Application of TRIM28-ERVs pathway negative regulator in treating myocarditis and heart failure

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a TRIM28-ERVs pathway negative regulator in treatment of myocarditis and heart failure. According to the invention, the important effect of the TRIM28-ERVs pathway negative regulator in treating myocarditis and heart failure is found for the first time, so that a new medicine for treating myocarditis and heart failure can be found.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedical technologies, and more particularly, to the use of negative regulators of the TRIM28-ERVs pathway in the treatment of myocarditis and heart failure. Background Art

[0002] Heart failure is a prevalent syndrome characterized by the heart's inability to pump enough blood to meet the body's needs. Heart failure is a major cause of morbidity and mortality worldwide, affecting over 64 million people globally. Due to the lack of effective treatment methods, the 5-year mortality rate of heart failure exceeds 50%, which is worse than most types of cancer. Acute heart failure (AHF) is the most dangerous subtype of heart failure, characterized by the sudden onset of heart failure or the deterioration of pre-existing heart failure, with a 1-year mortality rate of approximately 30%. Myocarditis, especially fulminant myocarditis, is a common cause of AHF, usually caused by exogenous viral infections such as HIV and COVID-19, but the role of endogenous viruses, a type of provirus embedded and integrated in the host genome, in heart failure remains unknown.

[0003] The mammalian genome contains a large number of transposable elements, which are classified as DNA transposons and retrotransposons based on their intermediates. Retrotransposons without long terminal repeats (LTRs) include autonomous long interspersed elements (LINEs) and non-autonomous short interspersed elements (SINEs). Retrotransposons with LTRs are mainly ERVs, accounting for approximately 8% of the human genome and 10% of the murine genome. ERVs contain four basic viral packaging genes (gag, pol, env, and pro), and are surrounded by 5' and 3' untranslated regions (UTRs) on both sides, which is similar to exogenous retroviruses and can be packaged into active viral particles. Human and rodent endogenous retroviruses are classified into three classes based on their clustering with lentiviruses, retroviruses, and spumaviruses: class I (ERV1), class II (ERVK), and class III (ERVL). Some studies have investigated the expression of various ERV populations in diseased tissues, but no clear correlation has been established between specific ERV subtypes and human diseases.

[0004] To maintain genomic stability, ERVs are silenced by various epigenetic modification mechanisms. In various disease states such as cancer and neurodegenerative diseases, ERVs have been reported to reactivate, but the underlying molecular mechanisms are unclear. Whether abnormal activation of ERVs occurs in other types of pathological processes, especially in cardiovascular diseases and heart failure, remains a mystery. Summary of the Invention

[0005] The present disclosure encompasses the following technical solutions:

[0006] (1) Use of a negative regulator of the TRIM28-ERVs signaling pathway in the preparation of a medicament for treating myocarditis and / or heart failure.

[0007] (2) The use according to (1), wherein the negative regulator comprises an inhibitor of ERVs, which inhibits the resurrection of ERVs or reduces the bioavailability of resurrected ERVs.

[0008] (3) The use according to (2), wherein the ERVs are ERV1.

[0009] (4) The use according to (3), wherein the ERVs are MMVL30-int and / or RLTR6_Mm.

[0010] (5) The use according to any one of (2) to (4), wherein the inhibitor comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozymes, which are designed to reduce or block the expression of the ERVs.

[0011] (6) The use according to any one of (2) to (4), wherein the inhibitor reduces or blocks the expression of the ERVs through epigenetic modification.

[0012] (7) The use according to any one of (2) to (4), wherein the inhibitor comprises a reverse transcriptase inhibitor, which reduces or blocks the expression of the ERVs.

[0013] (8) The use according to (7), wherein the reverse transcriptase inhibitor comprises one or more of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, and abacavir.

[0014] (9) The use according to any one of (2) to (4), wherein the inhibitor knocks down or knocks out the ERVs gene or the gene regulating the expression of the ERVs by ZFN technology, TALEN technology, or CRISPR technology.

[0015] (10) The use according to (1), wherein the negative regulator comprises an agonist of TRIM28, which increases the amount or activity of TRIM28 protein in host cells.

[0016] (11) The use according to (10), wherein the agonist is a vector capable of expressing TRIM28 protein in host cells, which comprises a polynucleotide capable of encoding TRIM28.

[0017] (12) The use according to (11), wherein the agonist is a viral vector.

[0018] (13) The agonist according to the application described in (12) is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector or a retrovirus vector.

[0019] (14) The AAV vector according to the application described in (13) is selected from the group consisting of: AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, AAVDJ, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB and Anc80.

[0020] (15) The agonist according to the application described in (10) is formulated in the form of a delivery system; the delivery system comprises a polynucleotide capable of encoding TRIM28, and a delivery vehicle.

[0021] (16) The delivery vehicle according to the application described in (15) includes one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more inorganic nanoparticles, one or more cell-penetrating peptides, a gene gun, one or more plasmids, and the group composed of them.

[0022] (17) The negative regulator according to any one of (1) to (16) is formulated alone or in combination with other drugs.

[0023] (18) The negative regulator according to any one of (1) to (17) forms a pharmaceutical composition with a pharmaceutically acceptable excipient.

[0024] (19) The form of the pharmaceutical composition according to the application described in (18) is selected from one or more of solutions, injections, sprays, nasal drops, aerosols, powder aerosols, tablets, capsules and granules.

[0025] (20) The cardiomyopathy according to any one of (1) to (19) is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy or specific cardiomyopathy.

[0026] (21) The heart failure according to any one of claims (1) to (20) is heart failure with preserved ejection fraction or heart failure with reduced ejection fraction.

[0027] (22) The patient of the drug according to any one of claims (1) to (21) is a mammal.

[0028] (23) For the application according to (22), the patient of the drug is a primate.

[0029] (24) For the application according to (23), the patient of the drug is a human.

[0030] (25) For the application according to (1), the negative regulator does not specifically bind to and regulate any one of TLR7, TLR9, NFKB.

[0031] Beneficial effects:

[0032] The present disclosure has for the first time discovered the important role of the negative regulator of the TRIM28-ERVs pathway in the treatment of myocarditis and heart failure, thereby enabling the search for new drugs for the treatment of myocarditis and heart failure. Description of the drawings

[0033] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 : Reactivation of retroviral proviruses in heart failure (HF). a. Box plot showing increased expression of HERV (human endogenous retrovirus), especially HERV1, in human heart failure hearts. 7 control subjects and 14 heart failure patients. b. Heat map showing significant upregulation of HERV genes in human heart failure hearts. Data cited from the GEO database (GSE135055). 7 control subjects and 14 heart failure patients. c. P-adj value ranking showing that HERV1 is the most significantly changed in human heart failure hearts. The dots indicate: P-adj = 0.05. LTR61, LTR25, LTR30, LTR24C of HERV1 are ranked in the top ten in red. d. Box plot indicating upregulation of ERV (endogenous retrovirus), especially ERV1, after myocardial ischemia-reperfusion (I / R) in mice. e. Heat map showing ERV reactivation one day after myocardial ischemia-reperfusion in mice. N = 3 mice per group. f. P-adj value ranking suggesting that ERV1 is the most significantly changed in the hearts of mice with myocardial ischemia-reperfusion. MMVL30-int, RTL6_Mm, RTL6-int, RTL6C_Mm, MuLV-int, RTL30D_RN of mouse ERV1 are ranked in the top ten in red.

[0035] Figure 2: Myocardial cell-specific deletion of TRIM28 leads to ERV reactivation and heart failure. a, b. Immunohistochemistry validates the depletion of TRIM28 in human DCM hearts. TRIM28 signals (a) are quantified using ImageJ (b). N = 3 hearts per group. Mean ± SD, two-tailed Student's t-test. c. RNA-seq shows ERV reactivation in TRIM28iCKO hearts. The box plot on the right shows the highest increase in RTL6-Mm and MMVL30-int expression levels (right). d. P-value ranking indicates that ERV1 shows the most significant changes in TRIM28iCKO hearts. The dotted line indicates: P-adj = 0.05. MMVL30-int, RLTR6_Mm, RLTR6-int, RLTR6C_Mm, RLTR30D_RN are among the top ten ranked in red. e. Structure of the MMVL30 provirus in the mouse genome. LTR: Long terminal repeat; Gag, Pol proteins are used for virus packaging. f. RNA-FISH shows the activation of MMVL30 in TRIM28iCKO cardiomyocytes. The intensity of MMVL30 (left) is quantified using ImageJ (right). N = 3 hearts per group, Mean ± SD, two-tailed Student's t-test. g. Survival curve of TRIM28iCKO mice. N = 18 - 22 mice per group. Statistical significance is calculated using the log-rank test. P < 0.05 indicates significance. h - j. M-mode echocardiography shows a decrease in ejection fraction (EF, h), an increase in left ventricular end-diastolic diameter (LVIDd, i), and a decrease in left ventricular posterior wall thickness at end-diastole (LVPWd, j) in TRIM28iCKO mice. N = 10 mice per group. Mean ± SD, two-tailed Student's t-test. k. Ratio of heart weight (HW) to tibia length (TL). N = 4 - 7 mice, Mean ± SD, two-tailed Student's t-test. l. Dissection shows a significantly enlarged heart in TRIM28iCKO mice. m, n. WGA staining shows an increase in cardiomyocyte size in TRIM28iCKO hearts. The area of cardiomyocytes (m) is calculated using ImageJ (n). N = 110 - 118 cardiomyocytes from 3 measured hearts. Mean ± SD, two-tailed Student's t-test. o. Hematoxylin and eosin (H&E) histology shows dilated cardiomyopathy. p. H&E histology shows sarcomere misalignment, cell death, and inflammatory cell infiltration in TRIM28iCKO hearts. q. Trichrome staining shows an increase in collagen deposition (blue area) in TRIM28iCKO hearts. r. Trichrome staining reveals an increase in collagen deposition in TRIM28iCKO hearts. N = 6 mice. Mean ± SD, two-tailed Student's t-test.

[0036] Figure 3: TRIM28 controls ERV activation by manipulating H3K9me3 and m6A. a-c. ChIP-seq results show that TRIM28 knockout leads to a decrease in H3K9me3 signals of some upregulated ERVs. d. Signals of H3K9me3, RNA, and MeRIP-seq at the upregulated MMVL30-int_dup726 genomic locus. TRIM28 knockout results in a decrease in H3K9me3 signals at this ERV locus without affecting m6A, suggesting that TRIM28 inhibits ERV activation by promoting H3K9me3 modification at ERV loci. e. Signals of H3K9me3, RNA, and MeRIP-seq at the upregulated MMVL30-int_dup810 genomic locus, indicating that TRIM28 knockout leads to a decrease in m6A signals at this ERV locus without affecting H3K9me3. f. TRIM28 knockout results in a decrease in m6A signals of upregulated ERVs. g. TRIM28 knockout results in a decrease in m6A signals of upregulated MMVL30 in the ERV1 family. h. Gene overlap analysis shows that m6A modification and H3K9me3 marks affect the upregulation of different groups of ERVs in TRIM28 knockout respectively. i, j. Co-immunoprecipitation experiments show that TRIM28 can interact with the m6A methyltransferase METTL3. k. RNA immunoprecipitation experiments show that TRIM28 deletion inhibits the binding of METTL3 to MMVL30, RLTR6, and MuLV in upregulated ERVs. l. RT-qPCR experiments show that overexpression of METTL3 can inhibit the expression of MMVL30, RLTR6, and MuLV in TRIM28 knockout cardiomyocytes, suggesting that METTL3 can inhibit ERV activation by m6A-modifying ERVs.

[0037] Figure 4:The revival of ERVs leads to myocarditis and heart failure by activating TLR7 / 9. a. GO analysis of upregulated differentially expressed genes (upDEGs). b-e. GSEA analysis of gene enrichment in the TLR (b), cGAS (c), NFKB (d), and IFN (e) nucleic acid sensing pathways. FDR: false discovery rate; NES: normalized enrichment score. There was significant gene enrichment in the TLR, cGAS, and NFKB pathways, but not in the IFN pathway. f. Heatmap of DEGs in the cardiac hypertrophy, nucleotide sensing, and NFKB pathways, but not in the IFN pathway. g. Western blot showed activation of the PRR (cGAS, TLR7 / 9, MyD88, TAK1, p-TAK1) and NFKB (p-P65, P65) pathways, but not the IFN (p-IRF7, IRF7) pathway. h. RT-qPCR indicated that MMVL30 siRNA inhibited the activation of the TLR7 / 9 and cGAS pathways in TRIM28iCKO cardiomyocytes. Mean ± SD, n = 6. One-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. i-k. Immunohistochemistry revealed infiltration of CD68+ macrophages and CD3+ lymphocytes (i, arrows). The intensity of CD68+ macrophages (j) and CD3+ lymphocytes (k) was calculated using ImageJ. Mean ± SD, CD68+ n = 6 hearts, CD3+ n = 5 hearts, two-tailed Student's t test.

[0038] Figure 5:Blocking the ERVs-TLR-NFκB pathway improved myocarditis and cardiac dysfunction in TRIM28iCKO mice. a. Administration strategies of different small molecule inhibitors. NSC4375 (20 mg / kg), JSH23 (6 mg / kg), and C176 (8 mg / kg) as well as vehicle (5% DMSO + 40% PEG300 + 5% Tween80 + 50% ddH2O) were administered by intraperitoneal injection, while RTi (zidovudine) was administered via drinking water. b. Survival rates of TRIM28iCKO mice with or without inhibitor treatment. n = 16 - 39 mice. Statistical significance was calculated using the log-rank test. P < 0.05 indicates significance. c - e. Echocardiography showed that NSC4375, JSH23, and RTi significantly alleviated pathological remodeling of the TRIM28iCKO heart. NSC4375, JSH23, and RTi but not C176 improved the systolic function (c) of TRIM28iCKO mice, reduced LVIDd (d), and increased LVPWd (e). Mean ± SD, n = 8 - 9 mice, one-way ANOVA followed by Tukey's post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference. f. The HW / TL ratio verified that NSC4375, JSH-23, and RTi but not C176 reduced the heart mass of TRIM28iCKO mice. Mean ± SD; n = 7 mice; one-way ANOVA followed by Tukey's post hoc test. g. M-mode echocardiogram of TRIM28iCKO mice after 9 days of inhibitor treatment. h. Dissection (upper) and H&E staining (lower) revealed that NSC4375, RTi, and JSH23 improved dilated cardiomyopathy of the TRIM28iCKO heart. i. Immunohistochemistry showed that NSC4375, JSH23, and RTi alleviated infiltration of CD68+ macrophages.

[0039] Figure 6:Suppression of ERV reactivation alleviates heart failure. a. Western blot showed that AAV9-TRIM28 inhibited the activation of TLR7 / 9 and NFKB pathways in the I / R heart. b. Administration strategy of AAV9-TRIM28. 8- to 10-week-old mice were intrapleurally injected with 2.5X10^11 vg AAV9-CTR and AAV9-TRIM28 7 days before I / R. c. AAV9-TRIM28 inhibited the expression of ERV1 and cytokines in the I / R heart by RT-qPCR. N = 6. Mean ± SD, one-way ANOVA followed by Tukey's post hoc test. d. Evans blue / TTC (triphenyl-2H-tetrazolium chloride) staining showed that AAV9-TRIM28 reduced the infarct area. Blue: non-ischemic area; Red: ischemic area or at-risk area; White: infarct area. e, f. Calculation of infarct area. IF: infarct, AAR: at-risk area, LV: left ventricle. The infarct area (IF / AAR) of the AAV9-TRIM28-treated heart was smaller than that of the control group. n = 6 mice, Mean ± SD, two-tailed Student's t-test, **P < 0.01, ns: not significant. g. Echocardiography showed that AAV9-TRIM28 significantly improved the systolic function of the I / R heart. EF was measured continuously for three weeks. n = 8 mice. Mean ± SD, two-way ANOVA followed by Sidak's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. h, i. Trichrome staining showed that AAV9-TRIM28 improved scar formation three weeks after injection. The ratio of the fibrotic area (blue) to the total area (blue + red) was calculated using Image J. n = 6 mice. Mean ± SD, one-way ANOVA followed by Tukey's post hoc test. ***P < 0.001.

[0040] Figure 7: Inhibition of ERV-mediated innate inflammation alleviates heart failure. a. Administration strategy of NSC4375. NSC4375 at 20 mg / kg was intraperitoneally injected three times a week for 2 days before I / R surgery. b. Western blot showed that NSC4375 inhibited the activation of TLR7 / 9 and NF-κB pathways in I / R hearts. c. RT-qPCR showed that NSC4375 inhibited the activation of cytokines rather than ERV1 in I / R hearts. Mean ± SD, n = 6, one-way ANOVA followed by Tukey's post hoc test. d. Evans blue / TTC staining showed that NSC4375-treated I / R mice had a reduced infarct area. e, f. Infarct area was calculated. The infarct area (IF / AAR) of NSC4375-treated hearts was smaller compared with the control group. n = 6 mice, Mean ± SD, two-tailed Student's t-test, *P < 0.05, ns: not significant. g. Echocardiography showed that NSC4375 significantly improved the systolic function of I / R hearts. PBS was used as a control. n = 8 mice. Mean ± SD, two-way ANOVA followed by Sidak's post hoc test. **P < 0.01, ***P < 0.001. h, i. Trichrome staining revealed that NSC4375 reduced fibrosis in I / R hearts. Mean ± SD, n = 5 hearts, two-tailed Student's t-test.

[0041] Figure 8 : MMVL30 RTE activates the TLR-NF-κB pathway. a. RT-qPCR showed that MMVL30 transduction activated the TLR7 / 9 and CGAS pathways in cardiomyocytes. Mean ± SD, n = 6, two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. b. LDH content in the sera of WT and TRIM28 iCKO mice. Mean ± SD, n = 4 mice, two-tailed Student's t-test.

[0042] Figure 9 : AAV-TRIM28 alleviates heart injury and heart failure in I / R mice. a. In I / R hearts, the fold ranking showed that RTEs related to ERV1 increased most significantly. The dotted line indicates: log2(fold change) = 0.585. The top ten of red ERV1 were RLTR6C_Mm, LTR45C, MMVL30-int, RLTR6_Mm, MER50B, LTR34, LTR58. b. RNA-seq heatmap showed increased cytokine expression in I / R hearts. N = 3 per group. c. AAV9-TRIM28 decreased serum LDH. N = 6 mice. Mean ± standard deviation, one-way ANOVA followed by Tukey's post hoc test. **p < 0.01, **p < 0.001. Detailed implementation manners

[0043] Reference will now be made in detail to embodiments of the disclosure, one or more examples of which are described below. Each example is provided by way of explanation, and not limitation, of the disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the disclosure without departing from the scope or spirit of the disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0044] Unless otherwise noted, all terms used to disclose the disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Through further guidance, the following definitions are used to better understand the teachings of the disclosure. The terms used in the specification of the disclosure herein are for the purpose of describing particular embodiments only and are not intended to limit the disclosure.

[0045] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures used herein are terms and conventional procedures widely used in the respective fields. At the same time, to better understand the present disclosure, definitions and explanations of relevant terms are provided below.

[0046] As used herein, the term "and / or", "or / and", "and / or" includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, said any and all combinations including any two of the related listed items, any more of the related listed items, or all of the related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in the present disclosure, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, includes any combination of two or three of A, B, C, D, and also includes the four-item combination of A, B, C, D (that is, the technical solution connected by "logical AND").

[0047] As used in this disclosure, the terms "comprising", "including" and "containing" are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0048] Numeric ranges represented by endpoints in this disclosure include all the values and fractions contained within the range, as well as the recited endpoints.

[0049] As used herein, the mention of "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, the description of "about X" includes the description of "X".

[0050] In this disclosure, when referring to concentration values, their meanings include fluctuations within a certain range. For example, it can fluctuate within the corresponding precision range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger values or values that do not require overly precise control, larger fluctuations are also allowed. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. When referring to molecular weight, fluctuations within the range of ±10% are allowed.

[0051] As used herein, unless otherwise indicated, the singular forms of the articles "a", "an" and "the" include plural referents.

[0052] In this disclosure, descriptions such as "a plurality of" and "a variety of", unless otherwise specified, refer to a quantity greater than or equal to 2.

[0053] In this disclosure, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the recited features, as well as an open technical solution containing the recited features.

[0054] In this disclosure, "preferred", "better", "more preferred", "preferably" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this disclosure. In this disclosure, "optionally", "optional", "optional" mean that it can be either present or absent, that is, it refers to any one of the two alternative options of "present" or "absent". If "optional" appears multiple times in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent.

[0055] In this disclosure, the term "TRIM28-ERVs signaling pathway" or "TRIM28-ERVs pathway" refers to the signaling pathway that regulates the reactivation of ERVs through TRIM28. A "negative regulator of the TRIM28-ERVs signaling pathway" refers to a substance that can inhibit the reactivation of ERVs or directly reduce / block the biological activity caused by their reactivation.

[0056] In the present disclosure, the term "inhibitor" refers to a molecule that partially or completely inhibits the effects of other molecules by any mechanism. An inhibitor of ERVs refers to a substance that can inhibit the reactivation of ERVs or reduce the bioavailability of reactivated ERVs. As is known to those skilled in the art, an inhibitor can inhibit a molecule at the genomic level, transcriptional level, translational level, post-translational modification level, etc., as long as it can reduce the content of the functional molecule. Examples of post-translational modification include the maturation of gene products or proteins, post-translational modifications (e.g., glycosylation, disulfide bonding, myristoylation, protease cleavage, binding to other proteins, ubiquitination, etc.). The processing, transport, and release of proteins may also be modified, for example, by being placed in storage organelles before release, by binding to other proteins that affect release, etc.

[0057] In the present disclosure, the term "agonist" refers to a molecule that activates the effects of other molecules by any mechanism. An agonist of TRIM28 is a substance that increases the bioavailability of TRIM28 protein in host cells, including increasing the quantity or activity of TRIM28 protein. In some examples, an agonist of TRIM28 can activate TRIM28 by phosphorylation. In some examples, an agonist of TRIM28 is an exogenously administered TRIM28 protein. In some examples, an agonist of TRIM28 is an exogenously administered nucleic acid capable of expressing TRIM28.

[0058] As used herein, the term "heart failure" (HF), also known as heart failure or cardiac failure, generally refers to a clinical syndrome characterized by the inability of the heart to pump out enough blood to meet tissue needs due to initial myocardial damage (such as myocardial infarction, excessive hemodynamic load, inflammation, etc.) that causes changes in myocardial structure and function, resulting in cardiac systolic and diastolic dysfunction; usually accompanied by an increase in blood natriuretic peptide levels, and / or objective evidence of cardiogenic pulmonary or systemic congestion on imaging examination at rest or during exercise, or an increase in ventricular filling pressure on hemodynamic examination. According to the rate of progression over time, heart failure can be divided into acute heart failure and chronic heart failure (congestive heart failure) in this article; according to anatomical location, it can be divided into left heart failure, right heart failure, and total heart failure; according to physiological dysfunction, it can be divided into systolic heart failure and diastolic heart failure; or heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF); according to the course of the disease, it can be divided into stage A (risk period of heart failure), stage B (pre-heart failure), stage C (symptomatic heart failure), and stage D (advanced heart failure).

[0059] As used herein, the term "cardiomyopathy" refers to a general term for a heterogeneous group of myocardial diseases with abnormal cardiac mechanical and electrical activities caused by different etiologies, characterized by abnormal myocardial morphology, structure, and function. Generally according to the WHO classification criteria for cardiomyopathy, the cardiomyopathy in this article can be classified into dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and specific cardiomyopathy. According to the AHA classification criteria for cardiomyopathy, the cardiomyopathy in this article can also be divided into primary cardiomyopathy and secondary cardiomyopathy. The former includes hereditary, mixed, and acquired cardiomyopathies, and the latter includes infiltrative, storage, toxic, endocardial, inflammatory, endocrine, cardio-facial syndrome, neuromuscular disease, nutritional deficiency, autoimmune, electrolyte, and cardiomyopathies caused by cancer treatment. According to the ESC classification criteria for cardiomyopathy, the cardiomyopathy in this article can also be divided into familial and non-familial cardiomyopathies.

[0060] The term "specific binding" as used herein refers to a higher affinity of a binding molecule for a target molecule compared to its affinity for non-target molecules. A binding molecule that specifically binds to a target molecule essentially does not recognize or bind to non-target molecules. For example, an antibody "specifically binds" and / or "specifically recognizes" another molecule, meaning that such an interaction depends on the presence of binding specificity of the molecular structure, such as an epitope.

[0061] In this specification, "treatment" and its variants or "amelioration" refer to therapeutic treatment, where the aim is to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of a disease (such as myocarditis and / or heart failure), its related conditions, and / or symptoms. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a disease (such as myocarditis and / or heart failure). More specifically, it can refer to (1) delaying the onset of myocarditis and / or heart failure; (2) slowing down or stopping the progression, exacerbation, or aggravation of the symptoms of myocarditis and / or heart failure; (3) bringing about remission of the symptoms of myocarditis and / or heart failure; or (4) referring to a method or process for curing myocarditis and / or heart failure. As a preventive measure, treatment can be carried out before the onset of a disease or condition, or treatment can be carried out after the onset of the disease.

[0062] As used in this disclosure, "therapeutically effective" and "effective dose" refer to a substance or amount that elicits a desired biological activity or effect.

[0063] Unless otherwise indicated, the terms "subject" or "patient" are used interchangeably and refer to mammals such as primates (more particularly human patients), as well as laboratory animals such as rabbits, rats, and mice, and other animals. Thus, the terms "subject" or "patient" as used in this disclosure refer to any mammalian patient or subject to whom the compounds of this disclosure can be administered. In an exemplary embodiment of this disclosure, in order to identify the target patient to be treated according to the methods of this disclosure, acceptable screening methods are employed to determine risk factors associated with a target or suspected disease or disorder or to determine the existing disease or disorder of the subject. These screening methods include, for example, routine examinations to determine risk factors that may be associated with a target or suspected disease or disorder. These and other routine methods allow clinicians to select patients in need of treatment using the methods and compounds of this disclosure.

[0064] Throughout the specification, when a composition is described as having, comprising, or including a particular component, or when a method is described as having, comprising, or including a particular process step, it is contemplated that the compositions of this disclosure also consist essentially of or consist of the following: the methods of this disclosure also consist essentially of or consist of the recited process steps.

[0065] All documents mentioned in this disclosure are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the inventive purpose and / or technical solution of this disclosure, the cited documents related to this disclosure are incorporated for all purposes and in their entirety. When this disclosure refers to cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated. When this disclosure refers to cited documents, examples and preferred modes of the relevant technical features cited can also be incorporated into this disclosure as references, but only to the extent that this disclosure can be implemented. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or be amended adaptively according to the description of this disclosure.

[0066] A first aspect of this disclosure relates to the use of a negative regulator of the TRIM28-ERVs signaling pathway in the preparation of a medicament for treating myocarditis and / or heart failure.

[0067] According to the description of this disclosure, TRIM28 manipulates histone and RNA methylation to silence ERVs, and the reactivation of ERVs induces myocarditis through the TLR7 / 9-NFKB pathway. On the premise that the relevant mechanisms have been fully explained in this disclosure, those skilled in the art can freely select negative regulators.

[0068] In some embodiments, the negative regulator does not specifically bind to and regulate any one of TLR7, TLR9, NFKB.

[0069] In some embodiments, the negative regulator includes an inhibitor of ERVs that inhibits the reactivation of ERVs or reduces the bioavailability of reactivated ERVs.

[0070] In some embodiments, the ERVs are ERV1.

[0071] In some embodiments, the ERVs are MMVL30-int and / or RLTR6_Mm.

[0072] In some embodiments, the inhibitor comprises at least one of an antisense oligonucleotide, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozyme, which is designed to reduce or block the expression of the ERVs.

[0073] siRNA refers to double-stranded RNA that inhibits the expression of a target nucleic acid, and means "RNAi reagent", "short interfering RNA", "short interfering nucleic acid", and "siRNA". The nucleic acid molecule can inhibit or down-regulate gene expression or viral replication through sequence-specific RNA interference (RNAi) or gene silencing. This can consist only of RNA or can be a fusion of DNA and RNA. siRNA can be prepared by conventional methods according to information obtained by searching the target nucleic acid and the NCBI database.

[0074] shRNA, that is, a small hairpin or short hairpin RNA, is an RNA sequence with a tight hairpin turn that contains a sense strand fragment, an antisense strand fragment, and a stem-loop structure connecting the sense strand fragment and the antisense strand fragment, and is often used to silence the expression of a target gene by RNA interference. The sequences of the sense strand and the antisense strand are complementary, and the sequence of the antisense strand fragment is the same as 10-30 consecutive nucleotide sequences in the target mRNA. The hairpin structure of shRNA can be cleaved into siRNA by the cellular mechanism, and then the siRNA binds to the RNA-induced silencing complex (RISC), which can bind to the target mRNAs and degrade them.

[0075] A ribozyme is an RNA molecule that specifically cleaves other single-stranded RNA molecules through a mechanism similar to that of DNA restriction endonucleases. A ribozyme for recognizing and cleaving a specific base sequence in a single strand of RNA can be manufactured by appropriately modifying the nucleic acid sequence of RNA using known techniques (Science, 239, p. 1412-1416, 1988).

[0076] In some embodiments, the inhibitor reduces or blocks the expression of the ERVs through epigenetic modification.

[0077] According to some specific embodiments of the present disclosure, the epigenetic modification is the regulation of H3K9me3 and / or m6A modification; these epigenetic modifications can induce the silencing of ERVs.

[0078] In some embodiments, the inhibitor includes a reverse transcriptase inhibitor, which reduces or blocks the expression of the ERVs. In some embodiments, the negative regulator includes a reverse transcriptase inhibitor.

[0079] Examples of reverse transcriptase inhibitors include one or more of Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, and Abacavir. In many cases, it can be a combination drug, such as Combivir, which contains Zidovudine and Lamivudine.

[0080] Knockdown or knockout can be achieved using any known existing technology, such as zinc finger nuclease (ZFN) technology, TALEN (transcription activator-like effector nucleases) technology, or CRISPR technology.

[0081] In some embodiments, the inhibitor comprises a gRNA and a CRISPR enzyme, or a vector capable of expressing them. In some embodiments, the antagonist is a CRISPR-CAS gene editing composition / combination product. In some embodiments, it comprises a gRNA and a CRISPR enzyme (such as Cas9) or a vector capable of expressing them. There are two common gene knockout strategies. One is single sgRNA-mediated gene knockout, which causes frameshift mutations in the coding gene exons through InDels generated by the CRISPR-Cas9 system, thereby achieving the purpose of gene knockout. Therefore, this gene knockout strategy is limited to the knockout of coding genes. The other is two-sgRNA-mediated gene knockout, which acts on both sides of the target fragment through two sgRNAs to delete the target fragment sequence, thereby achieving the knockout of the target gene.

[0082] In some embodiments, the negative regulator includes an agonist of TRIM28, which increases the amount or activity of TRIM28 protein in the host cell.

[0083] In some embodiments, the agonist is a vector capable of expressing the TRIM28 protein in a host cell, which contains a polynucleotide capable of encoding TRIM28.

[0084] The polynucleotide may contain regulatory elements, and the regulatory elements are operably associated with the portion encoding the mutant. In any of the embodiments described herein, the polynucleotides of the present disclosure can be operably associated with a variety of regulatory elements for expression in cells. Thus, in some embodiments, the regulatory elements include one or more of the following elements: promoter, intron, enhancer, terminator, 5' and 3' untranslated regions, nuclear localization signal (NLS) sequence, or nuclear export signal (NES). And the number of each can be one or more; preferably at least includes a promoter. The choice of promoter can vary according to the temporal and spatial requirements of expression and can also vary according to the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the existing knowledge in the art, a suitable promoter can be selected for a particular host organism of interest. Thus, for example, much is known about the promoters upstream of highly constitutively expressed genes in model organisms (such as Arabidopsis thaliana, Caenorhabditis elegans, yeast, Drosophila melanogaster, mouse, rat, etc.), and this knowledge can be easily obtained and implemented in other systems as appropriate. In some preferred embodiments, the polynucleotide is codon-optimized for expression in an organism (or host cell).

[0085] As used herein, the term "vector" refers to a macromolecule or macromolecular complex associated with or containing a polynucleotide, which can be used to mediate the transfer, delivery, or introduction of a polynucleotide into a cell. Vectors for transforming host organisms are well known in the art. Non-limiting examples of general vector classes include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, minicircle vectors, or Agrobacterium binary vectors, in double-stranded or single-stranded linear or circular form, which may or may not be self-transmissible or mobile. In some embodiments, the agonist is a viral vector. In some embodiments, the viral vector may include, but is not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, or retrovirus vectors. In some embodiments, the agonist is an AAV vector.

[0086] Taking the vectors used in the present disclosure application as an example, AAV is a parvovirus that infects humans and some other primate species. It is currently unknown whether adeno-associated virus causes disease, and upon infection, it has been shown to cause only a mild immune response. Adeno-associated virus is capable of infecting both dividing and non-dividing cells and can integrate its genome into the genome of the host cell. In addition, adeno-associated virus mostly remains episomal (that is, it can replicate in the host without integrating its payload into the host chromosome); for long-term stable expression. These characteristics make adeno-associated virus a suitable candidate for creating viral vectors for gene therapy. Thus, in one example, the viral vector is an adeno-associated virus (AAV) vector. In another example, the AAV vector is but not limited to AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV9, AAV10, AAV11, AAV12, AAV13, rh10, AAVDJ, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB, and Anc80. The AAV vector serotype can be matched to the target cell type. For example, Table 2 of WO 2018002719A1 lists exemplary cell types that can be transduced by the AAV serotypes that can be specified (incorporated herein by reference).

[0087] Recombinant AAV can be produced using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (containing the gene of interest) to be packaged into AAV particles, an AAV helper function vector, and a helper function vector. The AAV helper function vector encodes "AAV helper function" sequences (e.g., rep and cap), which act in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient production of AAV vectors without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). The helper function vector encodes nucleotide sequences for non-AAV-derived viral and / or cellular functions (e.g., "helper functions") upon which AAV replication depends. Helper functions include those functions required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. The virus-based helper functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0088] In some embodiments, the agonist is formulated in the form of a delivery system; the delivery system comprises a polynucleotide capable of encoding TRIM28, and a delivery vehicle.

[0089] In some embodiments, the delivery vehicle includes one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more inorganic nanoparticles, one or more cell-penetrating peptides, gene guns, one or more plasmids, one or more viral vectors (in some embodiments, the viral vectors are as defined above), and the groups composed thereof.

[0090] Liposomes can be cationic liposomes or neutral liposomes, which can be prepared or modified by known methods. For example, adding polyethylene glycol (PEG)-modified liposomes can effectively prevent the aggregation of liposome carriers and increase their stability. Liposomes or lipofection formulations can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, and each document is incorporated herein by reference in its entirety.

[0091] Dendrimers are generally a special family of polymers with a well-defined molecular structure, precisely controllable chemical structure, and unique multivalent properties, and are gradually becoming non-viral vectors for gene delivery. Typical dendrimers such as poly(amidoamine) (PAMAM) dendrimers can be further modified. For example, derivatives AP-PAMAM are constructed by modifying the nucleobase analogue 2-amino-6-chloropurine on the surface of PAMAM, or CS-PAMAM is prepared by coupling chondroitin sulfate (CS) with PAMAM, etc.

[0092] Inorganic nanoparticles can be selected from gold nanoparticles (AuNPs), magnetic nanoparticles, mesoporous silica nanoparticles (MSNs), etc.

[0093] Cell-penetrating peptides (CPPs) are generally a class of small peptides with strong transmembrane transport ability, which can carry various macromolecules such as polypeptides, proteins, and nucleic acids into cells. They can be cationic CPPs (such as TAT, Penetratin, Polyarginine, P22N, DPV3, and DPV6, etc.), amphiphilic CPPs (which can be formed by covalently linking hydrophobic peptide sequences and NLSs, or isolated from natural proteins, such as pVEC, ARF(1-22), and BPrPr(1-28)), and hydrophobic CPPs (generally containing only non-polar amino acid residues, with a net charge of about less than 20% of the total charge of the amino acid sequence).

[0094] In some embodiments, the delivery is via a plasmid. The dose can be a sufficient number of plasmids to elicit a response. In some cases, a suitable amount of plasmid DNA in the plasmid composition can be from about 0.1 to about 2 mg. The plasmid will generally include (i) a promoter; (ii) a sequence encoding the TRIM28 protein, each sequence operably linked to a promoter (e.g., the same promoter or different promoters); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator located downstream of (ii) and operably linked thereto. The frequency of administration is within the scope of a medical or veterinary practitioner (e.g., a physician, a veterinarian) or a person skilled in the art.

[0095] The delivery can be carried out by any means well known to those skilled in the art, such as transfection, lipofection, electroporation, gene gun, microinjection, ultrasound, calcium phosphate transfection, cationic transfection, viral vector delivery, etc.

[0096] In some embodiments, the negative regulator is formulated alone or in combination with other drugs.

[0097] For heart failure, the other drugs exemplary include: diuretics (such as loop diuretics, for example furosemide, torasemide, bumetanide, tolvaptan), renin-angiotensin system (RAS) inhibitors (such as ACEI, ARB and ARNI), vasodilators (such as hydralazine, isosorbide dinitrate, nitroglycerin patch or spray), aldosterone antagonists, β-adrenergic receptor blockers (such as bisoprolol or metoprolol succinate sustained release tablets or carvedilol), MRA (the representative drugs are spironolactone, eplerenone), sodium-glucose cotransporter 2 inhibitors (such as dapagliflozin), sinoatrial node pacemaker current inhibitors (such as ivabradine), digitalis drugs (such as digoxin), guanylate cyclase stimulators (such as vericiguat), myosin activators (such as Omecamtiv mercabil), at least one of them.

[0098] For myocarditis, the other drugs exemplary include: corticosteroids, drugs to prevent blood clots formation [diuretics, β-blockers, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARB)], heart disease drugs, and drugs for treating chronic conditions (such as chronic diseases like lupus that may cause myocarditis).

[0099] In some embodiments, the negative regulator and a pharmaceutically acceptable excipient form a pharmaceutical composition. The pharmaceutical composition can be formulated by methods well known to those skilled in the art. For example, it can be used parenterally in the form of an injection of a sterile solution or suspension with water or a pharmaceutically acceptable solution other than water. For example, appropriately combine pharmaceutically acceptable carriers or media, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. It is considered to be formulated by mixing in the unit dosage forms required for generally accepted drug implementation. The amount of the active ingredient in these preparations is set to obtain an appropriate volume within the indicated range.

[0100] The sterile composition for injection can be formulated according to the usual preparations using excipients such as distilled water for injection.

[0101] As an aqueous injection solution, isotonic solutions containing, for example, physiological saline solution, lactose, glucose and other supplements (such as D-sorbitol, D-mannose, D-mannitol, sodium chloride) can be cited. Appropriate solubilizing aids can be used in combination, such as alcohols (such as ethanol), polyols (such as propylene glycol, polyethylene glycol, etc.) and nonionic surfactants (such as polysorbate80(TM), HCO-50, etc.).

[0102] As oily liquids, sesame oil and soybean oil can be cited, and benzyl benzoate and / or benzyl alcohol can be used in combination as solubilizing aids. The composition can be mixed with buffering agents (e.g., phosphate buffer solution and sodium acetate buffer solution), painless agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection is usually filled in a suitable ampoule.

[0103] The pharmaceutical composition is preferably administered by parenteral administration. For example, in some embodiments, the form of the pharmaceutical composition is selected from one or more of solutions, injections, sprays, nasal drops, aerosols, powder aerosols, tablets, capsules, and granules. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0104] According to the above application of the present disclosure, in the second aspect of the present disclosure, a drug / drug composition for treating myocarditis and / or heart failure can be determined.

[0105] The drug contains a negative regulator of the TRIM28-ERVs signaling pathway as the main active ingredient.

[0106] The pharmaceutical composition comprises:

[0107] A pharmaceutical composition for treating myocarditis and / or heart failure, which contains a negative regulator of the TRIM28-ERVs signaling pathway and other drugs, more specifically other drugs for treating myocarditis and / or heart failure.

[0108] Alternatively, a pharmaceutical composition for treating myocarditis and / or heart failure, which contains a negative regulator of the TRIM28-ERVs signaling pathway and pharmaceutically acceptable excipients.

[0109] Alternatively, a pharmaceutical composition for treating myocarditis and / or heart failure, which contains a negative regulator of the TRIM28-ERVs signaling pathway and other drugs (more specifically other drugs for treating myocarditis and / or heart failure), and pharmaceutically acceptable excipients.

[0110] The third aspect of the present disclosure relates to a method for treating a patient with myocarditis and / or heart failure, comprising administering to the patient a pharmaceutical composition, the active ingredient of which contains an effective amount of at least one negative regulator of the TRIM28-ERVs signaling pathway.

[0111] The administration method can be appropriately selected according to the age and symptoms of the patient. The dose of the active ingredient can be set within the range of, for example, 0.0001 mg to 1000 mg per kg body weight per administration. Alternatively, for example, the dose can be 0.001 - 100000 mg (such as about 0.01, 0.1, 1, 10, 15, 25, 50, 100, 125, 150, 175, 200, 250, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 mg or higher) / patient, but the present disclosure is not necessarily limited to these values. The administration frequency can be once a day, twice a day, three times a day, four times a day, once a week, twice a week or three times a week to treat the disease or disorder of the subject. The dose and administration method vary according to the body weight, age, symptoms, etc. of the patient, but those skilled in the art can set appropriate dose and administration method considering these circumstances.

[0112] Those skilled in the art will recognize that in vivo and in vitro tests using suitable, known and widely accepted cell and / or animal models can predict the ability of a negative regulator of the TRIM28 - ERVs signaling pathway to treat or prevent myocarditis and / or heart failure. Those skilled in the art will further recognize that human clinical trials can be completed according to methods well known in the clinic, including first-in-human trials, dose range trials and efficacy trials in healthy subjects and / or subjects with a given disease.

[0113] In some embodiments, the pharmaceutical composition is used in combination with other drugs (preferably drugs for treating myocarditis and / or heart failure other than negative regulators of the TRIM28 - ERVs signaling pathway) to treat patients with myocarditis and / or heart failure. The pharmaceutical composition and other drugs can be administered together (such as forming a new pharmaceutical composition), or administered separately at intervals.

[0114] In some embodiments, the pharmaceutical composition is used in combination with at least one method for treating heart failure other than drug therapy to treat patients with myocarditis and / or heart failure.

[0115] Examples of methods for treating heart failure include: exercise training (such as through supervised exercise training for 3 - 6 months), cardiac implantable electronic device therapy [such as implantable cardioverter defibrillator (ICD), cardiac resynchronization therapy (CRT), cardiac contractility modulation (CCM)], lifestyle management (including avoiding excessive drinking of water, over-infusion, restricting sodium intake, adopting a low-fat or diabetic diet, alcoholism, infection and other inducing factors), heart transplantation, coronary artery bypass grafting, ventricular assist device (VAD), etc.

[0116] Examples of methods for treating myocarditis include: exercise training, ventricular assist device (VAD), intra-aortic balloon pump (IABP), heart transplantation, extracorporeal membrane oxygenation (ECMO), etc.

[0117] Moreover, the descriptions in the above first aspect also apply to the second aspect and the third aspect as described above.

[0118] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. For the experimental methods without specific conditions noted in the following examples, the guidelines given in the present disclosure are preferably referred to, and it is also possible to follow the experimental manuals or conventional conditions in the art, and it is also possible to refer to other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0119] In the following specific examples, for the measurement parameters of raw material components, unless otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0120] Example 1 ERV Reactivation in Human and Animal Heart Failure Models

[0121] To explore the possible function of retrotransposons (RTEs) in heart failure (HF), we analyzed the total RNA sequencing data from the public database (GSE135055)22. Clustering and high-quality data of 14 heart failure patients and 7 healthy donors were retrieved by principal component analysis (PCA) and subjected to RTE analysis. Among all RTEs, only ERVs containing LTRs were significantly upregulated in heart failure patients (P = 0.00011, Figure 1 in a), accounting for 65% of all differentially expressed RTEs. In addition, among four human endogenous retrovirus (HERV) subfamilies (HERV1, HERVK, HERVL, and HMaLR), HERV1 showed significant activation (P = 0.00048, Figure 1 in a). Consistently, 26 ERV genes were upregulated in failing hearts, most of which belonged to HERV1 (n = 15), and only a few belonged to HERVK (n = 2), HERVL (n = 6), and HMaLR (n = 3) ( Figure 1 in b). Ranking the differential expression of ERVs further confirmed the dominant position and the highest ranking of ERV1 RTEs in reactivated ERVs ( Figure 1In c). A significant proportion of the activated ERV1 RTEs contain retroviral genes gag and pol, such as MER52-int and MER61-int, indicating that they may have potential functions. HERV-H23 and HERV-W24, 25 in ERV1 were previously reported to be associated with neurodegenerative and inflammatory diseases, but did not change in heart failure patients.

[0122] To further verify the resurrection of ERVs in heart failure, we analyzed the expression of ERVs in the I / R mouse model. Consistent with the human results, ERVs were significantly upregulated in the rodent I / R heart compared with other RTEs ( Figure 1 In d). Both ERV1 and ERVK were upregulated ( Figure 1 In d), but the increase in ERV1 was more significant ( Figure 1 In e-f). The most significantly upregulated RTE genes were MMVL30-int (P-adj 2.52e-200) and RLTR6_Mm (P-adj 2.68e-171), both of which belong to the ERV1 family ( Figure 1 In f), and their functions have not been explored. Detection by RT-qPCR at multiple time points showed that their activation occurred immediately after injury and lasted for two weeks. Further discovery by RT-qPCR indicated the resurrection of ERV1 but not ERVK in the heart failure models of myocardial infarction (MI) in mice and non-human primates (NHP). Therefore, these results together imply that the resurrection of ERVs, especially ERV1, is a hallmark of heart failure.

[0123] Example 2 Deletion of TRIM28 leads to ERV resurrection and acute heart failure

[0124] Inhibition of epigenetic modification can silence the transcription of genomic ERVs. TRIM28 is a major regulator of epigenetic repression. We found that TRIM28 was significantly downregulated in human heart failure samples, indicating that it may mediate the activation of ERVs in heart failure ( Figure 2 In a-b). To further test this, we crossed TRIM28 flox mice with Myh6-MerCreMer mice to generate inducible cardiomyocyte-specific TRIM28 knockout mice, Myh6-MerCreMer:TRIM28f / f (TRIM28iCKO) mice. Next, we analyzed the expression of ERVs in wild-type and TRIM28iCKO hearts by total RNA-seq and found that 12 ERV genes were significantly upregulated in TRIM28iCKO hearts, a number much higher than that of LINE and SINE (3 and 0 genes, respectively) ( Figure 2 In c). Some ERVK genes were also upregulated, but IAPEZ26 and MMERVK10C14, which are known to have functions in immunity, did not change.Figure 2 in c). The most upregulated ERVs were ERV1, accounting for half of the upregulated ERVs and ranking first ( Figure 2 in c-d). Similar to the results of the mouse I / R model, the most significantly upregulated ERV1 RTEs in the TRIM28iCKO heart were MMVL30-int (P-adj 2.32e-24) and RLTR6_Mm (P-adj 1.15e-18) ( Figure 2 in d). The expressions of the MMVL30-int and RLTR6_Mm transcripts increased by 48.2% and 25.7% respectively, both exceeding 2-fold. MMVL30-int is a non-autonomous ERV, and its LTR element is provided by RLTR6_Mm 27, 28( Figure 2 in e), and has been associated with stress responses such as cerebral ischemia. RNA fluorescence in situ hybridization (RNA-FISH) verified the increase in MMVL30 RNA in TRIM28iCKO cardiomyocytes ( Figure 2 in f). MMVL30 showed a punctate pattern in the cytoplasm, indicating that their main functional location in cardiomyocytes may be in endosomes (sub-organelles of TLR7 / 9 and innate immune responses) or P-bodies (sub-organelles of RNA degradation).

[0125] Meanwhile, we found that TRIM28iCKO mice began to die only 4 days after tamoxifen induction, and the mortality rate within 2 weeks was 46% (P = 0.0037; Figure 2 in g). Echocardiographic evaluation performed on the 7th day after tamoxifen induction showed a significant reduction in cardiac systolic function (EF: CTR vs TRIM28iCKO, 52.37 ± 3.47 vs 19.81 ± 5.60, P < 0.001). The cardiac chamber was significantly enlarged (LVIDd: CTR vs TRIM28iCKO, 3.90 ± 0.09 vs 4.81 ± 0.28, P < 0.001), and the posterior ventricular wall was thinned (LVPWd: CTR vs TRIM28iCKO, 0.87 ± 0.05 vs 0.64 ± 0.16, P < 0.001), all of which indicated the dilated cardiomyopathy and heart failure phenotypes of TRIM28iCKO mice ( Figure 2 in h-j).

[0126] We dissected the heart 7 days after TAM induction and found that the TRIM28iCKO heart was much larger than the control heart, the ratio of heart weight to tibia length increased, and cardiomyocytes were enlarged (P = 0.0013; Figure 2 in k-n). Histological examination further illustrated the enlarged cardiac chamber and thinned myocardial wall of the TRIM28iCKO heart ( Figure 2In o). Most of the muscle bundles were ruptured and misaligned, and there was a large amount of blood and monocyte infiltration, indicating cell death and inflammation in the myocardium. Figure 2 In p). Although TRIM28 was only knocked out for 7 days, significant fibrosis appeared in the TRIM28iCKO heart. Figure 2 In q - r). The heart failure biomarkers atrial natriuretic peptide A (Nppa) and B (Nppb) were upregulated in the TRIM28iCKO heart, and at the same time, the ratio of adult myosin heavy chain α (Myh6) / fetal myosin heavy chain 7 (Myh7) was significantly decreased. Collectively, these results indicate that the deletion of TRIM28 in the myocardium leads to acute and severe heart failure. At the same time, obvious congestion and edema appeared in the lungs and livers of TRIM28iCKO mice, which is similar to the clinical manifestations of AHF patients.

[0127] Example 3 TRIM28 Manipulates Histone and RNA Methylation to Silence ERV

[0128] As a core member of the methyltransferase complex, the deletion of TRIM28 may lead to the resurrection of ERV, and this process may be achieved by disrupting H3K9 methylation. To verify this hypothesis, we analyzed the chromatin occupancy of H3K9me3 around genomic transposable elements (TEs) in wild - type and TRIM28iCKO hearts by chromatin immunoprecipitation sequencing (ChIP - seq). Figure 3 In a - b). 27.8% of ERV RTEs were occupied by H3K9me3, mainly in the coding regions, which was generally reduced in the TRIM28iCKO heart. Figure 3 In a). Among the upregulated ERVs (upERVs, a total of 1723 transcripts), 38.9% were significantly occupied by H3K9me3. In the hearts with TRIM28 deletion, 59.6% of the H3K9me3 - targeted upERVs lost their occupancy. Figure 3 In b - c). And the IGV image processing software showed that the occupancy of H3K9me3 on the int_dup726 genome of the upregulated MMVL30 transcript in the TRIM28iCKO heart was reduced, but the occupancy of H3K9me3 on another transcript of MMVL30 - int_dup810 did not change significantly. Figure 3 In d - e). The above results indicate that the loss of H3K9me3 mediated by TRIM28 may be only a partial mechanism for ERV resurrection.

[0129] To further evaluate this hypothesis, we generated transgenic mice with inducible knockout of SETDB1 (SETDB1iCKO) in cardiomyocytes. SETDB1 is a methyltransferase with H3K9me3 modification function in the TRIM28 complex. Consistent with the TRIM28 knockout results, the expression of ERVs increased in SETDB1iCKO hearts, but to a lesser extent than in TRIM28iCKO hearts. SETDB1iCKO mice developed impaired cardiac systolic function and dilated cardiomyopathy phenotypes 7 days after tamoxifen induction, characterized by increased left ventricular end-diastolic diameter and decreased left ventricular posterior wall at end-diastole, as shown by echocardiogram, which was similar to that of TRIM28iCKO mice (CTR vs SETDB1iCKO hearts, EF: 54.53±2.28 vs 30.11±4.97, P<0.001; FS: 27.79±1.43 vs 14.12±2.54, P<0.001; LVIDd: 3.88±0.15 vs 4.64±0.35, P = 0.0019; LVPWd: 0.85±0.09 vs 0.63±0.0, P<0.001), but these symptoms were all milder. Autopsy further verified these cardiac phenotypes, showing a milder dilated cardiomyopathy phenotype. Consistent with the phenotypic results, all SETDB1iCKO mice survived until 1 month after tamoxifen induction, in sharp contrast to TRIM28iCKO mice, 46% of which died within two weeks after induction. Therefore, these results support our hypothesis that H3K9me3 modification only leads to the activation of some ERVs.

[0130] RNA methylation (N6-methyladenosine, m6A) can promote the degradation of RNAs including RTE. The reactivation of upERVs not regulated by H3K9me3 may be attributed to changes in m6A modification34,35. Therefore, we analyzed m6A modification by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and identified 11,300 and 9,612 m6A peaks in control and TRIM28iCKO hearts, respectively. Consistent with what has been reported in other cells, m6A was highly enriched on the 3' and 5' UTRs of coding mRNAs, and no significant difference was observed between wild-type and TRIM28iCKO hearts. In contrast, after TRIM28 depletion, m6A in the internal regions of upregulated ERVs including MMVL30 was significantly reduced ( Figure 3 in e-g). The m6A of some upERVs, such as MMVL30-int_dup810, MuLV-int_dup62, and RLTR6-int_dup581, decreased, but the signal of H3K9me3 on their genomes did not change, indicating that m6A can function independently of H3K9me3 modification in ERV activation ( Figure 3e). This conclusion was further supported by gene association analysis. The Venn diagram showed that most of the m6A-modified and H3K9me3-labeled upERV transcripts were different (1723 transcripts, P = 0.9161, Figure 3 h). To further understand the molecular mechanism, we performed co-immunoprecipitation and found that TRIM28 could interact with METTL3 (the catalytic subunit of the m6A enzyme), and this interaction was independent of RNA. Figure 3 i, j). RNA immunoprecipitation (RIP) analysis showed that TRIM28 depletion weakened the binding ability of METTL3 to upERVs MMVL30, RLTR6, and MuLV. Figure 3 k). Conversely, overexpression of METTL3 in TRIM28iCKO cardiomyocytes inhibited their activation. Figure 3 l). In summary, TRIM28 can simultaneously manipulate H3K9me3 and m6A to control the silencing of ERVs. Disrupting these two epigenetic modifications will lead to the resurrection of ERVs in heart failure.

[0131] Example 4 ERV resurrection leads to innate inflammation and myocarditis

[0132] To explore the possible role of ERV activation in the pathogenesis of heart failure, we analyzed the mRNA transcriptome in TRIM28iCKO hearts by RNA-seq. Principal component analysis (PCA) showed that there were significant differences in the transcriptomes of wild-type and TRIM28iCKO hearts. A total of 6311 genes were differentially expressed (DEGs): 2894 genes were downregulated, and 3417 genes were upregulated (fold change > 1.5, P-adj < 0.05). Gene ontology (GO) analysis revealed that among the upregulated DEGs, terms related to inflammation accounted for the majority, P-adj < 9.75e-19, indicating significant immune activation. Figure 4 a lists the top 10 terms). Among the downregulated DEGs, terms related to energy and metabolism were enriched, further confirming the heart failure phenotype of TRIM28iCKO hearts. To further investigate the activated immune pathways in TRIM28-deficient hearts, we performed gene set enrichment analysis (GSEA), and the results showed that the Toll-like receptor signaling pathway and the cytoplasmic DNA sensing pathway related to pattern recognition receptors (PRRs) and innate immunity were significantly enriched. Figure 4In Fig. 2b-c). We also detected double-stranded RNA, a classical immunogen for antiviral and innate immunity, which further supported the activation of innate immunity in TRIM28iCKO cardiomyocytes. The NFKB and IFN I pathways are two typical pathways downstream of activated PRRs. Interestingly, GSEA analysis revealed that the NFKB rather than the IFNI pathway was activated upon TRIM28 deletion, suggesting a unique pattern of innate immune activation in the TRIM28iCKO heart ( Figure 4 In Fig. 2d-e).

[0133] To further analyze which of these innate immunity-related PRR pathways was activated, we detected the protein levels of TLR7 (a TLR that detects single-stranded [ss]RNA), TLR9 (a TLR that detects unmethylated CpG-containing ssDNA), MyD88 (a key signal transduction adapter that forms the supramolecular organizing center myddosome downstream of TLRs), and Cyclic GMP-AMP (cGAS, a sensor of cytoplasmic DNA), and found that they were all significantly increased ( Figure 4 In Fig. 2f-g). The phosphorylation of P65 and TAK1 (the upstream kinase of P65) was also significantly elevated, indicating the activation of the NFKB pathway. In contrast, the phosphorylation of the transcription factor IRF7 (a key regulator of type I IFN gene expression) remained almost unchanged, further confirming the inactive state of the IFN pathway shown by GSEA ( Figure 4 In Fig. 2g). Consistent with these results, the inflammatory cytokines IL6, IL1b, TNFa, VCAM1, and ICAM1 (downstream of NFKB) were all significantly increased, while the IFN-stimulated genes (ISGs) IFIT1 and IFI44 did not increase (IFIT1: P-adj = 0.62; IFI44: P-adj = 0.91, Figure 4 In Fig. 2f).

[0134] To further evaluate whether the activation of the innate immune response was attributed to the resurrection of ERVs, we overexpressed MMVL30-int (the most significantly upregulated ERVs) in cardiomyocytes and found that TLR7 / 9, MyD88, and cGAS were all activated ( Figure 8 In contrast, suppressing the resurrection of MMVL30-int with siRNA in TRIM28iCKO cardiomyocytes attenuated the activation of TLR7 / 9, MyD88, and cGAS ( Figure 4 In Fig. 2h). Therefore, both of these experiments demonstrated that it was indeed the resurrection of ERVs that stimulated innate immunity in the TRIM28iCKO heart. The abnormal innate immune response led to the activation of adaptive cellular immunity and the characteristics of myocarditis, manifested as increased infiltration of CD68+ macrophages and CD3+ lymphocytes in the myocardium ( Figure 4in (i-k), and further leading to cell death, which was confirmed by the increase in the content of lactate dehydrogenase in the serum ( Figure 8 in (b). Collectively, these results indicate that abnormal ERV reactivation stimulates innate immunity, thereby triggering the development of myocarditis and heart failure in TRIM28iCKO mice.

[0135] Example 5 ERV induces myocarditis through the TLR7 / 9-NFκB pathway

[0136] In the above results, both TLR7 / 9 and cGAS in the antiviral and exogenous nucleoside pathways were activated. Next, we attempted to analyze their possible functions in ERV-mediated myocarditis and heart failure. We treated TRIM28iCKO mice with the cGAS-STING inhibitor C176, the TLR7 / 9 inhibitor NSC4375, or the NFκB inhibitor JSH23 for 21 days ( Figure 5 in (a)), and evaluated cardiac function using echocardiography and histological examination. To further verify the role of ERV in myocarditis and heart failure in vivo, we also treated TRIM28iCKO mice with the reverse transcriptase inhibitor (RTi) zidovudine to inhibit the reverse transcription and immunogenicity of ERV. After 3 weeks of treatment with JSH23, NSC4375, and RTi (but not C176), the survival rate of TRIM28iCKO mice increased ( Figure 5 in (b), JSH23 85.7% P = 0.013; NSC4375 79.2% P = 0.041; RTi 80.9% P = 0.032; C176 62.5% P = 0.619 vs TRIM28iCKO 53.8%). This indicates that JSH23, NSC4375, and RTi can all significantly improve the systolic function of the TRIM28iCKO heart and alleviate dilated cardiomyopathy ( Figure 5 in (c-h). C176 had a tendency to improve cardiac function and reduce remodeling, but not significantly. Consistent with its anti-immune function, JSH23 and NSC4375 significantly reduced macrophage infiltration and cytokine expression, but did not affect ERVs, which confirmed the function of TLR7 / 9 and NFκB as downstream signaling effectors of ERVs ( Figure 5 in (i). RTi did not affect ERV expression, but significantly inhibited cytokine elevation and macrophage infiltration, which confirmed that ERV actually mediates the immune response and myocarditis in the TRIM28iCKO heart. Figure 5in i). C176 had no significant effect on ERV expression and inflammatory response, further excluding the dominant function of the cGAS pathway in ERV-mediated innate immunity and heart failure. Collectively, these results define the TLR7 / 9-NF-κB axis as the downstream signaling pathway induced by ERV reactivation in myocarditis and heart failure.

[0137] Example 6 Restricting ERV Reactivation Alleviates Heart Failure in the I / R Model

[0138] Percutaneous coronary intervention (PCI) commonly causes ischemia-reperfusion, which induces acute cardiac inflammation and heart failure. We found that ERVs, especially ERV1 including MMLV30-int, were significantly activated in the hearts of I / R mice ( Figure 9 in a). Western blot showed that TRIM28 was downregulated in the I / R hearts, and the TLR7 / 9 and NF-κB pathways were activated in the I / R hearts, similar to the situation in TRIM28 iCKO hearts ( Figure 6 in a). Cytokines (IL6, IL1β, and TNFα) downstream of ERV and NF-κB were also significantly increased ( Figure 9 in b). These results suggest that the ERV-TLR7 / 9-NF-κB pathway may also play a role in ischemia-induced heart failure. To test this, we constructed adeno-associated virus 9 (AAV9-cTNT-TRIM28) and injected 2.5×10 11 viruses intrathoracically 7 days before the I / R surgery to restore the expression of TRIM28 in the I / R myocardium ( Figure 6 in b). The reintroduction of TRIM28 significantly inhibited the activation of ERVs, further confirming the role of TRIM28 in imprinting ERVs ( Figure 2 and Figure 6 in c). In addition, AAV9-cTNT-TRIM28 alleviated the activation of the TLR7 / 9-NF-κB pathway and the expression of IL6, IL1β, and TNFα ( Figure 6 in a, c). AAV9-cTNT-TRIM28 also reduced cardiomyocyte death, as shown by serum lactate dehydrogenase assay ( Figure 9 in c). These results indicate that AAV9-cTNT-TRIM28 can inhibit the reactivation of ERVs and the inflammatory response in the I / R model.

[0139] Next, we evaluated whether AAV9-cTNT-TRIM28 could maintain cardiac function after I / R. We first measured the ischemic and damaged areas by Evans blue perfusion 24 hours after the I / R surgery and found that AAV9-TRIM28 did not change the area at risk (AAR), but significantly reduced the infarct area / area at risk (IF / AAR) - the area of myocardial injury induced by I / R ( Figure 6 in d-f, from 41.5% to 31.6%, P = 0.0024). Time-course echocardiography showed that AAV9-TRIM28 significantly improved cardiac systolic function from 24 hours to 3 weeks after I / R ( Figure 6 in g). Autopsy showed that AAV9-TRIM28 also significantly reduced the scar size 3 weeks after I / R ( Figure 6 in h-i, IR+CTR vs IR+TRIM28, from 14.5% to 7.9%, P<0.001). Therefore, these results together indicate that AAV9-mediated TRIM28 expression can inhibit ERV activation and protect the heart from heart failure.

[0140] Example 7 Intercepting ERV-mediated innate immunity can alleviate heart failure in the I / R model

[0141] To evaluate the role of innate immunity in I / R-induced cardiac inflammation and dysfunction. We treated mice with 20 mg / kg of NSC4375 two days before I / R, the same dose used in TRIM28iCKO mice ( Figure 7 in a), and found that it effectively inhibited the activation of the MyD88 and NFKB pathways, as manifested by the attenuation of TAK1 and P65 phosphorylation ( Figure 7 in b). NSC4375 also inhibited the expression of cytokines (IL6, IL1b, TNFa), but had no effect on ERVs (MMVL30, RLTR6, and MuLV), which further consolidated the signal transduction sequence and function of the ERV-TLR7 / 9-NFKB molecular axis in I / R ( Figure 7 in c). Similar to the results of AAV-TRIM28, the small molecule inhibitor NSC4375 significantly reduced the infarct area of the I / R heart ( Figure 7 in d-f, from 43.5% to 36.6%, P = 0.035), and improved systolic function ( Figure 7 in g). As expected, NSC4375 also significantly reduced the scar size ( Figure 7From 12.2% to 5.7% (P = 0.0052). In summary, these results suggest that the activation of the ERV-TLR7 / 9-NFκB axis in the I / R heart exacerbates cardiac function, while intercepting this activated axis can alleviate I / R-induced inflammation and heart failure.

[0142] The embodiments described above merely represent several implementation manners of the present disclosure, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present disclosure. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can still be made, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of a negative regulator of the TRIM28-ERVs signaling pathway in the preparation of a medicament for treating myocarditis and / or heart failure.

2. The use according to claim 1, wherein the negative regulator comprises an inhibitor of ERVs, which inhibits the reactivation of ERVs or reduces the bioavailability of reactivated ERVs.

3. The use according to claim 2, wherein the ERVs is ERV1.

4. The use according to claim 3, wherein the ERVs is MMVL30-int and / or RLTR6_Mm.

5. The use according to any one of claims 2 to 4, wherein the inhibitor comprises at least one of antisense oligonucleotides, dsRNA, siRNA, shRNA, miRNA, RNAi, and ribozymes, which is designed to reduce or block the expression of the ERVs.

6. The use according to any one of claims 2 to 4, wherein the inhibitor reduces or blocks the expression of the ERVs by epigenetic modification.

7. The use according to any one of claims 2 to 4, wherein the inhibitor comprises a reverse transcriptase inhibitor, which reduces or blocks the expression of the ERVs.

8. The use according to claim 7, wherein the reverse transcriptase inhibitor comprises one or more of zidovudine, didanosine, zalcitabine, stavudine, lamivudine, and abacavir.

9. The use according to any one of claims 2 to 4, wherein the inhibitor knocks down or knocks out the ERVs gene or the gene regulating the expression of the ERVs by ZFN technology, TALEN technology, or CRISPR technology.

10. The use according to claim 1, wherein the negative regulator comprises an agonist of TRIM28, which increases the amount or activity of TRIM28 protein in host cells.

11. The use according to claim 10, wherein the agonist is a vector capable of expressing TRIM28 protein in host cells, which comprises a polynucleotide capable of encoding TRIM28.

12. The use according to claim 11, wherein the agonist is a viral vector.

13. The use according to claim 12, wherein the agonist is an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, or a retrovirus vector.

14. The use according to claim 13, wherein the AAV vector is selected from the group consisting of AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, AAVDJ, AAV-PHP.S, AAV-PHP.B, AAV-PHP.eB, and Anc80.

15. The use according to claim 10, wherein the agonist is formulated in the form of a delivery system; the delivery system comprises a polynucleotide capable of encoding TRIM28, and a delivery vehicle.

16. The application according to claim 15, wherein the delivery vehicle comprises one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more inorganic nanoparticles, one or more cell-penetrating peptides, a gene gun, one or more plasmids, and a group composed of them.

17. The application according to any one of claims 1 to 4, 8, 10 to 16, wherein the negative regulator is formulated as a single drug or in combination with other drugs.

18. The application according to any one of claims 1 to 4, 8, 10 to 16, wherein the negative regulator and a pharmaceutically acceptable excipient form a pharmaceutical composition.

19. The application according to claim 18, wherein the form of the pharmaceutical composition is selected from one or more of a solution, an injection, a spray, a nasal drop, an aerosol, a powder aerosol, a tablet, a capsule, and a granule.

20. The application according to any one of claims 1 to 4, 8, 10 to 16, 19, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, or specific cardiomyopathy.

21. The application according to any one of claims 1 to 4, 8, 10 to 16, 19, wherein the heart failure is heart failure with preserved ejection fraction or heart failure with reduced ejection fraction.

22. The application according to any one of claims 1 to 4, 8, 10 to 16, 19, wherein the patient of the drug is a mammal.

23. The application according to claim 22, wherein the patient of the drug is a primate.

24. The application according to claim 23, wherein the patient of the drug is a human.

25. The application according to claim 1, wherein the negative regulator does not specifically bind to and regulate any one of TLR7, TLR9, and NFKB.

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