values ​​in each group.

[0022] Figure 7 shows how a single dose of JV001 reversed cardiac dysfunction and prevented cardiac fibrosis in mice with heart failure induced by aortic arch constriction (TAC). a. Changes in left ventricular ejection fraction: the sham-operated group remained stable, the model group gradually decreased, while the JV001 group showed an increasing trend. b. Trends in left ventricular fractional shortening: the sham-operated group remained stable, the model group decreased, and the JV001 group increased. c. Changes in left ventricular end-systolic diameter. d. Changes in the ratio of heart weight to tibia length. e. Histological images of the three groups under Gram staining and Masson staining, further revealing the physiological differences between the different treatment groups at the microstructural level.

[0023] Figure 8 shows that JV001 can effectively reduce the persistently elevated N-terminal pro-brain natriuretic peptide (NT-proBNP) levels in TAC mice.

[0024] Figure 9 shows the survival rate of TAC mice treated with JV001. Control group: wild-type TERT (100% survival rate after 12 weeks of administration); Model group: TAC model mice (26.1% survival rate after 12 weeks of administration); JV001: JV001 alone (43.5% survival rate after 12 weeks of administration); Entresto: Entresto alone (30.4% survival rate after 12 weeks of administration); JV001 + Entresto: combined administration of JV001 + Entresto (65.2% survival rate after 12 weeks of administration).

[0025] Figure 10 shows the cardiac function index in mice with DMD-induced heart failure. a. Trends in left ventricular ejection fraction at different days after JV001 injection in the three groups of mice. b. Changes in left ventricular fractional shortening over time after JV001 injection in the three groups of mice. c. Changes in left ventricular end-systolic diameter over time on day 12 after JV001 injection in the three groups of mice.

[0026] Figure 11 shows the left ventricular ejection fraction (LVEF) of mice in the DMD-induced heart failure mouse model sequence screening experiment. a. The LVEF of the wild-type genotype was significantly higher than that of other treatment groups; b. The LVEF of the model group decreased significantly over time, while the LVEF of the wild-type, Y707F+D868A, and Noxintu groups remained relatively stable, with the Y707F+D868A group showing better performance in the later stages.

[0027] Figure 12 shows the cardiac function index of mice in a myocardial infarction (MI) mouse model. a. Trends in left ventricular ejection fraction at different time points after injection in different treatment groups; b. Changes in left ventricular short-axis shortening rate over time after injection in different treatment groups.

[0028] Figure 13 shows the cardiac function index of mice in the HFpEF mouse model. a. Changes in the E / E' ratio at three time points in different model groups; b. Trends in the E / A ratio in different model groups from 0 to 9 weeks.

[0029] Figure 14 shows the cardiac function index in a mouse model of renal failure induced by unilateral nephrectomy + Ang II + high-salt diet. a. Changes in left ventricular ejection fraction at different time points after surgery in each treatment group (sham surgery group, sham surgery + JV001, unilateral nephrectomy group, unilateral nephrectomy + JV001). The decrease in left ventricular ejection fraction in the unilateral nephrectomy + JV001 group was relatively smaller. b. Trends in left ventricular short-axis shortening rate at the same time points. Similarly, the decrease in left ventricular short-axis shortening rate in the unilateral nephrectomy + JV001 group was also more gradual than in other groups.

[0030] Figure 15 shows the cardiac function indices in a mouse model of renal failure induced by nephrectomy. a. Changes in left ventricular ejection fraction (LVEF) at different time points post-surgery in different treatment groups. The LVEF in the nephrectomy + JV001 group was significantly higher than in other groups, especially maintaining a high LVEF level for a longer period post-surgery. b. Changes in left ventricular fractional shortening at different time points post-surgery in different treatment groups. The LVEF in the nephrectomy + JV001 group also showed a significantly better left ventricular fractional shortening rate than in other groups, indicating better cardiac function recovery.

[0031] Figure 16 shows the cardiac function index of mice in the STZ-induced type 1 diabetes model. a. Trends in the E / E' ratio of different treatment groups in the STZ-induced type 1 diabetes model at different time points; b. Changes in the E / A ratio of different treatment groups in the STZ-induced type 1 diabetes model at different time points.

[0032] Figure 17 shows the cardiac function index of mice in the STZ-induced type 2 diabetes model. a. Trends in the E / E' ratio of different treatment groups in the STZ-induced type 2 diabetes model at different time points; b. Changes in the E / A ratio of different treatment groups in the STZ-induced type 2 diabetes model at different time points.

[0033] Figure 18 shows the cardiac function index of hypertrophic cardiomyopathy (HCM) mice treated at week 8. a. Trends in left ventricular end-systolic diameter in different treatment groups; b. Changes in left ventricular posterior wall thickness at end-systolic diameter in different treatment groups; c. Dynamic changes in left ventricular end-diastolic diameter in different treatment groups; d. Differences in left ventricular posterior wall thickness at end-diastolic diameter in different treatment groups.

[0034] Figure 19 shows the cardiac function indices of hypertrophic cardiomyopathy (HCM) mice treated at week 40. a. Changes in left ventricular end-systolic diameter among the three groups. b. Changes in left ventricular end-diastolic diameter among the three groups. c. Comparison of left ventricular posterior wall end-systolic thickness among the three groups. d. Changes in left ventricular posterior wall end-diastolic thickness among the three groups.

[0035] Figure 20 shows the off-target toxicity assessment of JV001 in the non-target organ, the liver. a. Sirius red staining: There was no difference in the degree of liver fibrosis between the model group (DEN) and the JV001 group. b. Western blotting experiment: There was no difference in p53 protein among the groups. p-p53 protein was upregulated in both the model group and the JV001 group, and there was no difference between the model group and the JV001 group. Detailed Implementation

[0036] In this disclosure, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0037] Telomeres and telomerase

[0038] Telomeres are DNA-protein complexes located at the ends of eukaryotic chromosomes, primarily functioning to maintain the integrity and stability of the genome during replication. Human telomeres are composed of TTAGGG repeat sequences that bind to Shelterin, a protective protein, forming a unique "cap" structure. The Shelterin protein complex includes TRF1, TRF2, TPP1, TIN2, RAP1, and POT1. These proteins bind to the telomere repeat sequences, forming a DNA-protein bridge structure that protects telomeric DNA from recognition by DNA damage responses. TRF1 and TRF2 bind to double-stranded telomeric DNA, while POT1 binds to single-stranded telomeric DNA. TPP1 interacts with POT1 to help it bind to telomeres. TIN2 acts as a bridge in the complex, connecting TRF1, TRF2, TPP1, and POT1 together. RAP1 interacts with TRF2 and participates in regulating telomere length and protective function.

[0039] Telomere damage triggers a DNA damage response (DDR) within cells, leading to cell cycle arrest, apoptosis, or senescence. DDR activation plays a crucial role in the pathological process of heart disease. Telomere-induced DDR affects cardiomyocyte survival and function by activating the ATM / ATR signaling pathway and regulating downstream effectors such as p53 (Figure 1). In heart disease patients, telomere damage and its resulting abnormal activation of the DDR signaling pathway contribute to further disease progression. DDR is primarily regulated through two signaling pathways: ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related). ATM mainly responds to DNA double-strand breaks, while ATR responds to single-stranded DNA. Telomere damage can activate these two pathways, thereby activating downstream effectors such as p53, Chk1, and Chk2, which regulate cell cycle checkpoints, DNA repair, apoptosis, and senescence.

[0040] Telomerase, composed of the telomerase RNA component (TERC) and the telomerase reverse transcriptase component (TERT), primarily functions to elongate telomeres. Although telomerase activity is extremely low in most somatic cells, the protective effect of telomerase expression and activity regulation on cardiomyocytes in heart disease has attracted considerable attention. Studies have found that overexpression of hTERT (human telomerase reverse transcriptase gene) can slow down cardiomyocyte apoptosis and improve cardiac function. hTERT is the catalytic subunit of telomerase, and its main function is to catalyze telomere elongation. Overexpression of hTERT can protect cardiomyocytes from damage caused by various stressors, such as oxidative stress and ischemia-reperfusion injury. hTERT exerts its protective effect through multiple mechanisms, including slowing down apoptosis, promoting cell proliferation and repair, and regulating antioxidant responses. Oxidative stress is one of the important factors in the occurrence and development of heart disease. Overexpression of hTERT can reduce the level of oxidative stress in cardiomyocytes and alleviate oxidative damage by regulating the expression and activity of antioxidant enzymes. Regulating telomere homeostasis can provide new ideas and targets for the prevention and treatment of heart disease.

[0041] Inactivation of telomerase

[0042] Telomerase reverse transcriptase (TERT) is a key protein for maintaining the length and stability of telomeres in chromosomes. Mutations in the TERT gene are closely related to a variety of human diseases, including cancer and cardiovascular disease. The term "inactive telomerase" refers to telomerase reverse transcriptase with a mutated amino acid sequence. The NCBI protein ID for wild-type TERT is NP_937983.

[0043] Mutations in the TERT gene have been reported in various diseases. Mutations C228T and C250T in the TERT promoter region add new transcription factor binding sites (Ets / TCF), enhancing the transcriptional activity of the TERT gene. Mutations in the TERT coding region directly affect the function of the TERT protein. In fact, the TERT protein possesses multiple domains, including the N-terminal domain (TEN), nuclear localization signal (NLS), telomerase RNA binding domain (TRBD), reverse transcriptase domain (RT), nuclear export signal (NES), and C-terminal extension domain (CTE).

[0044] Furthermore, Judith Haendel et al. disclosed that the Y707F point mutation introduced into TERT can restrict it to the nucleus, thereby enhancing the cell's anti-apoptotic ability. Hidemasa et al. disclosed that the D868A mutation, generated through site-directed mutagenesis, leads to the loss of TERT's catalytic activity, and compared the effects of wild-type TERT and D868A mutant TERT in cardiac muscle cells, concluding that only active TERT can delay cardiac cell cycle exit and promote cell survival. In contrast, D868A mutant TERT does not have these effects.

[0045] The applicant of this invention has creatively prepared an inactive telomerase with double mutations of Y707F and D868A (CN112063601B). The Y707F mutation causes the telomerase to remain in the cell nucleus, and the restriction of its departure allows the telomerase to be permanently attached to the telomere tail to form a protective structure. The D868A mutation causes the telomerase to lose its activity, which prevents uncontrolled telomere elongation and the possibility of inducing cancer.

[0046] According to embodiments of the present invention, in heart failure, the telomere DNA of cardiomyocytes is in a naked, damaged, and unprotected state, and continues to shorten as the disease progresses. JV001 is delivered to cardiomyocytes via an AAV9 vector, where it expresses nuclear-localized and inactivated telomere reverse transcriptase TERT. By binding to TPP1, TERT locates and attaches to the damaged telomere DNA tail, preventing further telomere shortening. Through blocking telomere DNA damage signals, cardiomyocytes are able to resume mitochondrial generation, increasing the number of mitochondria and improving the energy supply to cardiomyocytes, thereby breaking the vicious cycle of "telomere shortening - cardiomyocyte function decline" in heart failure. While cardiomyocytes are provided with energy and their beating function is restored, the restoration of mitochondrial homeostasis also prevents further apoptosis of cardiomyocytes and gradually improves their contractile function, thus improving cardiac function. In particular, the combined use of JV001 and Enterostat significantly improves the survival rate of the disease-affected mouse model, demonstrating superior efficacy in treating heart failure.

[0047] As the research progressed, the inventors of this application unexpectedly discovered that a double-mutated inactive telomerase with the first mutation site being Y707 and the second mutation site being D868 can be used to prepare drugs for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome, and improving the contractile ability of myocardial cells.

[0048] In this invention, since the inactive telomerase lacks reverse transcription activity, it is also referred to as CI-TERT (Catalytically Inactive-Telomerase reverse transcriptase). In this invention, the terms "first," "second," etc., are used only to distinguish technical features and should not be construed as implying their relative importance or implicitly indicating the number or order of the indicated technical features. Furthermore, because the inactive telomerase of this invention has two mutation sites, it is also called a double-mutant inactive telomerase.

[0049] In this invention, the Y707 mutation can be any nonpolar hydrophobic amino acid, polar neutral amino acid, acidic amino acid, basic amino acid, or non-natural amino acid other than F, and the D868 mutation can be any nonpolar hydrophobic amino acid, polar neutral amino acid, acidic amino acid, basic amino acid, or non-natural amino acid other than A.

[0050] In a preferred embodiment, the double-mutant inactive telomerase is Y707L+D868V or Y707F+D868A.

[0051] Carrier and delivery method

[0052] As used in this invention, the term "vector" refers to a composition containing isolated nucleic acids and capable of delivering the isolated nucleic acids into the cell. Various vectors have been reported in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" can include autonomously replicating viruses. The term should also be interpreted to include non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, cationic liposomes, cationic polymers, lipid nanoparticles, peptides, exosomes, and nucleic acid conjugates. In some preferred embodiments, artificial mRNA (mmRNA) is used as the vector. Artificial mRNA is a modified or engineered artificially synthesized messenger RNA. The translation level of inactivated telomerase mRNA within the host cell can be improved by optimizing the codons or adding non-coding sequences to the ends of the coding sequences. Non-viral vectors that can be used in this invention include, but are not limited to, cationic lipids, cationic polymers, peptides, lipid nanoparticles, inorganic nanoparticles, exosomes, nucleic acid conjugates, or artificial mRNA.

[0053] In addition to viral vectors, the coding sequence of the inactivated telomerase of this invention can also be delivered to host cells via viral vectors to repair telomeres and achieve a new telomere homeostasis, block DNA damage, increase mitochondrial copy number, and enhance the function and metabolic state of cardiomyocytes, thereby achieving a reversible improvement in cardiac function. Delivery vectors and methods commonly used in mammals are well known in the art. The coding sequence of the inactivated telomerase of this invention can be inserted into a viral vector, such as a retrovirus, adenovirus, or adeno-associated virus. In some embodiments, the viral vector is, for example, a lentivirus. Lentiviral virus is a complex retrovirus that, in addition to the common retrovirus genes gag, pol, and env, contains other genes with regulatory or structural functions. Examples of lentiviral vectors include human immunodeficiency virus (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). Lentiviral vectors are generated by multiplex attenuated HIV virulence genes, for example, by deleting genes env, vif, vpr, vpu, and nef, thereby making the vector biosafety compliant. In a preferred embodiment, the viral vector is an adeno-associated virus (AAV) vector. The AAV genome consists of two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). The target gene can be inserted between the ITRs. In generating virus-like particles, co-transfection with a vector containing the viral genome is performed using an helper plasmid containing replication-related genes (E4, E2a, and VA). In a more preferred embodiment, a recombinant AAV vector of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 is used. In the most preferred embodiment, the viral vector is AAV9, thereby targeting the heart.

[0054] In a preferred embodiment, the expression cassette of the inactivated telomerase of the present invention can be inserted between the ITR sequences of a viral vector. The expression cassette includes one or more regulatory sequences: a promoter, a 5'-UTR, and a 3'-UTR. In some embodiments, the promoter may be a tissue-specific promoter, thereby achieving tissue-specific expression of the inactivated telomerase. In other embodiments, the promoter may be a broad-spectrum expression promoter for global regulation. Depending on the vector used, the administration route may be local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, etc. In a preferred embodiment, the inactivated telomerase of the present invention is administered to the subject via coronary injection, particularly via targeted cardiac injection.

[0055] By administering the inactivated telomerase of the present invention, the survival of patients can be prolonged.

[0056] Indications

[0057] HFrEF is mainly caused by myocardial ischemia and cardiomyopathy. Its incidence is high, especially in patients with coronary artery disease, characterized by left ventricular systolic dysfunction, leading to reduced cardiac output and insufficient tissue perfusion. Common causes of HErEF, besides cardiomyopathy and coronary artery disease, include hypertension, valvular heart disease, and others such as diabetes, chronic kidney disease, and congenital heart disease. Clinically, patients often present with decreased exercise tolerance, shortness of breath, and fluid retention. Current treatment strategies for HFrEF include pharmacological therapy (such as ACE inhibitors, beta-blockers, and aldosterone antagonists), cardiac resuscitation therapy (CRT), and left ventricular assist devices.

[0058] Heart failure with a reduced ejection fraction (HFmrEF), or heart failure with an ejection fraction between 40% and 49%, falls between heart failure with a reduced ejection fraction (HFrEF) and heart failure with a preserved ejection fraction (HFpEF). Its causes are diverse, including hypertension, diabetes, coronary artery disease, obesity, and metabolic syndrome. Epidemiological studies of HFmrEF are relatively limited, but data suggest its prevalence is approximately 10-20% of all heart failure patients. The pathophysiological characteristics of HFmrEF are not fully understood, but studies indicate that its pathological mechanisms may partially overlap with those of HFpEF and HFrEF. Its main features include left ventricular diastolic dysfunction and ventricular remodeling, leading to reduced cardiac output and insufficient tissue perfusion. Furthermore, patients with HFmrEF often have left ventricular hypertrophy and fibrosis. The clinical manifestations of HFmrEF are similar to other types of heart failure, with patients often experiencing decreased exercise tolerance, shortness of breath, fatigue, and fluid retention. However, due to the heterogeneity of HFmrEF, its clinical presentation and course can vary considerably. Currently, there are no clear guidelines for the treatment strategy of HFmrEF, and the treatment plans for HFrEF and HFpEF are mostly referenced. Drug therapy includes ACE inhibitors, ARBs, beta-blockers, aldosterone antagonists, and diuretics. In addition, cardiac resynchronization therapy (CRT) and left ventricular assist devices (LVADs) may also be used in some patients.

[0059] Heart failure with ventricular ejection fraction (HFpEF) is more common in elderly women, patients with hypertension, and those with diabetes. HFpEF is characterized by left ventricular diastolic dysfunction, leading to restricted ventricular filling and reduced cardiac output. Clinically, patients often present with shortness of breath, fatigue, and fluid retention, similar to heart failure with ventricular regurgitation (HFrEF), but with a normal ventricular ejection fraction. Currently, treatment for HFpEF primarily focuses on controlling symptoms and managing comorbidities such as hypertension and diabetes.

[0060] Myocardial infarction is mainly caused by coronary artery obstruction and is common in patients with atherosclerosis. Its pathological mechanisms include coronary plaque rupture, thrombosis, and myocardial ischemia and necrosis. The typical clinical manifestation of myocardial infarction is sudden chest pain, often accompanied by sweating, nausea, and dyspnea. Currently, the treatment of acute myocardial infarction includes reperfusion therapy (such as PCI), anticoagulation therapy, and supportive therapy.

[0061] Hypertrophic cardiomyopathy (HCM) is a hereditary disease commonly seen in young athletes. HCM is characterized by abnormal thickening of the ventricular muscle, leading to a reduction in ventricular cavity size and cardiac output. Clinically, patients often present with chest pain, syncope, and arrhythmias; in severe cases, it can lead to sudden death. Current treatments for HCM include beta-blockers, calcium channel blockers, and surgery (such as myocardectomy).

[0062] Dilated cardiomyopathy (DCM) can be caused by genetics, infection, and toxins such as alcohol. DCM is characterized by ventricular enlargement and impaired myocardial contractility, leading to reduced cardiac output and heart failure. Clinically, patients often present with shortness of breath, fatigue, and fluid retention. Current treatments for DCM include pharmacological therapy (such as ACE inhibitors and beta-blockers), cardiac resuscitation (CRT), and heart transplantation.

[0063] Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive inherited myopathy caused by mutations in the Dystrophin gene at X21.1. Mutations in this gene result in the loss of expression of Dystrophin protein, a protein essential for maintaining muscle cell stability. This leads to progressive muscle dysfunction in children with DMD, gradually affecting respiratory and cardiac muscles, eventually progressing to respiratory failure and heart failure. It primarily affects males. DMD is characterized by progressive muscle weakness and atrophy; cardiac involvement can lead to heart failure. Clinically, patients often present with movement disorders, dyspnea, and heart failure. Current treatments for DMD include gene therapy, hormone therapy, and supportive care.

[0064] Restrictive cardiomyopathy (RCM) is relatively rare, and its causes include genetics, amyloidosis, and fibrosis. RCM is characterized by ventricular wall stiffness, leading to restricted ventricular filling and reduced cardiac output. Clinically, patients often present with shortness of breath, fatigue, and fluid retention. Treatment for RCM primarily focuses on symptom management and heart transplantation.

[0065] Secondary cardiomyopathy can be caused by other diseases or conditions (such as hypertension, diabetes, and infections), and its characteristics vary depending on the primary disease, but generally include abnormalities in myocardial structure and function. Patients present with diverse clinical manifestations, depending on the primary disease. Currently, treatment for secondary cardiomyopathy mainly focuses on controlling the primary disease and managing symptoms.

[0066] The double-mutated inactive telomerase of the present invention or a carrier containing the double-mutated inactive telomerase can be used to prepare drugs for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome and / or improving myocardial cell contractility.

[0067] In some embodiments, the present invention relates to the use of double-mutated inactive telomerase or a carrier containing double-mutated inactive telomerase in the preparation of a medicament for treating heart failure. The heart failure may be caused by hypertension, renal failure, aortic stenosis, diabetes, obstructive epicardial coronary artery disease, coronary microvascular dysfunction, chronic kidney disease, chronic obstructive pulmonary disease, or metabolic syndrome.

[0068] Alternatively, the heart failure may be heart failure with reduced ejection fraction or heart failure with preserved ejection fraction. Preferably, the heart failure with reduced ejection fraction may be heart failure with reduced ejection fraction caused by hypertension, heart failure with reduced ejection fraction caused by renal failure, or heart failure with reduced ejection fraction caused by aortic stenosis. The heart failure with preserved ejection fraction may be heart failure with preserved ejection fraction caused by diabetes, heart failure with preserved ejection fraction caused by hypertension, heart failure with preserved ejection fraction caused by obstructive epicardial coronary artery disease, heart failure with preserved ejection fraction caused by coronary microvascular dysfunction, heart failure with preserved ejection fraction caused by chronic obstructive pulmonary disease, heart failure with preserved ejection fraction caused by renal failure, or heart failure with preserved ejection fraction caused by metabolic syndrome.

[0069] In some embodiments, the present invention relates to the use of double-mutated inactivated telomerase or a vector containing double-mutated inactivated telomerase in the treatment of myocarditis. Unwilling to be limited by theory, the double-mutated inactivated telomerase or vector of the present invention can restore or reduce the expression of cardiac anti-inflammatory factors, thereby treating myocarditis.

[0070] In some embodiments, the present invention relates to the use of double-mutated inactive telomerase or a carrier containing double-mutated inactive telomerase in the preparation of a treatment for myocardial infarction. The drug can be used during the acute or subacute phase of myocardial infarction.

[0071] In some embodiments, the present invention relates to the use of double-mutant inactive telomerase or a carrier containing double-mutant inactive telomerase in the preparation of a medicament for restoring or maintaining mitochondrial homeostasis.

[0072] In some embodiments, the present invention relates to the use of double-mutant inactive telomerase or a vector containing double-mutant inactive telomerase in restoring or maintaining mitochondrial homeostasis. In such applications, the double-mutant inactive telomerase or the vector can be administered via coronary injection, intravenous injection, or cardiac-targeted injection.

[0073] The implementation methods described in this article can be illustrated by the following numbered paragraphs:

[0074] 1. A double-mutant inactive telomerase, wherein the double-mutant inactive telomerase has two mutation sites, wherein the first mutation site is Y707 and the second mutation site is D868.

[0075] 2. The double-mutant inactivating telomerase as described in paragraph 1, wherein the double mutation is selected from Y707L and D868V, or Y707F and D868A.

[0076] 3. A vector containing a double-mutant inactive telomerase, the vector containing the coding sequence of the double-mutant inactive telomerase, wherein the double-mutant inactive telomerase has two mutation sites, wherein the first mutation site is Y707 and the second mutation site is D868.

[0077] 4. The vector as described in paragraph 3, wherein the double mutation is selected from Y707L and D868V; or Y707F and D868A.

[0078] 5. The vector as described in paragraph 3 or 4, wherein the vector is a non-viral vector.

[0079] 6. The carrier as described in paragraph 5, wherein the non-viral carrier is selected from cationic lipids, cationic polymers, polypeptides, lipid nanoparticles, inorganic nanoparticles, exosomes, or nucleic acid conjugates.

[0080] 7. The vector as described in paragraph 3, wherein the vector is a viral vector.

[0081] 8. The vector as described in paragraph 7, wherein the viral vector is selected from adeno-associated virus, lentivirus, or adenovirus.

[0082] 9. The vector as described in paragraph 7, wherein the viral vector is a retrovirus.

[0083] 10. The vector as described in paragraph 3, wherein the coding gene is encoded by artificial mRNA.

[0084] 11. The use of the double-mutant inactive telomerase as described in any one of paragraphs 1-2 or the vector as described in any one of paragraphs 3-10 in the preparation of medicaments for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome and / or improving the contractile capacity of myocardial cells.

[0085] 12. The application as described in paragraph 11, wherein the heart failure is selected from heart failure with reduced ejection fraction, heart failure with increased ejection fraction, and heart failure with preserved ejection fraction; preferably, the heart failure with reduced ejection fraction is selected from heart failure with reduced ejection fraction caused by hypertension, heart failure with reduced ejection fraction caused by renal failure, and heart failure with reduced ejection fraction caused by aortic stenosis; preferably, the heart failure with preserved ejection fraction is selected from heart failure with preserved ejection fraction caused by diabetes, heart failure with preserved ejection fraction caused by hypertension, heart failure with preserved ejection fraction caused by obstructive epicardial coronary artery disease, heart failure with preserved ejection fraction caused by coronary microvascular dysfunction, heart failure with preserved ejection fraction caused by chronic obstructive pulmonary disease, heart failure with preserved ejection fraction caused by chronic renal failure, or heart failure with preserved ejection fraction caused by metabolic syndrome.

[0086] 13. The application as described in paragraph 11, wherein the myocarditis is selected from myocarditis associated with the following inflammatory factors: IL-1α, IL-1β, IL-6, IL17A, TGF-β, TNF-α, CCL2, CCL3, CCL4, CCL5, RAGE, CXCL10, CXCL12, ICAM-1, VCAM-1, MMP-9 and / or IL-10.

[0087] 14. The application as described in paragraph 12, wherein the heart failure is selected from heart failure caused by hypertension, heart failure caused by renal failure, heart failure caused by aortic stenosis, heart failure caused by diabetes, heart failure caused by obstructive epicardial coronary artery disease, heart failure caused by coronary microvascular dysfunction, heart failure caused by chronic kidney disease, heart failure caused by chronic obstructive pulmonary disease, and heart failure caused by metabolic syndrome.

[0088] 15. The application as described in paragraph 12, wherein the cardiomyopathy is selected from hypertrophic cardiomyopathy, dilated cardiomyopathy, Duchenne muscular dystrophy, restrictive cardiomyopathy, and secondary cardiomyopathy.

[0089] 16. The use of the double-mutant inactive telomerase as described in any one of paragraphs 1-2 or the vector as described in any one of paragraphs 3-11 in the preparation of a drug for restoring or maintaining mitochondrial homeostasis.

[0090] 17. The application as described in any one of paragraphs 11-16, wherein the drug is administered by local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal injection, preferably coronary injection or intravenous injection.

[0091] 18. The application as described in any one of paragraphs 11-16, wherein the drug is administered via cardiac targeted injection.

[0092] 19. The application as described in any one of paragraphs 11-16, wherein the double-mutant inactive telomerase or the vector is co-administered with Entomo.

[0093] Example

[0094] The experimental method of this invention can be performed using or in accordance with methods known in the art.

[0095] Example 1: Double-mutant inactivating telomerase and JV001 vector

[0096] The gene therapy drug in this invention utilizes a three-plasmid co-transfection of host cells with the target gene GOI, adeno-associated virus Rep&Cap, and helper virus Helper (adenovirus) to complete the replication and packaging of recombinant adeno-associated virus (rAAV). A telomere / telomerase precision regulation technology platform is established, using viral vectors, non-viral vectors, or artificial mRNA to deliver genes for telomere regulatory proteins. This repairs telomeres and achieves a new telomere homeostasis, blocks DNA damage, increases mitochondrial copy number, and enhances the function and metabolic state of cardiomyocytes, thereby achieving a reversible improvement in cardiac function. Specifically, for conditions like heart failure, AAV9 can be used as a vector to specifically deliver the CI-TERT (Catalytically Inactive-Telomerase reverse transcriptase) gene into cardiomyocytes. CI-TERT binds to the damaged telomere tails of cardiomyocytes, upregulating the energy metabolism level of cardiomyocytes, thereby delaying the progression of heart failure, preventing myocardial fibrosis, restoring cardiac function, and improving the patient's quality of life. In this invention, CI-TERT containing the Y707F and D868A mutants is the CI-TERT mentioned in the following examples. In this invention, the gene therapy drug containing these two mutants is abbreviated as JV001 in the following pharmacodynamic verification. The abbreviations of gene therapy drugs containing other mutant combinations will be annotated in the following pharmacodynamic tests.

[0097] A search of the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / gene / ) yielded the TERT gene mRNA sequence (GenBank:mRNA:NM_198253.3). Analysis of this mRNA sequence revealed the coding region sequence of the TERT protein (https: / / www.ncbi.nlm.nih.gov / protein database: protein ID: NP_937983). Using this sequence as a starting point, we designed a target gene plasmid (GOI) containing a double mutation of catalytic inactivation (D868A) and extranuclear phosphorylation (Y707F) to inactivate telomerase. This plasmid was sent to Suzhou Genewise Biotechnology Co., Ltd. for synthesis. We also used AAV9 serotype plasmid and helper plasmids synthesized by Nanjing GenScript Biotechnology Co., Ltd. under contract from Guangzhou Paizhen Biotechnology Co., Ltd. Guangzhou Paizhen Biotechnology Co., Ltd. transformed E. coli JM108 competent cells to prepare the three-plasmids. Finally, Guangzhou Paizhen Biotechnology Co., Ltd. packaged the recombinant AAV virus using a three-plasmid packaging system. The AAV virus was purified and packaged by ultracentrifugation. HEK293 cells were transfected with the GOI plasmid (JV001) helper plasmid and AAV serotype plasmid (AAV Rep and Cap protein expression plasmids). After culturing for 72 hours, the cells and culture supernatant were harvested. The recombinant AAV virus was purified by ultracentrifugation and packaged to obtain the JV001 recombinant virus.

[0098] We also used the same method to construct mature viral particles from inactivated human telomerase that was double-mutated to Y707L and D868V.

[0099] Example 2: The protective effect of JV001 against AngII-induced myocardial dysfunction and its improvement of heart failure in hiPSC-CM.

[0100] Human induced pluripotent stem cells (hiPSCs) were cultured in pre-coated Matrigel (Corning, 356231) six-well plates, using Nutrigel hPSC XF medium (from Biological Industries) to provide necessary nutritional support. During the initial stage of culture, specifically day one, 5 μM of a Rock inhibitor (Y-27632 dihydrochloride, provided by Selleckchem, code S1049) was added to promote stable cell adhesion, and removed after 24 hours by replacing the medium with fresh medium. To guide cell differentiation towards the heart, when the hiPSCs reached 70-90% confluence, a cardiac differentiation program was initiated according to previously reported methods, aiming to cultivate cardiomyocytes (CMs) capable of beating. Specifically, cells were first treated with 4-6 μM CHIR-99021 (Selleck Chemicals, S2924) for two days, followed by two more days of treatment with 5 μM of the Wnt inhibitor IWR-1 (Sigma, I0161). The entire process was performed in RPMI 1640 medium supplemented with insulin-free B27 (Thermo Fisher Scientific, A1895601). On day 5, the medium was replaced with fresh RPMI 1640 medium containing only insulin-free B27 for two days, and then converted to RPMI 1640 medium supplemented with complete B27 until day 10. For purification of the obtained cardiomyocytes, a specially formulated metabolically selective medium was used, consisting of glucose-free RPMI 1640, B27 supplement (Thermo Fisher Scientific, 17504044), and 4 mM sodium lactate (Sigma, 72-17-3). Cardiomyocytes were maintained long-term with regular (every two days) medium changes. After being treated with 10 μM angiotensin II (Ang II) for 24 hours, hiPSC-CM cells were further treated with JV001 or a control vector, and the cells were collected 72 hours later for subsequent experimental studies.

[0101] To analyze the pathway by which JV001 regulates p53, KU-55933 (20 nM, APExBIO, A4605) and Ceralasertib (5 μM, MedChemExpress, 1352226-88-0) were used to inhibit ATM and ATR, respectively. Furthermore, Nutlin-3a (3 μM, MedChemExpress, 675576-98-4) was used to induce p53 activation, further investigating the regulatory effect of JV001 on p53.

[0102] Compared with the control group, Ang II stimulation significantly increased the beating rate of iPSC-CM and reduced the contraction amplitude; JV001 treatment alleviated these changes (Figure 2).

[0103] Figure 2 shows the effects of the control group, the AngII+shelled virus group, and the AngII+JV001 group on cardiomyocyte function. Regarding beat rate, the AngII+shelled virus group had the highest rate, indicating that AngII inhibited myocardial function. However, after treatment with JV001, the beat rate partially recovered, approaching the level of the control group. Simultaneously, the experimental results for contraction velocity showed a similar trend: the control group had the highest contraction velocity, the AngII+shelled virus group showed a significant decrease, and the contraction velocity of the AngII+JV001 group also recovered to a level close to that of the control group.

[0104] Furthermore, the velocity-time curves further illustrate the effect of JV001. The waveforms of the control group exhibited typical myocardial contractile characteristics, with both high amplitude and frequency; the waveforms of the AngII + empty shell virus group showed significantly impaired myocardial function, with significantly reduced amplitude and frequency; while the waveforms of the AngII + JV001 group were more stable and similar to those of the control group. These results indicate that JV001 can effectively improve AngII-induced cardiomyocyte dysfunction and shows significant potential in improving heart failure in the hiPSC-CM model.

[0105] Example 3: Screening for CI-TERT mutants in cardiomyocytes derived from human induced pluripotent stem cells (DCM-hiPSC-CM) with DCM disease.

[0106] To gain a more comprehensive understanding of the potential of CI-TERT mutants in cardiac therapy, we conducted a meticulous screening of CI-TERT mutants in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). The core objective of this experiment was to investigate and verify whether different combinations of CI-TERT mutants could produce similarly significant therapeutic effects. We hope that through this method, we can identify CI-TERT mutant combinations that perform optimally in cardiomyocytes and possess potential therapeutic efficacy, providing new insights and valuable experimental evidence for future treatment strategies for heart diseases. Through this screening process, we expect to discover more effective CI-TERT mutants, thereby advancing the development of cardiac disease treatment.

[0107] Disease-associated human induced pluripotent stem cells (DCM-hiPSCs) were cultured in Matrigel (Corning, 356231)-coated 6-well plates using Nutriristem hPSC XF medium (Biological Industries). 5 μM of the Rock inhibitor (Y-27632 dihydrochloride) was added on day 1 and removed after one day by replacing with fresh medium. For cardiac differentiation, when hiPSCs reached 70–90% confluence, cardiac differentiation to generate beating cardiomyocytes (CMs) was induced, as described in previous literature. Briefly, DCM-hiPSCs were first treated with 4–6 μM CHIR-99021 for 2 days, followed by treatment with the Wnt inhibitor IWR-1 (5 μM) for 2 days, all in RPMI 1640 medium supplemented with B27 and without insulin. On day 5, the culture medium was replaced with fresh RPMI 1640 supplemented with B27 (insulin-free) for 2 days, then replaced with RPMI 1640 supplemented with B27 until day 10. DCM-hiPSC-CM was then purified using a metabolically selective medium consisting of glucose-free RPMI 1640, B27 supplementation, and 4 mM sodium lactate. Cardiomyocytes were maintained for an extended period with the medium changed every two days.

[0108] To perform telomere analysis on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), cells were reseeded onto Matrigel-coated eight-chambered slides and fixed with 4% formaldehyde. Paraffin sections of the heart portion were dewaxed in xylene (Sigma-Aldrich, Missouri, USA) and dehydrated in a series of ethanol solutions. During immunofluorescence in situ hybridization (immuno-FISH), tissues or cells were further blocked using PBS solution containing 20% ​​fetal bovine serum and 0.1% Triton X-100, and then stained with the following pre-diluted primary antibodies: anti-rabbit 53BP1 (1:200, Cell Signaling Technology, 4937S), anti-rabbit phosphorylated histone H2AX (Ser 139) (1:200, Cell Signaling Technology, 9718S), anti-mouse cardiac troponin T (1:400, Abcam, ab8295), anti-mouse TRF2 (1:200, Novus Biologicals, IMG-124A), anti-mouse ACD / TPP1 (1:200, Santa Cruz, sc-100597), and anti-rabbit HA tag antibody (1:200, Cell Signaling Technology, 3724S), and incubated at room temperature for 4 hours. After washing, the sample was incubated with secondary antibody for 1 hour, and then stained with DAPI (4 μg / mL PBS solution) for 15 minutes as a control. It was then washed again, air-dried, and then... Mounting media (Vectorlabs) were used for encapsulation. For cells, confocal image stacks were collected using a Zeiss LSM880 confocal microscope with a 60x objective lens, with a total thickness of 4 micrometers. For tissues, stacks were collected at 1-micrometer intervals, with a total thickness of 5 micrometers, and maximum projection processing was performed using Zen software. Telomere length was determined by PNA signal intensity and quantification using Imaris (Bitplane) software.

[0109] In an experiment screening for CI-TERT mutants in disease-associated induced pluripotent stem cells (DCM-hiPSCs), cardiomyocytes were treated with two different mutant viruses at day 20 of differentiation, with an MOI of 1.0E+5. Samples were collected after 7 days, and the telomere length of the endpoint samples from each group was detected using the Q-FISH method. The effects of the two mutants (Y707F+D868A and Y707L+D868V) were compared with those of the control group (Figure 3, Table 1). The results showed that the control group had a concentrated data distribution and the lowest values, indicating that it had no significant effect on improving cardiomyocyte function. The values ​​of the Y707F+D868A and Y707L+D868V mutants were higher than those of the control group. The telomere length of cardiomyocytes in these two groups was longer than that in the control group, indicating that these two mutant viruses played a protective role in the telomere length of disease-associated cardiomyocytes. This indicates that both the Y707F+D868A mutant and the Y707L+D868V mutant are suitable for improving cardiomyocyte function in the DCM-hiPSC-CM model.

[0110] Table 1 Screening of CI-TERT mutants in DCM-related human induced pluripotent stem cells (DCM-hiPSCs)

[0111] Example 4: In vivo efficacy verification (animal model)

[0112] Eight-week-old male C57BL / 6J mice were provided by GemPharmatech Ltd. All animals were housed at constant room temperature under a 12-hour light / dark cycle and fed a standard rodent diet (Jiangsu Xietong Pharmaceutical Co., Ltd., 1010001). Mice were acclimatized to the laboratory environment for two weeks prior to the start of the experiment. Cardiac dysfunction was induced by Ang II perfusion, transverse aortic vasoconstriction (TAC), or ischemia-reperfusion (I / R). The methods used to establish these models and the measurement methods used in this invention are described in detail below.

[0113] Model 1 uses a subcutaneous osmotic micropump to perfuse angiotensin II (Ang II)

[0114] Mice were randomly assigned to three groups: untreated mice (saline, C57BL / 6J mice n=9); mice perfused with Ang II and injected with AAV9 empty shell virus (Ang II + empty shell virus, C57BL / 6J mice n=6-10); and mice perfused with Ang II and injected with JV001 (Ang II + JV001, C57BL / 6J mice n=10-12). Ang II was perfused via an osmotic micropump (RWD, 1004W) at a dose of 1000 ng / kg / min (Aladdin, 4474-91-3) for 4 weeks. Empty shell virus and JV001 treatment began 10 days after the start of Ang II injections. The injection dose of both empty shell virus and JV001 was 4.0E + 12 vg / kg. After four weeks, echocardiographic analysis was performed. At the end of the experiment, mice were euthanized by cervical dislocation, and their hearts were removed and weighed.

[0115] Model 2: Cardiac remodeling model of aortic arch stenosis (TAC)

[0116] Mice were randomly divided into three groups: untreated mice (sham surgery, C57BL / 6J mice n=6); TAC mice treated with the vector (TAC + empty shell virus, C57BL / 6J mice n=8-10); and TAC mice treated with JV001 (4.0E + 12 vg / kg) (TAC + JV001, C57BL / 6J mice n=8-10). During TAC surgery, mice were anesthetized by intraperitoneal injection of 1.2% 2,2,2-tribromoethanol solution (0.2 mL / 10 g body weight). Mice were placed in a supine position, with an endotracheal tube inserted, and ventilated using a volumetric recirculating rodent ventilator with a tidal volume of 0.4 mL of room air and a respiratory rate of 110 breaths per minute. The thoracic cavity was exposed by incising the proximal portion of the sternum. Isolate the portion between the brachiocephalic artery and the left common carotid artery in the aortic arch, and perform a three-time contraction treatment by tightly binding it with a 25-gauge blunt needle and 7-0 nylon suture to implement TAC.

[0117] The needle was withdrawn immediately after contraction. Mice in the sham-operated group underwent the same surgical procedure, but without aortic contraction. Treatment with the vector and JV001 began 15 days post-surgery. After 8 weeks of echocardiographic analysis, mice were euthanized by cervical dislocation, and their hearts were quickly removed and weighed.

[0118] Model 3 Ischemia / Reperfusion (I / R)

[0119] Eight-week-old male C57BL / 6J mice were randomly assigned to three groups: untreated mice (sham surgery, n=6); ischemia-reperfusion mice treated with a vector (I / R + empty shell virus, n=6); and ischemia-reperfusion mice treated with JV001 (4.0E + 12 vg / kg) (I / R + JV001, n=6). JV001 treatment began one day after the I / R surgery. Mice were anesthetized by intraperitoneal injection of 1.2% 2,2,2-tribromoethanol solution (0.2 mL / 10 g body weight) before the I / R surgery. Mice were placed in a supine position, endotracheal tubes were inserted, and they were ventilated using a volumetric circulatory rodent ventilator with a tidal volume of 0.4 mL of room air and a respiratory rate of 110 breaths per minute. The heart was exposed via thoracotomy, and the pericardium was dissected. The left anterior descending coronary artery (LAD) was wrapped with 5-0 silk suture and tightly secured to the surface of the heart via a small plastic tube. Ischemia was induced by tightly binding the tube for 30 minutes. After 30 minutes, the tube used for myocardial infarction was removed, and the chest cavity was closed.

[0120] Model 4: DEN / CCl4-induced mouse liver injury model

[0121] Mice were treated twice intraperitoneally at 4 and 5 weeks of age with diethylnitrosamine (DEN). Subsequently, starting at 8 weeks of age, they were administered 10% (v / v, dissolved in corn oil) carbon tetrachloride (CCl4) weekly via intraperitoneal injection at a dose of 5 mL / kg until 20 weeks of age. Additionally, JV001 injection (4E+12 vg / kg) was administered via a single tail vein injection during the first CCl4 induction at 8 weeks of age. Liver samples were collected at 20 weeks of age.

[0122] Echocardiographic assessment of cardiac function

[0123] Weekly echocardiographic examinations of cardiac function were performed using a Visual Sonics Vevo 3100 system (Visual Sonics, FUJIFILM) before and 4 weeks after Ang II injection, and 8 weeks after TAC surgery. Briefly, mice were anesthetized with 2% isoflurane, and anesthesia was maintained by keeping the heart rate within the range of 0.5% to 1.0% isoflurane. Systolic function was measured on the long axis of the mid-ventricular segment using M-mode scanning while maintaining a heart rate of 425–475 beats per minute. Diastolic function of the mitral valve was measured using pulsed-wave tissue Doppler in the apical four-chamber field of view while maintaining a heart rate of 325–375 beats per minute. Technical triplet measurements were performed for each animal, and the mean values ​​were calculated. Subsequent statistical analysis was then performed using the mean values.

[0124] Contraction force assessment

[0125] To measure contractile force, video was captured using a fluorescence microscope (OLYMPUS IX83) of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Contraction velocity and frequency were then determined using MATLAB (MathWorks) according to the previously described method. For assessing the contractile force of atrial myocytes (AMCM), the IonOptix HTC system (IonOptix) was used. Briefly, atrial myocytes (AMCM) isolated using the Langendorff method were seeded on imaging dishes coated with laminin (Sigma) in a medium supplemented with DMEM and 10% FBS and incubated at 37°C for 30 min. Cells were subjected to field stimulation at a frequency of 1 Hz (10 V), and changes in sarcomere length were recorded and calculated.

[0126] Histological analysis

[0127] After embedding heart sections in paraffin, hematoxylin and eosin (H&E) staining and Masson's trichrome staining were used to observe cardiomyocyte hypertrophy and fibrosis. Wheat germ lectin (WGA, Solarbio, I3300) staining was used to determine cross-sectional area (CSA), while actin (Proteintech, PF00003) staining was used to determine morphological changes in cardiomyocytes. Additionally, Sirius Red staining was used to assess fibrosis in liver sections. Histological features were observed and captured using an optical microscope (Olympus).

[0128] Immunoblotting

[0129] Proteins were extracted from mouse tissues using radioimmunoprecipitation buffer containing protease and phosphatase inhibitors. The lysis buffer was centrifuged at 12,500 rpm for 15 minutes at 4°C, and the supernatant was collected and transferred to new Eppendorf tubes. Protein concentrations were determined using a BCA kit (Thermo Fisher Scientific, 23227). Protein analysis was performed by Western blotting. The following master antibodies were used: p53 (1:1000, Cell Signaling Technology, 2524S), phosphorylated p53 (Ser15) (1:1000, Cell Signaling Technology, 9284S). Secondary antibodies were then used at a 1:2000 dilution of IgG goat anti-mouse HRP (Proteintech, SA00001-1) or IgG goat anti-rabbit HRP (Proteintech, SA00001-2) as secondary antibodies for 1 hour. Results were visualized using an Amersham Imager 600 (General Electric).

[0130] Coprecipitation and immunoblotting

[0131] For immunoprecipitation, cells were lysed in lysis buffer (0.5% NP-40, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 50 mM Tris, pH 7.8) containing a mixture of protease inhibitors (Roche Applied Sciences) at 4°C for 30 min, and the lysate was collected by centrifugation (14,000 × g, 15 min). The lysate was then processed using Pierce... TM Anti-HA magnetic beads (Thermo Fisher Scientific, 88836) were immunoprecipitated overnight at 4°C. After washing, the lysis buffer was subjected to SDS-PAGE electrophoresis and immunoblotting using the indicated antibody.

[0132] RNA extraction and real-time quantitative PCR (RT-qPCR)

[0133] Total RNA was extracted from tissues using TRIzol (Invitrogen, 15596-026) according to the manufacturer's instructions. II. 100 ng of RNA was used for cDNA synthesis using the 1st Strand cDNA Synthesis Kit (Vazyme, R201-01 / 02). The ChamQ Universal SYBR qPCR Master Mix (Vazyme) was used in… RT-qPCR was performed on a 480II (Roche) scanner. Relative mRNA expression levels were determined by 2... -ΔΔCt The method was used to calculate and normalize the data to GAPDH mRNA. (In ABIQuantStudio) TM Real-time quantitative polymerase chain reaction was performed on a 6Flex machine (Applied Biosystems). OwerUp SYBR Green Master Mix (Applied Biosystems) was used. TM (A25742).

[0134] NT-proBNP and Profiler Mouse XL cytokine array panel

[0135] NT-proBNP was measured according to the manufacturer's instructions (NBP2-76775, Novus). To examine the effects of JV001 on SASP and inflammation, the actual concentrations of SASP inflammatory proteins in mouse model serum and heart tissue were analyzed using an inflammatory cytokine assay panel (RD.ARY028, R&D Systems) according to the manufacturer's instructions.

[0136] Statistical analysis

[0137] All quantitative data are presented as mean ± SEM. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's multiple comparison test. Statistical significance between two groups was determined using a two-tailed Student's t-test. All statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad Software, version 9.0). Statistical significance is expressed as ns, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, respectively.

[0138] 4.1 Echocardiography results showed that mice in the JV001 treatment group had significantly improved EF and FS, and reduced myocardial fibrosis and myocardial hypertrophy.

[0139] When angiotensin II (Ang II)-induced animals entered the heart failure stage (left ventricular ejection fraction (LVEF) decreased by 20%, i.e., day 10), the animals were randomly assigned to groups and intravenously injected with JV001 or empty shell virus (dose 4.0E+12vg / kg). Cardiac function was assessed by echocardiography. Measurements of left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) on days 0, 7, 14, 21, and 28 are shown. Heart weight as a percentage of body weight and heart weight as a percentage of tibia length were measured on day 28.

[0140] Compared to the control group, Ang II-positive animals receiving JV001 showed improved EF and FS (Figure 4). Furthermore, JV001 protected cardiac structures, including heart diameter, volume, ejection volume, and cardiac output, at 28 days. Animals were sacrificed at 28 days for sample collection. CI-TERT mRNA (Figure 5) showed good expression of JV001 in the heart. Complete blood count and blood biochemistry analyses indicated that JV001 treatment did not activate immunogenicity. Ang II injection resulted in increased cardiac hypertrophy indices (heart weight to body weight ratio, HW / BW) and heart weight to tibia length ratio (HW / TL), but these were not observed in JV001-treated animals (Figure 4). In summary, these data suggest that a single intravenous injection of JV001 protects telomeres, leading to durable prevention of ventricular remodeling in Ang II-induced heart failure.

[0141] 4.2 The distribution of JV001 in different organs of the Ang II mouse model and its comprehensive regulation of the inflammatory cytokine spectrum in the heart tissue of heart failure mice, effectively restoring myocardial homeostasis.

[0142] The relative quantification of viral mRNA in different organs was detected using RT-qPCR technology.

[0143] The experimental results (Figure 5) showed the relative quantitative distribution of viral mRNA in five organs—heart, liver, lung, brain, and muscle—in the control group, the AngII+ empty shell virus group, and the AngII+JV001 group. In the control group, mRNA expression in each organ was extremely low or undetectable, reflecting the state of no viral infection under basal conditions. In the AngII+ empty shell virus group, the mRNA levels in each organ remained low, indicating that the empty vector did not significantly alter mRNA expression. In the AngII+JV001 group, mRNA expression in the heart tissue was significantly increased, far exceeding that in other organs, indicating that JV001 can effectively and specifically act on cardiomyocytes, verifying its tissue targeting and efficient delivery capabilities.

[0144] Analysis of AngII- and TAC-induced heart failure models using the Proteome Profiler Mouse XL cytokine array revealed that JV001 treatment significantly reversed abnormal changes in multiple inflammation-related factors. In serum, JV001 treatment significantly downregulated the levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6, restored the expression of the anti-inflammatory factor IL-10, and inhibited the abnormal upregulation of chemokines MCP1 (CCL2) and CCL5. In the TAC model, a significant decrease in the levels of IL-1α and CXCL10 was also observed. In cardiac tissue, key pro-inflammatory factors RAGE, CCL3, and CCL4 in the AngII model significantly recovered after treatment, while the expression of CXCL12 also tended to normalize. In the TAC model, JV001 significantly downregulated the levels of factors such as ICAM-1, VCAM-1, MMP-9, and IL-17A, and effectively inhibited the expression of the fibrosis-related factor TGF-β. Overall, JV001 treatment restored myocardial homeostasis by significantly modulating the systemic and local inflammatory environment, further validating the potential of telomere remodeling for the treatment of heart failure and myocarditis.

[0145] 4.3 Screening for other double-mutant inactive telomerases

[0146] We conducted a systematic TERT mutant sequence screening experiment in an AngII-induced heart failure mouse model. We induced heart failure in mice using Ang II, and performed echocardiography before modeling and on days 7 and 14 post-modeling. On day 18 post-modeling, the mutant combination Y707L+D868V was administered. Cardiac function was assessed by echocardiography two weeks after administration. The results showed that, compared with the model group, the mutant combination significantly increased the left ventricular ejection fraction in mice, indicating that the Y707L+D868V mutant has an effect on improving cardiac function (Figure 6, Table 2).

[0147] Table 2. Left ventricular ejection fraction values ​​in mice after administration of the Y707L+D868V mutant.

[0148] 4.4 In the TAC-induced mouse heart failure model, JV001 was well distributed in the mouse model, and the EF and FS of mice in the JV001 treatment group were significantly improved, and myocardial fibrosis and myocardial hypertrophy were reduced; no significant side effects were found in long-term monitoring of JV001 in the TAC mouse model.

[0149] In this study, C57BL / 6J mice were used as the model animals. The aortic arch constriction model was established to induce transaortic stenosis (TAC). The sham-operated group did not undergo aortic arch ligation, but all other procedures were the same. The experimental model group and the experimental group (JV001) were modeled and enrolled according to standard procedures. The experimental animals were grouped according to specific criteria. Mice in the JV001 group received a single slow intravenous injection of 2.0E + 13 vg / kg. During the experiment, blood flow velocity at the aortic stenosis site and cardiac function parameters (left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), and left ventricular end-systolic diameter (LVESD)) were measured multiple times, and the treatment effect was evaluated at the experimental endpoint (12 weeks after administration).

[0150] As shown in Figure 7, the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) of the model group mice decreased significantly over time, while the left ventricular end-systolic diameter increased significantly, indicating significant deterioration of cardiac function. In contrast, the JV001 group mice showed a significant improvement trend in EF and FS, with EF increasing significantly starting at 4 weeks after administration and recovering by 19.5% at 12 weeks; FS also improved significantly at 4 and 12 weeks. The left ventricular end-systolic diameter (LVESD) in the JV001 group was significantly lower than that in the model group, indicating improved cardiac morphology. Furthermore, Gram staining and Masson's staining showed significant myocardial injury and fibrosis in the model group mice, while the JV001 group showed varying degrees of relief in both aspects. Heart weight to tibia length ratio (HW / TL) analysis further supported the ameliorative effect of JV001 on cardiac hypertrophy. These results collectively confirm that JV001 has a significant protective and ameliorative effect on myocardial injury and cardiac dysfunction induced by the TAC model.

[0151] Long-term monitoring of the TAC mouse model was conducted to evaluate the efficacy and potential side effects of gene therapy. Long-term monitoring results showed that the gene therapy was durable and no significant side effects were observed. In the experiment, we administered JV001 alone and in combination with luteolin, and observed changes in mouse survival rates. The results showed that the survival rate of TAC mice was significantly improved after combination therapy compared to luteolin or JV001 alone. This result strongly suggests that combination therapy may have a superior therapeutic effect in treating TAC-induced heart failure and can effectively improve the survival rate of affected mice (Figure 9).

[0152] 4.5 A single dose of JV001 improved cardiac dysfunction and prevented cardiac fibrosis in ischemia-reperfusion (I / R) mice.

[0153] The Ang II and TAC models represent only non-ischemic heart failure. We replicated the efficacy study in an ischemia-reperfusion model, which represents ischemic heart failure. Consistent with the results of Ang II and TAC, JV001 administered 1 day after ischemia-reperfusion provides cardioprotection.

[0154] Experimental results showed that in the I / R injury model, cardiac function significantly decreased after reperfusion, specifically manifested as a marked reduction in left ventricular ejection fraction (EF) and left ventricular short-axis systolic rate (FS) (EF decreased from 80% to 40%, and FS decreased from 60% to 20%). Simultaneously, left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic diameter (LVIDs) significantly increased (LVEDV increased from 25 μL to 75 μL, and LVIDs increased from 1.5 mm to 3.2 mm). These changes indicate that I / R injury has a significant negative impact on cardiac function.

[0155] However, these negative effects were significantly alleviated when JV001 was used in I / R injury model mice. In the I / R+JV001 group, cardiac function remained relatively stable after reperfusion, with EF increasing to 55% and FS increasing to 30%, while LVEDV decreased to 60 μL and LVIDS decreased to 2.5 mm. These results indicate that JV001 has a significant protective effect on cardiac function.

[0156] Further observation revealed that the I / R group exhibited more severe myocardial fibrosis and cellular structural disorder, while the I / R+JV001 group showed better myocardial structure and cell morphology. Quantitative analysis of cardiomyocyte size revealed that cardiomyocyte hypertrophy was reversed in the I / R+JV001 group, and the cardiomyocyte area was significantly reduced compared to the I / R group (260 μm in the I / R group). 2 I / R+JV001 group 200μm 2Furthermore, compared to the 55% infarct area in the I / R group, the myocardial infarction area in the I / R+JV001 group was significantly reduced to 10%. These results further confirm the positive effect of JV001 in protecting cardiac structure.

[0157] Furthermore, by comparing the Anp and Bnp mRNA expression levels in different treatment groups, it was found that the Anp and Bnp mRNA expression levels in the I / R group were also significantly increased (12 and 6, respectively), while the I / R+JV001 group showed relatively mild increase in heart weight and lower Anp and Bnp mRNA expression levels (4 and 2, respectively). These results further confirm the significant role of JV001 in mitigating the negative effects of I / R injury on cardiac structure and function.

[0158] 4.6 Effect of JV001 on mouse survival

[0159] Nine male C57BL / 6J mice, aged 8 weeks and weighing 22–25 g, were used in this experiment. The experimental design included the construction and evaluation of the TAC model, as well as grouping and drug administration. Mice were anesthetized with intraperitoneal injection of salbutamol and xylazine, followed by thoracic surgery to expose the aortic arch. A suture was threaded between the brachiocephalic trunk and the left common carotid artery, and ligation was performed using a 27G needle to create an aortic stenosis model. The thoracic cavity was sutured layer by layer postoperatively, and mice were monitored after awakening on a heated pad. Baseline assessments included preoperative ultrasound evaluation, blood flow velocity at the aortic arch stenosis site on postoperative day 7, and left ventricular ejection fraction (EF) assessment at week 4. Mice with EF < 45% ± 5% and blood flow velocity at the aortic stenosis site > 3 times the baseline were selected for inclusion in the group. Eligible mice were randomly divided into 4 groups, with 24 unoperated normal mice serving as a blank control.

[0160] Long-term monitoring of a TAC mouse model was conducted to evaluate the sustained efficacy and potential side effects of JV001. Long-term monitoring results showed that JV001 treatment had a durable effect, with no significant side effects, and that JV001 effectively reduced the persistently elevated NT-proBNP levels in TAC mice (Figure 8). In the experiment, we also used JV001 alone and in combination with oxaliplatin, and observed changes in mouse survival rates. The results showed that the survival rate of TAC mice was significantly improved after combination therapy. This result strongly suggests that combination therapy may have a superior therapeutic effect in treating TAC-induced heart failure and can effectively improve the survival rate of affected mice.

[0161] 4.7JV001 has a protective effect against Duchenne muscular dystrophy (DMD)-induced heart failure in mice and improves left ventricular ejection fraction.

[0162] The mouse model of heart failure induced by DMD is a commonly used experimental animal model for studying heart failure caused by human Duke-related muscular dystrophy (DMD). This model typically utilizes a mutation in the Dmd gene in the mouse genome, resulting in a deficiency of the important muscle protein dystrophin, thus mimicking the muscle atrophy and functional impairment experienced by human DMD-induced heart failure patients due to the same gene defect. Analyzing these mouse models allows for a deeper exploration of the pathophysiological mechanisms of DMD-induced heart failure, the development of new treatment strategies, and the evaluation of the efficacy of potential therapeutic drugs, providing important experimental evidence and theoretical support for the treatment of this genetic disease.

[0163] This study uses Mdx 4CV and mTR Het Mdx is obtained through mating 4Cv / mTR Het Mice, Mdx 4Cv / mTR Het Mdx was obtained through multiple generations of self-breeding in mice. 4Cv / mTR KO This refers to the model animal used in the examples. Detailed construction information can be found in the reference [Humanizing the mdx mouse model of DMD: the long and the short of it, https: / / doi.org / 10.1038 / s41536-018-0045-4].

[0164] Seventeen DMDmdx / mTR mice (female:male = 5:12) were randomly assigned to two groups based on body weight and cardiac function: the DMD-Saline group (n=5 females) and the DMD-JV001 group (n=6 males and 6 females). Ten C57BL / 6J mice were also included as... In the DMD-Saline group, mice were given a single dose of saline via tail vein at 8 weeks of age, while mice in the DMD-JV001 group were given a single dose of JV001 injection at a dose of 2.0E+13vg / kg via tail vein. Cardiac function was assessed by echocardiography before administration and on days 16, 30, 44, 58, 95, 130, 159, and 188 after administration. At day 203 after administration, a final dissection was performed, and the heart weight and tibia length were measured. The heart weight to body weight ratio (HW / BW) and the heart weight to tibia length ratio (HW / TL) were calculated to assess the cardiac hypertrophy index.

[0165] In this invention, when DMDmdx / mTR gene mice have just reached sexual maturity and have not yet shown a heart failure phenotype, a single intravenous administration of JV001 can effectively improve various cardiac function indices, prevent further development of heart failure, and protect normal cardiac function (Figure 10, Figure 11, Table 3).

[0166] Table 3 Left ventricular ejection fraction in DMD-induced heart failure model mice after using Y707L+D868V.

[0167] 4.8 Myocardial infarction (MI) model

[0168] The mouse model of myocardial infarction (MI) is an important tool in cardiovascular research, used to simulate the pathophysiological process of myocardial infarction in humans. In this model, myocardial ischemia and infarction are induced by ligation of the left anterior descending coronary artery (LAD) in mice, leading to cardiomyocyte death, inflammation, and fibrosis. This is highly similar to the symptoms and pathological changes that occur after myocardial infarction in humans, such as ventricular remodeling, decreased cardiac function, and heart failure. Therefore, the mouse MI model provides a crucial experimental platform for studying the mechanisms of myocardial infarction, evaluating potential treatment strategies, and developing novel drugs for treating heart disease.

[0169] Forty-five male C57BL / 6J mice were selected as the MI model and randomly divided into four groups according to the drug administration time points: model group, JV001-2h, JV001-D3, and JV001-D7, with enrollment numbers of 11, 10, 11, and 13 mice respectively. Ten additional male C57BL / 6J mice were selected as the model group. The model group served as a blank control. The model group received a single dose of saline via tail vein; the JV001-D7, JV001-D3, and JV001-2h groups received a single dose of JV001 at a dose of 4.0E+12vg / kg via tail vein. Cardiac function was assessed by echocardiography before administration and on days 7, 21, 35, and 63 after administration.

[0170] In this embodiment, C57 / BL / 6J mice were given the same dose of JV001 intravenously 2 hours, 3 days and 7 days after MI surgery. No statistically significant difference was found among the treatment groups, indicating that intravenous injection of JV001 within 7 days after MI modeling can alleviate cardiac function in mice and there is no difference in efficacy (Figure 12).

[0171] 4.9 Heart Failure Preservation of Ejection Fraction (HFpEF) Model

[0172] Mouse models of heart failure with preserved ejection fraction (HFpEF) are important tools for studying the pathological mechanisms and potential treatment strategies of human HFpEF. These models are typically induced through various methods, including high-fat diets, ovariectomy, and / or chronic stress overload (such as aortic coarctation), to mimic key features of human HFpEF, such as myocardial fibrosis, diastolic dysfunction, and cardiac hypertrophy. Despite species differences, mouse HFpEF models share high similarity with human HFpEF in pathophysiology, cardiac structure, and functional changes, providing a valuable research platform for a deeper understanding of the pathogenesis of HFpEF and the development of effective treatments.

[0173] Eight-week-old C57BL / 6J mice (n=50) were randomly divided into an experimental group (n=32) and a control group (n=18). The experimental group was fed a high-fat diet, and L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) was added to their drinking water daily to inhibit nitric oxide synthase, thereby inducing metabolic syndrome and hypertension. The control group was fed a standard diet and ordinary drinking water. After several weeks of treatment, the experimental group mice exhibited cardiac dysfunction, left ventricular hypertrophy, and fibrosis, thus establishing a HFpEF model. At week 8 post-modeling, mice were injected intravenously with JV001 at a dose of 2.0E+13vg / kg (n=16). Echocardiography was performed on all mice at weeks 0, 4, and 9 to assess cardiac function. The groupings were as follows: control group n=18, HFpEF model group n=16, and JV001-treated HFpEF mice n=16.

[0174] In a mouse HFpEF model study, the E / E′ ratio (the ratio of peak mitral valve velocity in early diastole to peak mitral valve annular velocity in early diastole) and the E / A ratio (the ratio of early diastolic peak to atrial systolic peak) were significantly improved, indicating a marked improvement in cardiac diastolic function (Figure 13). Since the E / E′ and E / A ratios are widely used clinically to assess cardiac diastolic function...

[0175] 4.10 Unilateral nephrectomy + Ang II + high-salt diet-induced mouse model of renal failure

[0176] A mouse model of renal failure induced by unilateral nephrectomy combined with angiotensin II (Ang II) and a high-salt diet successfully simulated the pathophysiological features of human heart failure with reduced ejection fraction (HFrEF). This model reproduces the main clinical manifestations of HFrEF by inducing key pathological changes such as renal insufficiency, hypertension, and cardiac remodeling, providing an ideal animal model for in-depth research into the mechanisms of heart failure and its potential treatments.

[0177] The left kidney was removed via a lateral ventricular incision (unilateral nephrectomy) in Ang II + unilateral nephrectomy + high-salt diet (AUH) mice. The control group underwent a sham operation, exposing only the left kidney without removal. One week after the unilateral nephrectomy / sham operation, AUH mice were infused with Ang II (1.2 mg / kg / day) via a subcutaneously implanted osmotic pump. The osmotic pump was replaced every four weeks during the experiment to maintain continuous infusion. Kinin II was dissolved in 0.9% saline. Control mice were implanted with osmotic pumps containing 0.9% saline. Postoperatively, AUH mice were fed a diet containing 1% sodium chloride. Control mice were given a regular diet. Food and tap water were provided without restriction throughout the experiment. Following surgical modeling, mice were injected via tail vein at week 6 with JV001 at a dose of 4.0E+12vg / kg. Cardiac function was assessed by echocardiography at weeks 0, 2, 4, 6, 8, 10, and 12. Grouping information was as follows: sham-operated control group (n=10), JV001-treated sham-operated group (n=9), unilateral nephrectomy control group (CKD group) (n=7), and JV001-treated unilateral nephrectomy group (n=8).

[0178] Echocardiography results showed that the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) were significantly improved in the JV001 treatment group (unilateral nephrectomy + JV001) compared to the model group (Figure 14). These findings indicate that the drug treatment has a significant protective effect on cardiac function in a mouse model of renal failure.

[0179] 4.11 Nephrectomy (5 / 6 nephrectomy) to induce mouse model of renal failure

[0180] The nephrectomy-induced renal failure mouse model is an experimental method used to simulate human heart failure (especially heart failure with reduced ejection fraction, HFrEF). By partially removing the kidneys of mice, researchers can induce chronic kidney disease, leading to renal failure. This model helps to study the interaction between renal and cardiac function, and to explore the mechanisms by which renal insufficiency affects the development of heart failure. This mouse model can provide an important experimental basis for the development of novel treatment strategies and drugs.

[0181] Male C57BL / 6 mice aged 10 to 12 weeks underwent a two-thirds nephrectomy of the left kidney, followed by a total nephrectomy of the right kidney one week later. The control group underwent a sham operation, exposing only the left or right kidney without removal. Following surgical modeling, mice were injected intravenously with 4.0E + 12 vg / kg of JV001 at week 12. Cardiac function was assessed by echocardiography at weeks 0, 2, 4, 6, 8, 10, and 12. Grouping information was as follows: sham operation control group (n=9), JV001-treated sham operation group (n=5), total nephrectomy control group (n=8), and JV001-treated total nephrectomy group (n=9).

[0182] Echocardiography results showed that the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) were significantly improved in the drug-treated group (nephrectomy + JV001) compared to the model group (Figure 15). These findings indicate that the drug treatment has a significant protective effect on cardiac function in a mouse model of renal failure.

[0183] 4.12STZ-induced mouse model of diabetes

[0184] Streptozotocin (STZ)-induced diabetic mouse models are commonly used to study diabetes-related complications, including heart failure. This model mimics the metabolic characteristics of human diabetes in mice, particularly elevated blood glucose and destruction of pancreatic β-cells. Despite differences in physiology and metabolic processes between mice and humans, STZ-induced diabetic mouse models remain valuable for studying diabetes-related heart failure, especially heart failure with preserved ejection fraction (HFpEF). Research on this model can lead to a better understanding of how diabetes contributes to the development of HFpEF and to explore potential therapeutic strategies.

[0185] STZ-induced mouse model of type 1 diabetes

[0186] A common method for inducing type 1 diabetes in mice with streptozotocin (STZ) involves intraperitoneal injection of STZ solution over several consecutive days. First, STZ is dissolved in cold citrate buffer (0.1M, pH 4.5) and injected once daily for 5 consecutive days at a dose of 40-50 mg / kg, depending on the mouse's body weight. Mice are fasted for 4-6 hours before injection but allowed free access to water. Following injection, starting from day 3, tail vein blood glucose levels are measured every 2-3 days. A blood glucose level above 16.7 mmol / L (300 mg / dL) indicates a successful diabetes model. During and after induction, mice should be allowed access to a 10% glucose solution to prevent hypoglycemia. At week 8, mice are administered JV001 via tail vein injection at a dose of 4.0E+12vg / kg. Cardiac function in type 1 diabetic mice is assessed by echocardiography at weeks 0, 4, 8, 10, 12, 14, and 16. The grouping information is as follows: control group n=6, type 1 diabetes model group n=6, and type 1 diabetes group treated with JV001 n=6.

[0187] The experimental results showed that after drug intervention, the E / E′ and E / A ratios measured by echocardiography were significantly improved compared with the model group (Figure 16). Specifically, the results of the mouse model showed that the E / E′ ratio of mice decreased significantly and the E / A ratio increased significantly after drug treatment, indicating a significant improvement in left ventricular diastolic function. These results indicate that drug intervention effectively improved cardiac dysfunction in the mouse model.

[0188] STZ-induced mouse model of type 2 diabetes

[0189] A common method for inducing type 2 diabetes in mice is to combine a high-fat diet (HFD) with low-dose streptozotocin (STZ) injections. First, mice are fed a high-fat diet (containing 45%-60% fat) for 8-12 weeks to induce insulin resistance. Subsequently, a low-dose STZ (e.g., 100 mg / kg body weight) is administered intraperitoneally to damage pancreatic β-cells. Before injection, the STZ is dissolved in cold citrate buffer (0.1 M, pH 4.5) and prepared fresh. This method successfully establishes a mouse model of type 2 diabetes for studying metabolic disorders and related treatments. At week 16, mice are administered JV001 at a dose of 4.0E+12 vg / kg via tail vein injection. Cardiac function in the type 2 diabetic mice is assessed by echocardiography at weeks 0, 4, 8, 12, 16, 18, 20, 22, and 24. The grouping information is as follows: control group n=8, type II diabetes model group n=8, and type II diabetes group treated with JV001 n=8.

[0190] Experimental results showed that cardiac function in mice was significantly improved by echocardiography after drug intervention, particularly in the E / E′ and E / A ratios (Figure 17). Specifically, drug treatment significantly decreased the E / E′ ratio and significantly increased the E / A ratio in the mouse model, indicating a significant improvement in left ventricular diastolic function. These findings suggest that drug intervention effectively alleviated cardiac dysfunction in the mouse model. Based on these results in the mouse model, it can be inferred that the drug may have similar effects in humans, potentially improving diastolic function and reducing the risk of heart failure and other heart-related diseases.

[0191] 4.13MYH6 R404Q mouse model

[0192] The MYH6 R404Q mouse model is a commonly used animal model for studying hypertrophic cardiomyopathy (HCM). In this model, the R404Q mutation in the MYH6 gene leads to abnormal proliferation and hypertrophy of cardiomyocytes in mice, resulting in myocardial hypertrophy. This mutation is also associated with HCM in humans, where patients typically exhibit left ventricular wall thickening, abnormal cardiac function, and even heart failure. By studying the MYH6R404Q mouse model, scientists can gain a deeper understanding of the pathogenesis of hypertrophic cardiomyopathy and its potential treatments, providing more effective treatment strategies for human HCM patients.

[0193] The mouse MYH6R404Q corresponds to the human MYH7R403Q (location on the human genome). 1) MYH6R404Q mice were injected with JV001 at week 8, and cardiac function was assessed by echocardiography at weeks 8, 12, 14, 16, 18, and 20. The grouping information was as follows: control group n=7, MYH6R404Q model group n=7, and JV001-treated MYH6R404Q group n=7. 2) MYH6R404Q mice were injected with JV001 at week 20, and cardiac function was assessed by echocardiography at week 32. The grouping information was as follows: control group n=8, MYH6R404Q model group n=8, and JV001-treated MYH6R404Q group n=8.

[0194] 1) JV001 was administered at a dose of 4.0E+12vg / kg at week 8, and the observation continued until week 20: After administration, the end-systolic and end-diastolic diameters of the left ventricle in hypertrophic cardiomyopathy (HCM) mice decreased, and the thickness of the left ventricular posterior wall also decreased at both end-systole and end-diastole (Figure 18). These changes indicate that the drug effectively alleviated myocardial hypertrophy and improved cardiac structure and function.

[0195] 2) JV001 was administered at a dose of 4.0E+12vg / kg at week 20, and echocardiography was performed on mice at week 32. After JV001 treatment, the end-systolic and end-diastolic diameters of the left ventricle in hypertrophic cardiomyopathy (HCM) mice were significantly reduced, and the thickness of the posterior wall of the left ventricle was also significantly thinned at both ends of systole and diastole (Figure 19). These significant changes fully demonstrate the powerful effect of the drug in reducing myocardial hypertrophy, and the structure and function of the heart were significantly improved.

[0196] 4.14 Comparison of mutant protein structure simulation

[0197] 4.15 Study on route of administration

[0198] Under the conditions of this experiment, after a single intravenous injection / coronary intervention in Bama miniature pigs with a dose of 2.0E+12vg / kg of JV001 injection, JV001 was mainly distributed in the liver, spleen, and blood. After intravenous injection and coronary intervention, except for the epididymis in the vein, JV001 was expressed to varying degrees in all other organs. The expression of mRNA in the blood was significantly higher than that in the tissues after both administration methods. The exposure level and expression of JV001 in the blood were higher after coronary intervention compared to intravenous injection. Both administration methods resulted in the shedding of JV001 virus through the digestive system.

[0199] 4.16JV001 Off-target toxicity assessment in the non-target organ liver

[0200] Nine male C57BL / 6J mice were randomly divided into three groups: a control group, a model group, and a JV001 group, with three animals in each group. The model group and the JV001 group were induced to develop liver fibrosis through intraperitoneal injection of DEN / CCl4. The modeling method was as follows: DEN was injected intraperitoneally once a week for two consecutive weeks starting at 6 weeks of age, followed by CCl4 injections once a week from 8 to 20 weeks of age. Additionally, JV001 injection solution (4E+12vg / kg) was injected intravenously via the tail vein during the first CCl4 induction at 8 weeks of age. Liver samples were collected from each group at 20 weeks of age for Sirius red staining to assess the degree of liver fibrosis; and the protein expression levels of p53 and p-p53 in the mouse liver were detected using Western blotting.

[0201] Sirius red staining results showed that the livers of both the model group and the JV001 group developed fibrotic lesions, while the degree of liver fibrosis in the JV001 group after injection was not significantly different from that in the model group. Western blotting showed no difference in p53 protein band signals in liver tissue among the groups; however, compared with the control group, the p-p53 protein band signals in the liver tissue of the model group and the JV001 group were upregulated, and there was no difference in p-p53 protein band signals between the JV001 group and the model group.

[0202] The results (Figure 20) showed that a single tail vein injection of JV001 at a dose of 4E+12vg / kg did not affect the activation of p53 in DEN / CCl4-induced damaged mouse livers. Phosphorylated p53 cells in the damaged apoptosis pathway were normally activated, suggesting that JV001 expressed the target protein in non-target organs without producing the expected biological effects. This indicates a low off-target risk associated with JV001 expressing the target protein in non-target organs.

Claims

1. A double-mutant inactive telomerase, wherein the double-mutant inactive telomerase has two mutation sites, wherein, The first mutation site is Y707, and the second mutation site is D868.

2. The double-mutant inactivating telomerase as described in claim 1, wherein, The first mutation site is Y707F, and the second mutation site is D868A.

3. The double-mutant inactivating telomerase as described in claim 1, wherein, The first mutation is Y707L, and the second mutation is D868V.

4. A vector containing a double-mutant inactive telomerase, said vector containing the coding sequence of the double-mutant inactive telomerase, wherein, The double-mutated inactive telomerase has two mutation sites, the first mutation site being Y707 and the second mutation site being D868.

5. The carrier as described in claim 4, wherein, The first mutation is Y707L and the second mutation is D868V; or the first mutation is Y707F and the second mutation is D868A.

6. The use of the double-mutant inactive telomerase of any one of claims 1-3 or the carrier of any one of claims 4-5 in the preparation of a medicament for treating myocardial fibrosis, heart failure, cardiomyopathy, myocardial infarction, myocarditis, renal-cardiac syndrome, cardio-renal syndrome and / or improving the contractile capacity of myocardial cells.

7. The use of the double-mutant inactive telomerase of any one of claims 1-3 or the vector of any one of claims 4-5 in the preparation of a drug for restoring or maintaining mitochondrial homeostasis.

8. The application as described in claim 6 or 7, wherein, The drug is administered via local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intravenous, or intraperitoneal injection.

9. The application as described in claim 6 or 7, wherein, The drug is administered via targeted injection into the heart.

10. The application according to claim 6 or 7, wherein, The double-mutant inactivating telomerase or the vector is co-administered with Entomologous.

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