Treatment of heart failure with preserved ejection fraction and drug screening

Abstract

The present invention relates to the field of disease treatment, and particularly to use of a reagent capable of inhibiting or knocking out Jun gene expression in the treatment of heart failure with preserved ejection fraction (HFpEF) and a method for drug screening.

Description

Treatment and drug screening for heart failure with preserved ejection fraction

[0001] This application is based on and claims priority to an application with CN application number 202410101243.8 and application date January 24, 2024. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of disease treatment, and specifically provides use of an agent capable of inhibiting or knocking out Jun gene expression in the treatment of heart failure with preserved ejection fraction (HFpEF) and a method for screening drugs. Background Art

[0003] Cardiovascular disease represents one of the greatest medical challenges. Heart failure is the leading cause of death in patients with cardiovascular disease and remains a major clinical challenge. It encompasses both systolic and diastolic heart failure, with the latter now more broadly defined as heart failure with preserved ejection fraction. Heart failure with preserved ejection fraction (HFpEF) is a leading cause of morbidity and mortality throughout the industrialized world, and its prevalence is increasing at an alarming rate. HFpEF currently accounts for 50% of all HF cases. Patients with this complex disease experience classic symptoms of heart failure, including exertion intolerance, dyspnea, extravascular fluid accumulation in the lungs, subcutaneous tissue, and peritoneal cavity, and intermittent cardiovascular decompensation that often leads to hospitalization for urgent diuresis. This definition of HFpEF excludes conditions with well-defined etiologies, such as cardiac amyloidosis, hereditary hypertrophic cardiomyopathy, valvular disease, and others. HFpEF more broadly refers to patients whose pathophysiology involves a multi-organ syndrome, with cardiac, pulmonary, renal, skeletal, immune, inflammatory, metabolic, and other components contributing to the disease. Importantly, HFpEF is a syndrome with high morbidity and mortality. According to statistics, the mortality rate due to HF is 35% worldwide, of which HFpEF accounts for more than 50%.

[0004] To date, basic research on HFpEF is almost blank, and there are few effective drug therapies proven to change the disease progression and prognosis of HFpEF patients, resulting in huge unmet clinical needs. Summary of the Invention

[0005] The inventors of the present application have discovered for the first time that Jun is an important regulatory factor in the development and progression of HFpEF, thereby providing the following invention.

[0006] Inhibit or knockout Jun gene expression

[0007] In one aspect, the present invention provides the use of an agent capable of inhibiting or knocking out Jun gene expression in the preparation of a medicament for preventing and / or treating heart failure with preserved ejection fraction (HFpEF). Also provided are methods for preventing and / or treating heart failure with preserved ejection fraction (HFpEF), comprising administering to a subject (e.g., a human) in need thereof an effective amount of an agent capable of inhibiting or knocking out Jun gene expression. In certain embodiments, the treatment includes at least improving cardiac diastolic function, such as reducing the diastolic function parameters E / E' and / or E / A.

[0008] The agents described herein that are capable of inhibiting or knocking out Jun gene expression can disrupt Jun gene expression by any mechanism, such as by inhibiting Jun expression at the RNA or protein level, such as knocking out the Jun gene, reducing or inhibiting gene transcription, and / or reducing or inhibiting translation of the gene's mRNA product.

[0009] In certain embodiments, the determination of expression levels can be implemented at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include, but are not limited to, Northern blotting, PCR, RT-PCR, or real-time RT-PCR. Methods for determining expression at the protein level include, but are not limited to, Western blotting or polyacrylamide gel electrophoresis combined with protein staining techniques such as Coomassie brilliant blue or silver staining, mass spectrometry, ELISA, and the like.

[0010] Gene editing system

[0011] In certain embodiments, the agent capable of inhibiting or knocking out Jun gene expression is a gene editing system. It is well known to those skilled in the art to use gene editing systems to knock down or knock out target genes (e.g., Jun).

[0012] In certain embodiments, the gene editing system can be any site-specific (sequence-specific) genome editing system known today. In certain embodiments, the genome editing system includes at least one site-specific nuclease, such as an RNA-guided nuclease (e.g., Cas nuclease), a zinc finger nuclease, a megabase wide range nuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof. In certain embodiments, the site-specific endonuclease targets the Jun gene, induces DNA breakage at the target site, and modification is completed by, for example, homologous recombination (HR) or non-homologous end joining (NHEJ) to cause destruction of the Jun gene.

[0013] In certain embodiments, the gene editing system is selected from CRISPR / Cas, TALEN, ZFN, transposon technology, PASTE technology, PE technology, base editor, and any combination thereof.

[0014] In certain embodiments, the gene editing system comprises an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence in a target locus. In certain embodiments, the gene editing system is present on one or more vectors.

[0015] In certain embodiments, the gene editing system is a CRISPR / Cas system, which includes Cas effector proteins (including but not limited to Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), C2c3, Cas13b) and corresponding guide RNAs (gRNAs). The CRISPR / Cas system recruits Cas enzyme proteins to the target locus to complete the modification by guide RNAs (gRNAs), and the gRNAs include guide sequences that are complementary to the target sequences in the target locus. In certain embodiments, the gRNAs can be chimeric guide RNAs or single guide RNAs (sgRNAs). In certain embodiments, the gRNAs include guide sequences and tracr pairing sequences (or direct repeat sequences). In certain embodiments, the gRNAs include guide sequences, tracr pairing sequences (or direct repeat sequences) and tracr sequences. In certain embodiments, the CRISPR-Cas system does not include and / or does not rely on the presence of a tracr sequence (e.g., if the Cas protein is Cas12a).

[0016] RNA interference agents

[0017] In certain embodiments, the agent capable of inhibiting or knocking out Jun gene expression comprises an RNA interference agent.

[0018] As used herein, the expression "RNA interference agent" refers to any agent that inhibits target gene expression through the RNA interference (RNAi) mechanism. "RNA interference (RNAi)" is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to that of a target gene leads to sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from the target gene, thereby inhibiting the expression of the target gene.

[0019] Therefore, those skilled in the art know that they can design small interfering RNA (siRNA) or small RNA (microRNA, miRNA) molecules based on the sequence of the Jun encoding gene or its transcribed mRNA. Such siRNA or miRNA molecules can interfere with gene transcription, translation, or transcriptional and post-translational modifications, thereby affecting protein expression.

[0020] In this article, the siRNA refers to Small interfering RNA, which is a small RNA molecule consisting of approximately 21-25 nucleotides and is processed by Dicer (an enzyme in the RNAase III family that is specific for double-stranded RNA). siRNA is the main member of siRISC and stimulates the silencing of the target mRNA that is complementary to it.

[0021] As used herein, microRNA (miRNA) refers to a naturally occurring non-coding RNA molecule of approximately 21-25 nucleotides in length. Based on sequence complementarity with target mRNAs, they can regulate gene expression post-transcriptionally by specifically pairing with target mRNAs, causing target mRNA degradation or inhibiting its translation. For more information on miRNAs, refer to the miRBase database (http: / / microrna.sanger.ac.uk / ).

[0022] Furthermore, to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed, annealed, and cloned into a vector. The transcription product of this recombinant vector, a short hairpin RNA (shRNA), folds and pairs into a stem-loop structure with a stem length of 19-21 bases. These 19-21 bases are derived from a specific sequence in the target gene mRNA. This stem-loop precursor is rapidly cleaved within the cell to form a functional siRNA. An shRNA typically comprises an "antisense" sequence (or "guide" sequence) that forms a "stem" structure and specifically binds to the target sequence; a "sense" sequence (or "passenger" sequence) that is complementary or partially complementary to the "antisense" sequence; and a "loop" sequence. The shRNA expressed from this vector, after cleavage, produces siRNAs that exhibit stable expression and a long shelf life, resulting in long-lasting inhibition of target gene expression.

[0023] In certain embodiments, the RNA interfering agent is selected from small interfering RNA (siRNA), small hairpin RNA (shRNA), or micro RNA (miRNA).

[0024] In certain embodiments, the RNA interfering agent is used or administered in the form of an expression vector (eg, an expression vector comprising a coding sequence for the RNA interfering agent).

[0025] In certain embodiments, the RNA interfering agent is shRNA.

[0026] In certain embodiments, the shRNA comprises a first region complementary to a target sequence in the Jun gene or its mRNA, a second region that is in the opposite direction and complementary to the first region, and a loop region.

[0027] In certain embodiments, the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 19. For example, the first region of the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 19.

[0028] In certain embodiments, the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 18.

[0029] In another aspect, the present application also provides an shRNA or its coding sequence, or a vector comprising the shRNA or its coding sequence. In certain embodiments, the shRNA comprises the nucleotide sequence shown in SEQ ID NO: 19. In certain embodiments, the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO: 18.

[0030] antisense oligonucleotides

[0031] In certain embodiments, the agent comprises an antisense oligonucleotide.

[0032] In this article, the expression "antisense oligonucleotide" refers to a molecule that is complementary to a sense nucleic acid, for example, complementary to the coding strand of the Jun gene or complementary to the mRNA sequence of the Jun gene. Therefore, the antisense oligonucleotide can form a hydrogen bond with the sense nucleic acid (i.e., anneal with it). The antisense oligonucleotide can be complementary to the entire coding strand of the nucleic acid sequence encoding Jun, or only complementary to a portion thereof, such as all or part of the protein coding region (or open reading frame). The antisense oligonucleotide can also be antisense to all or part of the non-coding region of the coding strand of the nucleic acid sequence encoding Jun. The length of the antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30 or more nucleotides.

[0033] The antisense oligonucleotides can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, naturally occurring nucleotides or various modified nucleotides can be used for chemical synthesis, which are designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine-substituted nucleotides. Alternatively, the antisense oligonucleotides can be produced biologically using an expression vector containing a target nucleic acid cloned therein, and the RNA transcribed from the inserted nucleic acid will have an antisense orientation to the target nucleic acid.

[0034] After obtaining the RNA interfering agent or antisense oligonucleotide, the inhibitory activity of the RNA interfering agent or antisense oligonucleotide on the mRNA or protein expression level of Jun can be further determined using the method described above.

[0035] Application

[0036] The agents described herein can be administered in the form of pharmaceutical compositions with pharmaceutically acceptable carriers and / or excipients. "Pharmaceutically acceptable carriers and / or excipients" refer to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and are well known in the art and include, but are not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0037] Reagents as described herein can be used by any suitable method known in the art.Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration refers to a mode of administration generally by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, non-parenteral routes can be used, such as topical, epidermal or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

[0038] In certain embodiments, the agents described herein are administered to a specific organ or tissue or cell (e.g., heart, myocardial tissue, myocardial cells) of a subject. For example, in certain embodiments, the agents described herein are administered in situ (e.g., heart orthotopic injection, myocardial tissue orthotopic injection) to the subject.

[0039] In certain embodiments, the agents described herein are administered in an organ or tissue or cell-specific (e.g., heart-specific, myocardial tissue-specific, myocardial cell-specific) manner. For example, in certain embodiments, the agents described herein are endowed with targeting capabilities (e.g., the ability to target the heart, myocardial tissue, or myocardial cells), e.g., the ability to accumulate and / or exert activity at a subject's target site (e.g., heart, myocardial tissue, or myocardial cells).

[0040] The reagents described herein can be formulated into a dosage form compatible with its intended route of administration. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: a reagent as described herein is mixed with the necessary dose in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use.

[0041] The agents described herein can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0042] The agents described herein are administered to a subject in need thereof. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0043] Drug screening

[0044] In another aspect, the present invention provides a method for screening a drug for preventing and / or treating heart failure with preserved ejection fraction (HFpEF), the method comprising the step of screening a Jun inhibitor. The screening step is performed in vitro.

[0045] In certain embodiments, the Jun inhibitor is capable of inhibiting or downregulating the expression of a Jun gene, or inhibiting or blocking the activity of a Jun protein.

[0046] In certain embodiments, the Jun inhibitor is selected from the agents described herein that are capable of inhibiting or knocking out Jun gene expression.

[0047] In certain embodiments, the Jun inhibitor is selected from an affinity molecule that specifically binds to the Jun protein, thereby inhibiting or blocking the activity of the Jun protein. In certain embodiments, the affinity molecule is selected from a small molecule compound.

[0048] In certain embodiments, the step of screening Jun inhibitors comprises: detecting whether the test agent can inhibit Jun gene expression or Jun protein activity; and selecting the test agent that can inhibit Jun gene expression or Jun protein activity as a candidate drug.

[0049] In certain embodiments, the inhibition of Jun gene expression comprises inhibition at the protein level and / or mRNA level.

[0050] In certain embodiments, the step of screening for Jun inhibitors comprises: (1) contacting a test agent with cells capable of expressing a Jun gene; (2) measuring the expression level of the Jun gene or the activity of the Jun protein; (3) comparing the measurement result of step (2) with the measurement result in the absence of the test agent; (4) selecting the test agent that has the ability to inhibit the expression of the gene or the activity of the protein as a candidate drug. In certain embodiments, the expression level includes protein level and / or mRNA level.

[0051] Definition of terms

[0052] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0053] As used herein, the term "heart failure with preserved ejection fraction (HFpEF)" refers to a clinical syndrome in which, in the presence of normal or mildly reduced ventricular systolic function, impaired ventricular diastolic function and reduced compliance lead to reduced ventricular filling and increased filling pressure, thereby causing congestion in the pulmonary and systemic circulations. HFpEF typically refers to heart failure with diastolic dysfunction. In certain embodiments, the clinical diagnostic criteria for HFpEF mainly include: (1) the presence of symptoms and / or signs of heart failure; (2) cardiac imaging examination (mainly TTE examination) indicating LVEF ≥ 50%; (3) objective evidence of cardiac structural and / or functional abnormalities consistent with left ventricular diastolic dysfunction and / or increased left ventricular filling pressure, wherein the structural and / or functional abnormality indicators of left ventricular diastolic dysfunction and / or increased ventricular filling pressure mainly include: (a) mean E / e' ratio > 15; (b) left atrial volume index > 40 ml / m 2 (Atrial Fibrillation).

[0054] As used herein, the term "Jun" refers to the Jun proto-oncogene, AP-1 transcription factor subunit (Jun proto-oncogene, AP-1 transcription factor subunit), also known as AP1, AP-1, cJUN, or c-Jun. Jun can be human or a homologous gene from another species (e.g., non-human mammals, fish, reptiles, or birds, such as rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, primates, etc.). The sequence of Jun is well known to those skilled in the art and can be found in various public databases. An exemplary gene sequence of human Jun can be found in GenBank: NM_002228.4, and an exemplary protein sequence can be found in NCBI: NP_002219.1; an exemplary gene sequence of mouse Jun can be found in Ensembl: ENSMUSG00000052684, NCBI Gene ID: 16476, and an exemplary protein sequence can be found in UniProtKB: P05627, NCBI: NP_034721.1.

[0055] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (if not receiving treatment).

[0056] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount is also the amount at which the therapeutic benefit exceeds any toxic or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following results, such as eliminating or reducing risk, alleviating severity, or delaying the onset of the disease, including the biochemistry of the disease, histology and / or behavioral symptoms, the intermediate pathological phenotype presented during its complication and disease formation. For therapeutic use, the beneficial or desired result includes clinical results, such as reducing one or more symptoms from the disease, improving the quality of life of those subjects suffering from the disease, reducing the dosage of other drugs needed for treating the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and / or prolonging survival. An effective amount can be used in one or more administrations. Beneficial effects

[0057] The inventors of this application have discovered for the first time that Jun is a key regulator of the development and progression of HFpEF. Inhibiting or knocking out Jun gene expression can effectively curb the development and progression of HFpEF and improve diastolic function, thereby providing important clinical value for the prevention and treatment of HFpEF. Furthermore, methods for screening drugs for the prevention and / or treatment of HFpEF are provided.

[0058] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1: Construction of the HFpEF model. A: Schematic diagram of the experimental process; B: Diastolic function (E / A) of mice after 5 weeks of feeding; C: Diastolic function (E / E') of mice after 5 weeks of feeding.

[0060] Figure 2: Relative expression of Jun in cardiomyocytes after 15 weeks of HFD+L-NAME feeding, indicating that Jun is highly expressed in HFpEF model mice.

[0061] Figure 3: Myh6-cre ERT2 / Jun-Rosa26 LSL / - Jun overexpression efficiency testing in mice. A: Schematic diagram of the experimental process; B: Real-time quantitative PCR analysis of Jun-OE overexpression on day five of tamoxifen therapy; C: Immunofluorescence staining demonstrating Jun overexpression in Jun-OE mice; D: Immunofluorescence staining of lung and heart tissues in Jun-OE mice.

[0062] Figure 4: Myh6-cre ERT2 / Jun-Rosa26 LSL / - Diastolic function test in mice. A: Schematic diagram of left ventricular diastolic function test in mice; B: Statistical results of left ventricular E / A ratio in mice; C: Statistical results of left ventricular E / E' ratio in mice.

[0063] Figure 5: Jun flox / flox Schematic diagram of the mouse construction strategy.

[0064] Figure 6: Jun flox / flox Electropherogram of PCR analysis of the 5' and 3' homology arms of F1 mice. Numbers: F1 mouse number; WT: wild-type control; M: 1 kb DNA ladder.

[0065] Figure 7: Myh6-cre ERT2 / Jun flox / flox Electrophoresis diagram of PCR identification of mouse (2% agarose gel).

[0066] Figure 8: Knocking out Jun can effectively alleviate the occurrence and development of HFpEF.

[0067] Figure 9: Schematic diagram of the Jun knockdown plasmid structure.

[0068] Figure 10: Knockdown of Jun can effectively alleviate the occurrence and development of HFpEF.

[0069] Sequence information

[0070] A description of the sequences involved in this application is provided in the table below.

[0071] Table 1: Sequence information Example

[0072] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit it.

[0073] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0074] Experimental materials and methods

[0075] Animals: C57BL / 6N wild-type mice were purchased from Beijing Weitonglihua; Myh6-cre ERT2 Mice were purchased from Shanghai Model Organisms Technology Co., Ltd. The full name of the strain is C57BL / 6Smoc-Myf6em1(CreERT2-pA)Smoc (Southern Model Organisms, NM-KI-200125); Jun-Rosa26 LSL / LSL Mice were purchased from Shanghai Model Organisms Technology Co., Ltd. They were Rosa26 site-directed knock-in heterozygous mice that conditionally overexpressed the CAG-LSL-Jun-IRES-EGFP-WPRE-pA gene using CRISPR / Cas9 technology; Jun flox / floxMice were purchased from Shanghai Model Organisms Science Co., Ltd. They were Jun gene flox heterozygous mice that can be conditionally knocked out using CRISPR / Cas9 technology.

[0076] The reagents are shown in the following table:

[0077] Table 2: Reagent information

[0078] Animal Experimentation Guidelines

[0079] In this example, all animal studies were conducted under the guidance of the Laboratory Animal Center, Fuwai Hospital Animal Care and Use Committee, National Center for Cardiovascular Diseases, China. All mice were propagated and housed under the same conditions and randomly assigned to groups during the experiment. Echocardiographic analysis was performed by an independent investigator who was unaware of the study objectives.

[0080] Conventional ultrasonic testing

[0081] All mice were fed under different conditions for five weeks and then underwent routine ultrasound examinations every two weeks until the end of the fifteen-week monitoring period. Specifically, transthoracic echocardiography was performed using a VisualSonics Vevo 2100 system equipped with an MS400 transducer (Visual Sonics). Left ventricular ejection fraction (LVEF) and other systolic function indicators were obtained from short-axis M-mode scans at the level of the mid-ventricle, as indicated by the presence of papillary muscles, in conscious, lightly restrained mice. Apical four-chamber views were obtained in anesthetized mice for diastolic function measurements using pulsed wave and tissue Doppler imaging at the level of the mitral valve. Anesthesia was induced by 2.5% isoflurane and confirmed by the lack of response to firm pressure on one of the hind paws. Isoflurane was reduced to 1.0-1.5% during echocardiographic acquisition (under temperature-controlled conditions) and adjusted to keep the heart rate within 500 beats per minute. Parameters collected included heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, end-diastolic interventricular septal wall thickness, left ventricular end-diastolic posterior wall thickness, left ventricular fractional shortening, left ventricular ejection fraction (LVEF), peak Doppler velocity across the mitral valve in early diastole, peak Doppler velocity across the mitral valve in late diastole, isovolumetric relaxation time, and tissue Doppler peak relaxation velocity at the mitral annulus during early diastole and early filling deceleration. At the end of the procedure, all mice recovered from anesthesia without any abnormalities. All parameters were measured at least three times, and the mean values ​​are presented. Ultrasound testing included both systolic and diastolic function.

[0082] Example 1: Jun expression is correlated with HFpEF

[0083] 1.1 Induction of heart failure model with preserved ejection fraction

[0084] Eight- to ten-week-old male C57BL / 6N wild-type mice were divided into two groups: a normal group (normal diet and water) and a model group (high-fat diet combined with N-nitro-L-arginine methyl ester). The model group was established using the method described in the following literature: Gabriele G. Schiattarella et al., Nitrosative stress drives heart failure with preserved ejection fraction, https: / / doi.org / 10.1038 / s41586-019-1100-z. Specifically, a high-fat diet (HFD) (60% kcal from fat (lard)) and N-nitro-L-arginine methyl ester (L-NAME, 0.5 g / L in drinking water) were used to induce heart failure with preserved ejection fraction, creating a HFpEF animal model.

[0085] The systolic function parameter LVEF of the mice tested in the fifth week of model induction did not change, while the diastolic function parameter (E / E') increased significantly in the fifth week of model induction, indicating that the heart failure model with preserved ejection fraction described in the aforementioned literature has been successfully obtained. At the same time, there was no significant difference in the diastolic function parameter (E / E') between the model control group and the model treatment group at five weeks, and subsequent drug administration was carried out under the same baseline conditions, as shown in Figure 1.

[0086] Correlation between Jun expression and HFpEF

[0087] At 15 weeks of model induction, myocardial cells from normal mice and the HFpEF model were extracted and separated using a perfusion method, and quantitative RCR detection was performed. The specific procedures are as follows:

[0088] 1.2.1. Isolation of adult mouse cardiomyocytes:

[0089] In order to isolate cardiomyocytes from the heart of adult mice, we used the classic perfusion method to isolate cardiomyocytes. Specifically, 100 μl of sodium heparin (1000 units in 50 ml) was injected into the mouse 20 minutes before being killed to prevent heart coagulation during the operation, which increased the difficulty of digestion. After that, the mouse was anesthetized and killed, the heart was removed and transferred to a calcium-free solution for washing. Then, the Langendorff method was used for digestion. The heart was perfused with calcium-free solution for 5 minutes using a Langendorff apparatus, and then digested with a digestive enzyme solution (0.7 mg / ml type II collagenase and 0.7 mg / ml bovine serum albumin calcium-free solution) for about 30 minutes. After about 20 minutes, the heart was constantly touched. When the heart became soft and slippery, it indicated that the digestion was basically completed. Then, the tissue from the ventricle was collected, chopped, and gently blown to dissociate into single cells. The cells were allowed to settle, the supernatant was taken, and the undigested and adherent tissues were removed. 100 g Centrifuge at 4°C for 2 minutes to obtain a myocardial cell pellet. The supernatant is mostly non-myocardial cells. Resuspend the myocardial cells in calcium-free solution containing 10% FBS for subsequent experiments. Non-myocardial cells can be re-selected with culture medium or PBS for subsequent experiments. To obtain purer myocardial cells and non-myocardial cells, centrifuge the cell suspension (100g, 2 minutes at room temperature) three times to separate myocardial cells from non-myocardial cells. Collect myocardial cells for further experiments.

[0090] 1.2.2. Quantitative PCR detection:

[0091] Total RNA was extracted from cells using a GeneJet RNA purification kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed using an iScript™ cDNA synthesis kit (Bio-Rad, 1708890) to generate cDNA. qPCR (primer F is SEQ ID NO: 1; primer R is SEQ ID NO: 2) was performed using iTaq Universal SYBR Green supermix (1725121, Bio-Rad) on an ABI Vii7 Real-Time System (Life Technologies, Q6). β-Actin was used for standardized quantitative analysis. As shown in Figure 2, compared with normal mice, significantly higher expression of Jun was observed in the HFpEF mouse model. This indicates that Jun expression in mice is correlated with HFpEF and that Jun is highly expressed in HFpEF.

[0092] Example 2: Jun overexpression leads to heart failure with preserved ejection fraction

[0093] 2.1 Jun-overexpressing transgenic mice

[0094] Using the cardiomyocyte-specific Cre tool mouse Myh6-cre ERT2 Jun-Rosa26 in Jun-overexpressing mice LSL / LSL Myh6-cre ERT2 / Jun-Rosa26 LSL / - Myh6-cre mice, which are tamoxifen-induced overexpression of Jun in cardiomyocytes. ERT2 Mice and Jun-Rosa26 LSL / LSL All mice were produced by Shanghai Model Organisms Technology Co., Ltd.

[0095] Myh6-cre ERT2 The full name of the strain is C57BL / 6Smoc-Myf6em1(CreERT2-pA)Smoc. CreERT2-pA is inserted into the start codon of the mouse Myf6 gene. Myf6-CreERT2 is hybridized with a mouse strain containing flanking sequences of loxP sites. Induced Cre-mediated recombination will lead to the deletion of flanking sequences in the myf6-positive cells (cardiomyocytes) of the offspring.

[0096] Jun-Rosa26 LSL / LSL The mice were constructed using CRISPR / Cas9 technology, and the CAG-LSL-Jun-IRES-EGFP-WPRE-pA expression cassette was inserted into the Rosa26 gene locus by homologous recombination.

[0097] Myh6-cre ERT2 With Jun-Rosa26 LSL / LSL Myh6-cre ERT2 / Jun-Rosa26 LSL / - The mice are conditional overexpression mice models, in which tamoxifen induces Cre recombinase cre ERT Jun is specifically expressed in cardiomyocytes, where it recognizes two LoxPs in LSL and cleaves the terminator between them, resulting in the constitutive expression of Jun in Myh6-positive cells.

[0098] 2.2 Expression efficiency detection

[0099] Our study found that mice died about 6 days after tamoxifen induction (Figure 3, A). First, we tested the overexpression efficiency of mice. On the fifth day of tamoxifen administration, we isolated the cardiomyocytes of mice and extracted RNA for real-time quantitative polymerase chain reaction (PCR) to detect Jun expression levels. We found that Myh6-cre ERT2 / Jun-Rosa26 LSL / -The expression of Jun in mouse cardiomyocytes was upregulated about tenfold compared with the control group (Figure 3, B), which was in line with expectations. ERT2 / Jun-Rosa26 LSL / - The expression of GFP was higher in mouse heart sections (Figure 3, C). In addition, by staining the lung tissue sections and heart sections of Myh6-creERT2 / Jun-Rosa26LSL / - mice with Tag, GFP expression was indeed only detected in heart sections (Figure 3, D). Based on these data, we concluded that Myh6-cre ERT2 / Jun-Rosa26 LSL / Jun can be overexpressed specifically in cardiomyocytes.

[0100] 2.3 Detection of changes in diastolic function

[0101] Through experiments, we have shown that there is no significant change in contractile function and structural parameters after overexpression of Jun in adult mouse cardiomyocytes. We have also ruled out death caused by fulminant myocarditis, atrial enlargement and congestion, and severe pulmonary congestion. Taking all these into consideration, we speculate that the mice may develop diastolic dysfunction heart failure, that is, heart failure with preserved ejection fraction. Therefore, we tested the diastolic function evaluation indicators (E / E' and E / A) of the mice. As we predicted, Myh6-cre ERT2 / Jun-Rosa26 LSL / - Tamoxifen induction in mice did not show significant changes on the third day, but on the fourth and fifth days, significant abnormalities in cardiac diastolic function occurred, with E / A and E / E' values ​​significantly increasing on the fourth day and worsening on the fifth day (Figure 4, AC). Therefore, we can conclude that overexpression of Jun in adult mouse cardiomyocytes leads to acute heart failure with preserved ejection fraction, leading to death.

[0102] Example 3: Jun knockout reverses heart failure with preserved ejection fraction

[0103] 3.1 Overview

[0104] Using the cardiomyocyte-specific Cre tool mouse Myh6-cre ERT2 (Southern Model Organisms, NM-KI-200125) and Jun gene-floxed mouse Jun flox / flox Myh6-cre ERT2 / Jun flox / flox Myh6-cre mice, which can be induced by tamoxifen to specifically knock out Jun in cardiomyocytes. ERT2 / Jun flox / flox The mice may be referred to herein as Jun-KO mice.

[0105] Myh6-cre, a cardiomyocyte-specific cre tool mouse ERT2 The strain is named C57BL / 6Smoc-Myf6em1(CreERT2-pA)Smoc. CreERT2-pA is inserted into the start codon of the mouse Myf6 gene. Myf6 (myogenic factor 6) is a DNA-binding protein involved in muscle development. Crossing Myf6-CreERT2 with a mouse strain containing loxp sites flanking the target gene (i.e., floxed mice) induces Cre-mediated recombination, resulting in deletion of the gene segment between the loxp sites in the myf6-positive offspring.

[0106] Jun gene of mice modified by flox flox / flox The schematic diagram of the mouse construction strategy is shown in Figure 5. The Jun gene was floxed by homologous recombination of fertilized eggs using the principle of homologous recombination. The brief process is as follows: Cas9 mRNA and gRNA were obtained by in vitro transcription; a homologous recombination vector (donor vector) was constructed by the In-Fusion cloning method. The vector contains a 3.1kb 5' homology arm, a 2.2kb flox region and a 3.0kb 3' homology arm. Cas9 mRNA, gRNA and donor vector were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. The F0 generation mice that were positive for PCR amplification and sequencing were mated with C57BL / 6J mice to obtain 9 positive F1 generation mice. The F1 generation mice are mice whose Jun gene is floxed. flox / flox .

[0107] 3.2 Targets and related sequences

[0108] Target gene name (Ensembl number): Jun (ENSMUSG00000052684);

[0109] Ensembl website link for target gene: http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000052684;r=4:95049034-95052222;t=ENSMUST00000107094;

[0110] Transcript targeted by the protocol (Ensembl number): Jun-201 (ENSMUST00000107094.1);

[0111] Exon targeted by Flox: exon 1;

[0112] gRNA1 is SEQ ID NO: 3;

[0113] gRNA2 is SEQ ID NO: 4;

[0114] The 5' homology arm sequence is SEQ ID NO: 5;

[0115] The flox region (loxp-Jun fragment-loxp) sequence is SEQ ID NO: 6;

[0116] The 3' homology arm sequence is SEQ ID NO:7.

[0117] 3.3 Genotype identification

[0118] The primers used for PCR identification of the 5' homology arm are: Primer I (Forward) is SEQ ID NO: 8; Primer II (Reverse) is SEQ ID NO: 9.

[0119] The 3' homology arm primers are: Primer III (Forward) is SEQ ID NO: 10; Primer IV (Reverse) is SEQ ID NO: 11.

[0120] The electrophoresis results of PCR identification of the 5' and 3' homology arms of F1 mice are shown in Figure 6. PCR-positive mice were 12, 13, 14, 15, 17, 18, 19, 21, and 22; all were confirmed to be positive by sequencing.

[0121] 3.4 Subsequent breeding

[0122] Using the cardiomyocyte-specific Cre tool mouse Myh6-cre ERT2 (Southern Model Organisms, NM-KI-200125) and Jun gene-floxed mouse Jun flox / flox Myh6-cre ERT2 / Jun flox / flox mice (i.e., Jun-KO mice).

[0123] During subsequent mouse mating and breeding, mouse genotypes can be identified using short-range PCR. The PCR conditions and primers are shown below. Example results are shown in Figure 7: WT is a single 221 bp band; heterozygotes are two bands of 221 and 289 bp; and homozygotes are a single 289 bp band.

[0124] Table 3: PCR identification conditions and primers

[0125] Cre activity can also be verified at the DNA level. This is typically done by extracting genomic DNA from a small sample of Cre-expressing tissue. The floxed region is then amplified using PCR. The presence or absence of the floxed region provides a qualitative assessment of Cre activity. The PCR conditions and primers are shown below. Cre activity is indicated by a 1386 bp band; inactivity by a 3638 bp band; and wild-type by a 3505 bp band.

[0126] Table 4: PCR identification conditions and primers

[0127] 3.5 Experimental Procedure

[0128] After the animal model was obtained, that is, from the fifth week of inducing the HFpEF model, tamoxifen was used to perform gene knockout in the model treatment group, while no gene knockout was performed in the model control group. Mice that were given normal diet and drinking water throughout the induction process served as negative controls.

[0129] 3.6 Experimental Results

[0130] The results are shown in Figure 8. First, diastolic function was assessed in mice at 5 weeks, and the diastolic function parameters E / E' and E / A were significantly elevated, demonstrating diastolic dysfunction and the successful establishment of a model as described in the aforementioned literature. Furthermore, during continuous monitoring from 5 to 15 weeks, the development and progression of HFpEF were significantly suppressed in the model treatment group (Jun gene knockout using tamoxifen). Specifically, after Jun knockout, mice treated with a high-fat diet combined with L-NAME (HFD + 0.5g / L L-NAME) showed significant improvement in diastolic function, which persisted until the 15th week. However, in the model control group, where Jun was not knocked out, continued deterioration of diastolic function was observed. Furthermore, Jun expression was downregulated in the model treatment group compared to the model control group. This suggests that inhibiting or knocking out Jun expression can have both preventive and therapeutic effects on HFpEF in this mouse HFpEF model.

[0131] Example 4: Jun knockdown reverses heart failure with preserved ejection fraction

[0132] 1. Construction of knockdown plasmid

[0133] The pAAV-EnCMV-MCS-U6-shRNA plasmid (Miaoling Biotechnology, P28262) was selected, and the JUN shRNA sequence was ligated to the plasmid as shown in FIG9 to construct a JUN knockdown plasmid.

[0134] shjun forward oligo(SEQ ID NO:16):

[0135] shjun Reverse oligo(SEQ ID NO:17):

[0136] 2. Virus Packaging and Titer Determination

[0137] 2.1 293T cell culture

[0138] 1. Passaging of 293T Cells

[0139] (1) Prepare complete culture medium according to the formula shown in Table 5. Preheat complete culture medium and PBS (Zhongshan Jinqiao, ZLI-9062) in a 37°C water bath.

[0140] Table 5: Complete medium (50 mL) formulation

[0141] (2) Taking a 10 cm cell culture dish as an example, cells (GNHu44, Chinese Academy of Sciences) can be passaged when the cell density reaches about 70% to 80%, and the cell culture medium is removed using a filter pump;

[0142] (3) Add 1 mL of preheated PBS and gently tilt the culture dish back and forth at 45° to cover all cells with PBS to wash away dead cells and impurities. Remove the PBS with a suction pump.

[0143] (4) Add 1 mL of 0.25% trypsin (Gibco, 25200056) and gently tilt the culture dish back and forth at 45° to allow the trypsin to cover all cells and digest the cells. Once cells begin to fall off, immediately remove the trypsin with a suction pump.

[0144] (5) Add 2 mL of preheated complete culture medium for neutralization, gently tilt the culture dish back and forth at 45° to cover all cells with the culture medium, and gently pipette the 293T cells down to form a single-cell suspension;

[0145] (6) Place the 293T cell suspension in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 3 min at room temperature, and discard the supernatant;

[0146] (7) Add 2 mL of preheated complete culture medium to resuspend the cell pellet, take one-third of the cells and re-plate them on a new cell culture dish, add 7 mL of complete culture medium and shake thoroughly, and culture in a cell culture incubator under 5% CO2, 95% humidity, and 37°C.

[0147] 2. Cryopreservation of 293T Cells

[0148] (1) Preheat complete culture medium and PBS in a 37°C water bath;

[0149] (2) Taking a 10 cm cell culture dish as an example, when the cells are cultured to a cell density of about 70% to 80%, they can be passaged and the cell culture medium can be removed using a suction pump;

[0150] (3) Add 1 mL of preheated PBS and gently tilt the culture dish back and forth at 45° to cover all cells with PBS to wash away dead cells and impurities. Remove the PBS with a suction pump.

[0151] (4) Add 1 mL of 0.25% trypsin and gently tilt the culture dish back and forth at 45° to allow the trypsin to cover all cells and digest the cells. Once cells begin to fall off, immediately remove the trypsin with a suction pump.

[0152] (5) Add 2 mL of preheated complete culture medium for neutralization, gently tilt the culture dish back and forth at 45° to cover all cells with the culture medium, and gently pipette the 293T cells down to form a single-cell suspension;

[0153] (6) Place the 293T cell suspension in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 3 min at room temperature, and discard the supernatant;

[0154] (7) Add 2 mL of freezing solution to resuspend the cell pellet, take 1 mL of cell suspension and dispense it into cryovials. Store the cryovials in a freezing box and slowly cool them at -80°C.

[0155] (8) The next day, transfer the cryovials to a -196°C liquid nitrogen tank for long-term storage.

[0156] 2.2 AAV packaging

[0157] (1) 293T cells were seeded into 15 cm cell culture dishes using 293T cell culture medium and cultured in a cell culture incubator at 5% CO2, 95% humidity, and 37°C.

[0158] (2) Passage was performed at a ratio of 2:3 one day in advance;

[0159] (3) 2 h before transfection, replace the medium with double-antibody-free low-serum medium (prepared according to the formula shown in Table 6) at 18 ml / dish and culture in a cell culture incubator under 5% CO2, 95% humidity, and 37°C. Adeno-associated virus (AAV) packaging can be performed when the cell density reaches 80%-90% (when observing the cell density, pay attention to the density difference between the center and the edge for judgment);

[0160] Table 6: Formula for low serum dual antibody-free culture medium (50 mL)

[0161] (4) Prepare the AAV packaging system (prepared and used immediately): the molar ratio of pAAV-ITR, pAAV-2 / 9n (Miaoling Biotechnology, P12267), and pAdDeltaF6 (Miaoling Biotechnology, P10945) is 1:1:1. Calculate the proportion of each plasmid in 80 μg of total plasmid according to the molar ratio, add 3 mL of Opti-MEM (Gibco, 31985070), and let it stand for 5 minutes to mix thoroughly.

[0162] (5) Prepare the transfection reagent using a PEI concentration of 3 times the amount of DNA. Add 3 mL of Opti-MEM to 240 μL of PEI (Polysciences, 24765-1). Tilt the tube 45° and gently invert it to mix thoroughly. After the system is prepared, let it stand for 5 minutes to mix thoroughly.

[0163] (6) After mixing for 5 minutes, add the plasmid mixture to the PEI liposome mixture, mix well, and let it stand for 15 minutes (note: do not blow or vibrate, PEI is a liposome, and vigorous shaking can easily cause structural damage. Tilt the test tube 45° and gently invert it back and forth to mix well) to obtain a plasmid PEI mixture;

[0164] (7) Aspirate the excess culture medium without double antibody in the 293T cell culture dish, leaving about 14 ml / dish, slowly add about 6 ml of the plasmid PEI mixture along the side wall of the cell culture dish, with a total volume of about 20 mL / dish, gently tilt the culture dish to mix, and culture in a cell culture incubator under 5% CO2, 95% humidity, and 37°C. Change the medium 24 hours after plasmid transfection, use double antibody-free low serum medium, 20 mL / 15 cm culture dish, 5% CO2, 95% humidity, and culture in a cell culture incubator under 37°C;

[0165] (8) After changing the medium, continue culturing for 48-72 hours. If CPE is observed, harvest the 293T cells promptly. Gently blow the cells off with a pipette and collect the culture medium and cell pellet in a 50 ml centrifuge tube. Centrifuge at 2000 rpm for 10 minutes at 4°C. After removing the supernatant, freeze the cell pellet at -80°C until lysis and AAV extraction.

[0166] 2.3 AAV extraction

[0167] (1) Take out the frozen AAV-packaged 293T cell pellet from the -80°C freezer and resuspend it in 9 mL of PBS (autoclaved);

[0168] (2) Add 1 mL of Lysis Buffer (0.5% Sodium deoxycholate + 0.1% SDS + 1% Triton TMX-100) and PF68 (MP Biomedicals, 092750016) at a final concentration of 0.001%, mixed well, and incubated in a 37°C water bath for 15 min (Sodium deoxycholate (Sigma-Aldrich, D6750-25G); SDS (VWR, 0227-1KG); Triton TM X-100(Sigma-Aldrich,T8787-100ML));

[0169] (3) The cell suspension was repeatedly frozen and thawed using -196°C liquid nitrogen and 37°C water bath, and the freeze-thaw cycles were repeated four times;

[0170] (4) Add Benzonase Nuclease (Jinan Technology, M046-01B) to a final concentration of 500 U / mL and add MgCl2 to a final concentration of 2 mM. Incubate in a 37°C water bath for 45 min (nucleic acid fragments disappear).

[0171] (5) Add 4 mL of 5 M NaCl and shake for 30 s (to prevent AAV loss);

[0172] (6) Centrifuge the AAV solution at 4000 rpm for 10 min at 4°C. Collect the supernatant in a new centrifuge tube and continue centrifugation at 4000 rpm for 10 min at 4°C. Collect the supernatant in a new centrifuge tube and store in a 4°C refrigerator until density gradient centrifugation is performed. Repeat the centrifugation several times until there is almost no precipitation in the supernatant.

[0173] (7) Density gradient centrifugation in a biosafety cabinet (Thermo): Use a 39 mL ultracentrifuge tube and a 10 mL syringe (1 mL syringe needle) to sequentially add iodixanol (Sigma, D1556-250 mL) gradient solution (its formula is shown in Table 7), 5.4 mL of 60% solution (can be slightly faster), 6.5 mL of 40% solution, 6.5 mL of 25% solution, and 8 mL of 15% solution (when adding different concentrations of iodixanol, tilt the centrifuge tube and push it in slowly to avoid mixing layers, especially when adding the 15% layer. Since the density difference between 15% and 25% iodixanol is small, the 15% layer is easy to mix with the 25% layer). Finally, add 12 mL of viral supernatant and fill the remaining volume with PBS (bubbles should be prevented during the injection process. For example, 7 mL of solution should be drawn when injecting 6.5 mL of solution to prevent disturbance and bubbles from remaining in the centrifuge tube. There should be no liquid in the neck of the ultracentrifuge tube).

[0174] Table 7: Preparation formula of iodixanol gradient solution (50 mL)

[0175] The 10× Gradient Buffer was prepared according to the formula shown in Table 8.

[0176] Table 8: 10× Gradient Buffer (100 mL)

[0177] (8) Seal the ultracentrifuge tube at high temperature and centrifuge it in an ultracentrifuge at 300,000 g for 2.5 h at 10°C.

[0178] (9) Take out the ultracentrifuge tube, fix it on the iron stand, and use a 10mL syringe needle to insert it into the upper part of the centrifuge tube at a 45-degree angle to guide the gas;

[0179] (10) Use a 1 mL syringe needle equipped with a 10 mL syringe to vertically insert the syringe into the side of the centrifuge tube at the junction of the 40% gradient solution and the 60% gradient solution. Draw out the AAV saline solution in the 40% gradient solution, collect it in the centrifuge tube, and store it in a 4°C refrigerator.

[0180] 2.4 Desalting and Purification of AAV

[0181] (1) Add PBS containing 1% PF68 to the ultrafiltration centrifuge tube for column equilibration. Centrifuge for 3 minutes at 3000 g for 3 minutes, and discard the waste liquid.

[0182] (2) Add PBS containing 0.1% PF68 to the ultrafiltration centrifuge tube for column equilibration, centrifuge for 3 minutes, and then discard the waste liquid at 3000g for 3 minutes;

[0183] (3) PBS containing 0.01% PF68 was added to the ultrafiltration centrifuge tube for column equilibration. After 3 min, the column was centrifuged at 3000 g for 3 min, and the waste liquid was discarded.

[0184] (4) Add four times the volume of PBS (autoclaved) to the AAV saline solution containing iodixanol to reduce the viscosity of the solution. Mix well and add the solution to an ultrafiltration centrifuge tube for centrifugation and desalination at 3000 g for 10 min at 4°C. Centrifuge several times until 1 mL of saline solution remains.

[0185] (5) Continue to add four times the volume of PBS (autoclaved) to the AAV saline solution containing iodixanol for centrifugal desalting at 3000 g for 20 min-30 min at 4°C until 250 μL of AAV solution remains (the content of the concentrate was observed every 5 min);

[0186] (6) Add 250 μL (100 μL if the virus is small) of 0.002% PF68 in PBS (autoclaved) to rinse the ultrafiltration centrifuge tube filter membrane, and use PCR tubes to divide the AAV solution into 50 μL / tubes and freeze them in a -80°C refrigerator.

[0187] 2.5 AAV titer determination

[0188] (1) Removal of free DNA molecules in AAV samples

[0189] Pipette 5 μL of AAV into 45 μL of PBS buffer and dilute the sample 10-fold. 2 times, 10 3 times, 10 4 times, 10 5 The virus dilution was performed at 1:1 and the reaction system was prepared according to Table 9 below.

[0190] Table 9: Reaction system

[0191] The prepared reaction system was incubated at 37°C for 30 min using a PCR reactor to degrade free DNA, and then incubated at 95°C for 5 min to inactivate the DNase.

[0192] (2) Removal of AAV capsid protein

[0193] 1 μL of proteinase K (Solebo, P9460) (5 μg / μL) was added to each reaction system and incubated at 37°C for 30 min to fully remove the AAV capsid protein.

[0194] Add 30 μL of ddH2O to each of the above reaction systems for dilution and incubate at 95°C for 5 min to inactivate proteinase K.

[0195] (3) Dilute the AAV titer standard

[0196] The AAV titer standard was serially diluted to 10 7 times, 10 6 times, 10 5 times, 10 4 times, 10 3 times, 10 2 10-fold dilution.

[0197] (4) Real-time PCR reaction

[0198] The AAV nucleic acid sample and the serially diluted standard in the above reaction system were aspirated to prepare the reaction system shown in Table 10 for Real-time PCR detection.

[0199] Table 10: Real-time PCR reaction system

[0200] The reaction was carried out using a fluorescent quantitative PCR instrument ABI Quant Studio 6 according to the program shown in Table 11.

[0201] Table 11: PCR reaction program

[0202] 3. JUN Knockdown Adeno-associated Virus Injection

[0203] (1) The purified adeno-associated virus obtained in the above step was diluted to 5×10 11 The dose of copies was injected in situ into the heart.

[0204] (2) Place the injected mice on a heating pad, wait for them to wake up, and then return them to their original cages for further testing.

[0205] 4. Experimental Results

[0206] The results are shown in Figure 10. First, the diastolic function of mice was tested at 8 weeks (0 weeks after knockdown treatment), and the diastolic function parameters E / E' and E / A were significantly increased, indicating that diastolic function was impaired, indicating that the model described in the aforementioned literature was successfully obtained. Furthermore, in the continuous testing for 13 weeks (5 weeks of knockdown treatment), the occurrence and development of HFpEF in the model treatment group (Jun gene knockdown using shRNA) were well curbed. Specifically, after Jun was knocked down, the diastolic function of mice treated with a high-fat diet combined with L-NAME (HFD + 0.5g / L L-NAME) was significantly improved; however, in the model control group mice where Jun was not knocked down, a continuous deterioration of diastolic function was observed; this indicates that inhibiting or knocking down Jun expression can have a preventive and therapeutic effect on HFpEF in the mouse HFpEF model.

[0207] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a reagent capable of inhibiting or knocking out Jun gene expression in the preparation of a medicament for preventing and / or treating heart failure with preserved ejection fraction (HFpEF).

2. The use according to claim 1, wherein, The reagent is a gene editing system.

3. The use according to claim 2, wherein, The gene editing system comprises at least one site-specific nuclease.

4. The use according to claim 3, wherein, The site-specific nuclease is selected from RNA-guided nucleases (such as Cas nucleases), zinc finger nucleases, megabase meganucleases, TALE-nucleases, recombinases (such as Cre recombinase), transposases, and any combination thereof.

5. The use according to claim 1, wherein, The reagent is an RNA interfering agent or an antisense oligonucleotide.

6. The use according to claim 5, wherein, The RNA interfering agent is siRNA, shRNA or miRNA.

7. Use according to any one of claims 1-6, wherein, The reagent knocks out the Jun gene, reduces or inhibits the transcription of the Jun gene, and / or reduces or inhibits the translation of the mRNA product of Jun.

8. A method for screening a medicament for preventing and / or treating heart failure with preserved ejection fraction (HFpEF), the method comprising the step of screening for a Jun inhibitor.

9. The method according to claim 8, wherein The Jun inhibitor is capable of inhibiting or downregulating the expression of the Jun gene, or inhibiting or blocking the activity of the Jun protein.

10. The method according to claim 8, wherein, The step of screening for a Jun inhibitor comprises: detecting whether a test reagent can inhibit Jun gene expression or Jun protein activity; selecting a test reagent capable of inhibiting Jun gene expression or Jun protein activity as a candidate drug.

11. The method of claim 9 or 10, wherein, The inhibition of Jun gene expression includes inhibition at the protein level and / or the mRNA level.

12. A method for preventing and / or treating heart failure with preserved ejection fraction (HFpEF), which comprises administering to a subject (such as a human) in need thereof an effective amount of a reagent capable of inhibiting or knocking out Jun gene expression; Preferably, the reagent is defined as in any one of claims 2-7.

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