Use of an agent for inhibiting or knocking out Jun gene expression for treating heart failure with preserved ejection fraction

Abstract

The present invention relates to the field of disease treatment, and specifically provides a method for inhibiting or knocking out Jun Application of gene expression reagents in the treatment of heart failure with preserved ejection fraction (HFpEF) and methods for drug screening.

Description

Technical Field

[0001] The present invention relates to the field of disease treatment, and specifically provides a method for inhibiting or knocking out Jun Application of gene expression reagents in the treatment of heart failure with preserved ejection fraction (HFpEF) and methods for drug screening. Background Art

[0002] Cardiovascular disease represents one of the most challenging medical problems. Heart failure is the leading cause of death in patients with cardiovascular disease and a major clinical problem. It includes heart failure with systolic dysfunction and heart failure with diastolic dysfunction. With the development of medicine, heart failure with diastolic dysfunction has been more broadly defined as heart failure with preserved ejection fraction. Heart failure with preserved ejection fraction (HFpEF) is the leading cause of morbidity and mortality throughout the industrialized world, and its prevalence is growing at an alarming rate. HFpEF currently accounts for 50% of all HF. Patients with this complex disease experience typical symptoms of heart failure, including exertion intolerance, dyspnea, extravascular fluid accumulation in the lungs, subcutaneous tissue and abdominal cavity, and intermittent cardiovascular decompensation that often leads to hospitalization for emergency diuresis. The definition of HFpEF excludes diseases such as cardiac amyloidosis, hereditary hypertrophic cardiomyopathy, valvular disease and other diseases with clear etiologies. HFpEF refers more to patients whose pathophysiology involves a multi-organ syndrome, in which the heart, lungs, kidneys, bones, immunity, inflammation, metabolism and other components are jointly responsible. 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%.

[0003] 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

[0004] The inventor of the present application first discovered Jun It is an important regulatory factor for the occurrence and development of HFpEF. The following invention is provided.

[0005] Inhibition or knockout Jun Gene expression

[0006] In one aspect, the invention provides a method for inhibiting or knocking out Jun Use of an agent for inhibiting or knocking out gene expression in the preparation of a medicament for preventing and / or treating heart failure with preserved ejection fraction (HFpEF). Also provided is a method for preventing and / or treating heart failure with preserved ejection fraction (HFpEF), the method comprising administering to a subject (e.g., a human) in need thereof an effective amount of a gene that can inhibit or knock out JunIn some embodiments, the treatment comprises at least improving cardiac diastolic function, such as reducing diastolic function parameters E / E' and / or E / A.

[0007] The invention relates to the ability to inhibit or knock out Jun Agents that disrupt gene expression by any mechanism Jun Gene expression, for example by inhibiting it at the RNA or protein level Jun expression, such as knockout Jun gene, reducing or inhibiting the transcription of the gene, and / or reducing or inhibiting the translation of the mRNA product of the gene.

[0008] In certain embodiments, the determination of expression level can be implemented at the nucleic acid level or the protein level. The method of measuring expression at the nucleic acid level includes but is not limited to Northern blotting, PCR, RT-PCR or real-time (real) RT-PCR. The method of measuring expression at the protein level includes but is 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, etc.

[0009] Gene editing system

[0010] In certain embodiments, the ability to inhibit or knock out Jun The reagent for gene expression is a gene editing system. Those skilled in the art are familiar with the use of a gene editing system to modify a target gene (e.g. Jun ) for knockdown or knockout.

[0011] In certain embodiments, the gene editing system can be any site-specific (sequence-specific) genome editing system currently known. 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 meganuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof. In certain embodiments, the site-specific endonuclease targets a Jun Genes are induced to break DNA at the target site, and modifications are completed by, for example, homologous recombination (HR) or non-homologous end joining (NHEJ) to result in Jun Gene disruption.

[0012] 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.

[0013] In certain embodiments, the gene editing system comprises an RNA-guided endonuclease and a guide RNA (gRNA), the gRNA comprising a guide sequence having complementarity with a target sequence in a target locus. In certain embodiments, the gene editing system is present on one or more vectors.

[0014] In certain embodiments, the gene editing system is a CRISPR / Cas system, which comprises a Cas effector protein (including but not limited to Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), C2c3, Cas13b) and a corresponding guide RNA (gRNA). The CRISPR / Cas system recruits the Cas enzyme protein to the target locus through a guide RNA (gRNA) to complete the modification, and the gRNA comprises a guide sequence that is complementary to the target sequence in the target locus. In certain embodiments, the gRNA can be a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr pairing sequence (or a direct repeat sequence). In certain embodiments, the gRNA comprises a guide sequence, a tracr pairing sequence (or a direct repeat sequence), and a tracr sequence. In certain embodiments, the CRISPR-Cas system does not include and / or does not rely on the presence of a tracr sequence (for example, if the Cas protein is Cas12a).

[0015] RNA interference agents

[0016] In certain embodiments, the ability to inhibit or knock out Jun Gene expression inhibitors include RNA interference agents.

[0017] In this article, the expression "RNA interference agent" refers to any agent that inhibits the expression of a target gene 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 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.

[0018] Therefore, those skilled in the art know that Jun The sequence of the coding gene or its transcribed mRNA is selected as the target sequence to design small interfering RNA (siRNA) or small RNA (microRNA, miRNA) molecules. The siRNA or miRNA molecule can interfere with the transcription, translation or transcription and post-translational modification of the gene, thereby affecting the expression of the protein.

[0019] 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 to double-stranded RNA). siRNA is a major member of siRISC and stimulates the silencing of complementary target mRNA.

[0020] In this article, microRNA (miRNA) refers to a naturally occurring non-coding RNA molecule with a length of about 21-25 nucleotides. Based on sequence complementarity with the target mRNA, it can cause target mRNA degradation or inhibit its translation through specific base pairing with the target mRNA, thereby regulating gene expression after transcription. miRNA can refer to the miRBase database (http: / / microrna.sanger.ac.uk / ).

[0021] In addition, in order to prolong the inhibitory effect of siRNA on target gene expression, a pair of specific oligonucleotide sequences can be designed and cloned into a vector after annealing. The transcription product of the recombinant vector is short hairpin RNA (shRNA), which can fold and pair into a stem-loop structure with a stem length of 19-21 bases, wherein the 19-21 bases are derived from a specific sequence of the target gene mRNA. The precursor of this stem-loop structure is quickly cleaved in the cell to form a functional siRNA. The shRNA expressed by this vector is sheared to form a siRNA with stable expression and long duration, thus causing long-term inhibition of target gene expression.

[0022] In certain embodiments, the RNA interfering agent is selected from small interfering RNA (siRNA), small hairpin RNA (shRNA) or micro RNA (miRNA).

[0023] Antisense Oligonucleotides

[0024] In certain embodiments, the agent comprises an antisense oligonucleotide.

[0025] As used herein, the expression "antisense oligonucleotide" refers to a molecule complementary to a sense nucleic acid, such as Jun The coding strand of a gene is complementary or Jun The antisense oligonucleotide can form hydrogen bonds with the sense nucleic acid (i.e., anneal with it). Jun The antisense oligonucleotides may be complementary to the entire coding strand of a nucleic acid sequence, or only to a portion thereof, such as all or part of a protein coding region (or open reading frame). Jun The antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30 or more nucleotides in length.

[0026] The above 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, and the modified nucleotides are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as thiophosphate derivatives and acridine substituted nucleotides. Alternatively, the above antisense oligonucleotides can be produced biologically using an expression vector comprising 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.

[0027] After obtaining the RNA interfering agent or antisense oligonucleotide, the method described above can be used to further determine the effect of the RNA interfering agent or antisense oligonucleotide on Jun The inhibitory activity at the mRNA or protein expression level.

[0028] Application

[0029] 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, which 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.

[0030] Reagents as described herein can be applied 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 administration methods usually by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, non-parenteral routes of administration may be used, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0031] The reagents described herein can be formulated into dosage forms compatible with their intended routes 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: the reagents described herein are 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.

[0032] The agents described herein may 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.

[0033] 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.

[0034] Drug Screening

[0035] 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 screening Jun The screening step is performed in vitro.

[0036] In certain embodiments, the Jun Inhibitors can inhibit or downregulate Jun Gene expression, inhibition or blocking Jun Activity of protein.

[0037] In certain embodiments, the Jun The inhibitor is selected from the group consisting of the proteins described herein that can inhibit or knock out Jun Gene Expression Reagents.

[0038] In certain embodiments, the Jun Inhibitors are selected from the group consisting of Jun Affinity molecules for proteins, thereby inhibiting or blocking Jun In certain embodiments, the affinity molecule is selected from small molecule compounds.

[0039] In certain embodiments, the screening Jun The inhibitor step includes: testing whether the test agent can inhibit Jun Gene expression or JunProtein activity; select for inhibition Jun Gene expression or Jun In some embodiments, the expression level includes protein level and / or mRNA level.

[0040] In certain embodiments, the screening Jun The inhibitor step includes: (1) making the test agent interact with the Jun Gene cell contact; (2) Determination Jun The expression level of the gene or Jun (3) comparing the assay result of step (2) with the assay result in the absence of the test agent; (4) selecting a test agent that has the ability to inhibit the gene expression or protein activity as a candidate drug.

[0041] Definition of terms

[0042] 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 the relevant terms are provided below.

[0043] 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 slightly 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 circulation and systemic circulation. 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 examinations (mainly TTE examinations) 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 abnormalities 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).

[0044] As used herein, the term “ Jun ” refers to 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. JunIt may be of human origin 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, cows, sheep, pigs, goats, primates, etc.). Jun The sequences are well known to those skilled in the art and can be found in various public databases. Jun An exemplary gene sequence of can be found in GenBank: NM_002228.4, and an exemplary protein sequence can be found in NCBI: NP_002219.1; Mouse Jun An exemplary gene sequence 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.

[0045] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is 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 progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0046] 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. The effective amount is also the amount of any toxic or adverse effect of the treatment that the therapeutic benefit exceeds. For preventive use, the beneficial or desired result includes the following result, 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 complications 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 objects suffering from the disease, reducing the dosage of other drugs required for the treatment of the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and / or prolonging survival. The effective amount can be used in one or more administrations. Beneficial Effects

[0047] The inventor of the present application first discovered Jun It is an important regulatory factor in the development of HFpEF. JunGene expression can effectively inhibit the occurrence and development of HFpEF and improve diastolic function, thereby being used for the prevention and treatment of HFpEF and having important clinical value. In addition, a method for screening drugs for the prevention and / or treatment of HFpEF is also provided.

[0048] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present 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

[0049] Figure 1 :Construction of HFpEF model. A: Schematic diagram of the experimental process; B: Detection results of diastolic function (E / A) of mice after 5 weeks of feeding; C: Detection results of diastolic function (E / E') of mice after 5 weeks of feeding.

[0050] Figure 2 :Myocardial cells after 15 weeks of HFD+L-NAME feeding Jun The relative expression level of Jun Highly expressed in HFpEF model mice.

[0051] Figure 3 : Myh6 -cre ERT2 / Jun -Rosa26 LSL / - Mouse Jun Overexpression efficiency detection. A: Schematic diagram of the experimental process; B: Real-time quantitative PCR detection Jun- OE overexpression reached the fifth day after tamoxifen; C: Immunofluorescence staining proved Jun exist Jun -OE mice overexpress; D: Jun Immunofluorescence staining of lung and heart tissues in OE mice.

[0052] Figure 4 : Myh6-cre ERT2 / Jun-Rosa26 LSL / - Diastolic function test of mice. A: Schematic diagram of left ventricular diastolic function test of mice; B: Statistical results of E / A of left ventricle of mice; C: Statistical results of E / E' of left ventricle of mice.

[0053] Figure 5 : Jun flox / flox Schematic diagram of the mouse construction strategy.

[0054] Figure 6: Jun flox / flox Electrophoresis of PCR identification of 5' homology arms and 3' homology arms of F1 generation mice. Numbers: F1 generation mouse number; WT: wild type control; M: 1kb DNA ladder.

[0055] Figure 7 : Myh6-cre ERT2 / Jun flox / flox Electrophoresis diagram of PCR identification of mice (2% agarose gel).

[0056] Figure 8 : Knockout Jun It can effectively alleviate the occurrence and development of HFpEF.

[0057] Sequence information

[0058] A description of the sequences involved in this application is provided in the table below.

[0059] Table 1: Sequence information.

[0060]

[0061]

[0062]

[0063]

[0064] DETAILED DESCRIPTION

[0065] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0066] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0067] Experimental Materials and Methods

[0068] 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- Myf6 em1(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 obtained using CRISPR / Cas9 technology; Jun flox / flox The mice were purchased from Shanghai Model Organisms Technology Co., Ltd. They were Jun gene flox heterozygous mice that could be conditionally knocked out using CRISPR / Cas9 technology.

[0069] The reagents are shown in the table below.

[0070] Table 2: Reagent information.

[0071]

[0072] Animal Experimentation Guidelines

[0073] In this example, all animal studies were conducted under the guidance of the Laboratory Animal Center of the Animal Care and Use Committee of the Fuwai Hospital of the National Center for Cardiovascular Diseases of China. All mice were propagated and raised in the same environment, and the mice were randomly divided into groups during the experiment. Echocardiographic analysis was performed by an independent researcher who was unaware of the research objectives.

[0074] Conventional Ultrasonic Testing

[0075] All mice were routinely examined by ultrasound starting after five weeks of feeding under different conditions and every two weeks until the end of the fifteenth week of monitoring. 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 indices 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, gently 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 in the range of 500 beats per minute. The 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, left ventricular fractional shortening, LVEF, peak Doppler blood flow velocity across the mitral valve in early diastole, peak Doppler blood flow velocity across the mitral valve in late diastole, isovolumetric relaxation time, tissue Doppler peak of myocardial relaxation velocity at the mitral annulus in early diastole and early filling deceleration time. At the end of the procedure, all mice recovered from anesthesia without any abnormalities. All parameters were measured at least 3 times, and the average value was given. Ultrasound testing included systolic and diastolic function testing.

[0076] Embodiment 1: Jun The expression of TNF-α was correlated with HFpEF.

[0077] 1.1 Induction of heart failure model with preserved ejection fraction

[0078] Eight to ten-week-old male C57BL / 6N wild-type mice were divided into three groups, namely a normal group (normal diet and drinking water) and a model group (high-fat diet combined with N-nitro-L-arginine methyl ester). The model group was modeled by the following method described in the 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 (abbreviated as L-NAME, 0.5 g / L in drinking water) were used to induce heart failure with preserved ejection fraction to obtain a HFpEF animal model.

[0079] The systolic function parameter LVEF of mice detected 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 recorded in the above 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. Subsequent drug administration was carried out under the same baseline, such as Figure 1 shown.

[0080] 1.2 Jun Correlation between expression of β-catenin and HFpEF

[0081] When the model was induced for 15 weeks, the cardiomyocytes of normal mice and the mouse HFpEF animal model were extracted and separated by perfusion method, and quantitative RCR detection was performed as follows.

[0082] 1.2.1. Isolation of adult rat cardiomyocytes:

[0083] 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 coagulation of the heart during the operation, which increased the difficulty of digestion. After that, the mouse was anesthetized, killed, and 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, cut into pieces, and gently blown to dissociate into single cells. The sediment was allowed to stand, and the supernatant was taken to remove the undigested and adherent tissues. 100 g 4 ° C Centrifuge for 2 minutes to obtain cardiomyocyte sedimentation, and the supernatant is mostly non-cardiomyocytes. Cardiomyocytes are resuspended in calcium-free solution containing 10% FBS for subsequent experiments, and non-cardiomyocytes can be reselected with culture medium or PBS for subsequent experiments. If you want to obtain purer cardiomyocytes and non-cardiomyocytes, you can centrifuge the cell suspension (100g, 2 minutes at room temperature) three times to separate cardiomyocytes from non-cardiomyocytes. Collect cardiomyocytes for further experiments.

[0084] 1.2.2. Quantitative PCR detection:

[0085] Total RNA was extracted from cells using the GeneJet RNA purification kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed using the iScript TM cDNA synthesis kit (Bio-Rad, 1708890) to generate cDNA, which was then subjected to qPCR using iTaqUniversl SYBR Green supermix (1725121, Bio-Rad) on an ABI Vii7 real-time system (Life Technologies, Q6) (primer F is SEQ ID NO: 1; primer R is SEQ ID NO: 2), and b-Actin was used for normalization of quantitative analysis. Figure 2 As shown, compared with normal mice, significant changes were observed in the mouse HFpEF animal model. Jun This indicates that in mice Jun The expression of is correlated with HFpEF. Jun It is highly expressed.

[0086] Embodiment 2: Jun Overexpression leads to heart failure with preserved ejection fraction

[0087] 2.1 Jun Overexpression transgenic mice

[0088] Using cardiomyocyte-specific Cre tool mice Myh6-cre ERT2 and Jun Overexpressing mice Jun-Rosa26 LSL / LSL Hybridization Myh6-cre ERT2 / Jun-Rosa26 LSL / - Mice, namely tamoxifen-induced Jun In cardiomyocyte-specific overexpression mice. Myh6-cre ERT2 Mice and Jun-Rosa26 LSL / LSL All mice were constructed by Shanghai Model Organisms Technology Co., Ltd.

[0089] Myh6-cre ERT2 The full name of the strain is C57BL / 6Smoc- Myf6 em1(CreERT2-pA)Smoc , CreERT2-pA was inserted into the start codon of the mouse Myf6 gene, and Myf6-CreERT2 was crossed with a mouse strain containing the flanking sequence of the loxP site. Inducing Cre-mediated recombination will lead to the deletion of the flanking sequence in the myf6-positive cells (cardiomyocytes) of the offspring.

[0090] Jun-Rosa26 LSL / LSL The mouse was constructed using CRISPR / Cas9 technology, and the CAG-LSL-Jun-IRES-EGFP-WPRE-pA expression frame was inserted into the Rosa26 gene locus by homologous recombination.

[0091] Myh6-cre ERT2 and Jun-Rosa26 LSL / LSL Hybridization Myh6-cre ERT2 / Jun-Rosa26 LSL / - Mouse, which is a conditional overexpression mouse model, in which tamoxifen induces Cre recombinase cre ERT Specifically expressed in cardiomyocytes, it recognizes the two LoxPs in LSL and cuts the terminator between them, making Myh6 Positive cells continue to express Jun .

[0092] 2.2 Expression efficiency detection

[0093] 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 cardiomyocytes from mice and extracted RNA for real-time quantitative polymerase chain reaction (PCR) detection. Jun Expression levels, found Myh6 -cre ERT2 / Jun -Rosa26 LSL / - Mouse cardiomyocytes Jun The expression level of Figure 3 , B) is consistent with expectations. Next, we used immunofluorescence staining to demonstrate that Jun exist Myh6-cre ERT2 / Jun-Rosa26 LSL / - Higher expression in mouse heart slices ( Figure 3 , C). In addition, by staining the lung tissue sections and heart sections of Myh6-creERT2 / Jun-Rosa26LSL / - mice with Tag, namely GFP, it was indeed only detected in the heart sections ( Figure 3 , D). Through these data, we conclude that Myh6- cre ERT2 / Jun-Rosa26 LSL / It can be achieved JunSpecific overexpression in cardiomyocytes.

[0094] 2.3 Detection of changes in diastolic function

[0095] We have demonstrated that adult mouse cardiomyocytes Jun After overexpression, there was no significant change in contractile function and structural parameters, and death caused by fulminant myocarditis was ruled out. Atrial enlargement and congestion occurred, and severe pulmonary congestion occurred. Taking all factors into consideration, we speculated that the mice had heart failure with diastolic dysfunction, 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 / - There was no significant change on the third day after tamoxifen induction in mice, but from the fourth to the fifth day, the diastolic function of the mice showed obvious abnormalities, that is, the E / A value and the E / E' value increased significantly on the fourth day and intensified on the fifth day ( Figure 4 , AC). Therefore, we can conclude that adult mouse cardiomyocytes Jun After overexpression, the mice developed acute heart failure with preserved ejection fraction and died.

[0096] Embodiment 3: Jun Knockout reverses heart failure with preserved ejection fraction.

[0097] 3.1 Overview

[0098] Using cardiomyocyte-specific Cre tool mice Myh6-cre ERT2 (Southern Model Organisms, NM-KI-200125) and mice with Jun gene modified by flox Jun flox / flox Hybridization Myh6-cre ERT2 / Jun flox / flox Mice, which can be induced by tamoxifen Jun Knockdown specifically in cardiomyocytes. Myh6-cre ERT2 / Jun flox / flox Mice may be referred to herein as Jun -KO mice.

[0099] Cardiomyocyte-specific Cre tool mouse Myh6-cre ERT2 The full name of the strain is C57BL / 6Smoc- Myf6 em1 (CreERT2-pA)Smoc, insert CreERT2-pA into the start codon of the mouse Myf6 gene. Myf6 (myogenic factor 6) is a DNA binding protein involved in muscle development. Myf6-CreERT2 is hybridized with a mouse strain (i.e., flox mice) that has loxp sites inserted on both sides of the target gene, and inducing Cre-mediated recombination will lead to the deletion of the gene fragment between the loxp in the myf6-positive cells of the offspring.

[0100] Mice with Jun gene modified by flox Jun flox / flox Schematic diagram of the mouse construction strategy Figure 5 As shown, 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 In-Fusion cloning, which contained a 3.1 kb 5' homology arm, a 2.2 kb flox region, and a 3.0 kb 3' homology arm. Cas9 mRNA, gRNA, and donor vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 mice. F0 mice that were positive for PCR amplification and sequencing were mated with C57BL / 6J mice to obtain 9 positive F1 mice. F1 mice are mice with the Jun gene modified by flox. Jun flox / flox .

[0101] 3.2 Targets and related sequences

[0102] Target gene name (Ensembl number): Jun (ENSMUSG00000052684);

[0103] Target gene Ensembl website link: http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000052684;r=4:95049034-95052222;t=ENSMUST00000107094;

[0104] Transcripts targeted by the protocol (Ensembl number): Jun-201 (ENSMUST00000107094.1);

[0105] Exons targeted by Flox: exon 1;

[0106] gRNA1 is SEQ ID NO: 3;

[0107] gRNA2 is SEQ ID NO: 4;

[0108] The 5' homology arm sequence is SEQ ID NO: 5;

[0109] The flox region (loxp-Jun fragment-loxp) sequence is SEQ ID NO: 6;

[0110] The 3' homology arm sequence is SEQ ID NO: 7.

[0111] 3.3 Genotype identification

[0112] The primers for 5' homology arm PCR identification are: Primer I (Forward) is SEQ ID NO: 8; Primer II (Reverse) is SEQ ID NO: 9.

[0113] The 3' homology arm primers are: Primer III (Forward) is SEQ ID NO: 10; Primer IV (Reverse) is SEQ ID NO: 11.

[0114] The results of PCR electrophoresis for the 5' and 3' homology arms of F1 mice are as follows: Figure 6 The positive mice identified by PCR were No. 12, 13, 14, 15, 17, 18, 19, 21, and 22, and all were confirmed to be positive by sequencing.

[0115] 3.4 Subsequent breeding

[0116] Using cardiomyocyte-specific Cre tool mice Myh6-cre ERT2 (Southern Model Organisms, NM-KI-200125) and mice with Jun gene modified by flox Jun flox / flox Hybridization Myh6-cre ERT2 / Jun flox / flox Mice (i.e., Jun -KO mice).

[0117] In the subsequent mouse mating and breeding process, the mouse genotype can be identified by short fragment PCR. The PCR identification conditions and primers are shown below. Figure 7 As shown, WT is a band of 221 bp; heterozygotes are two bands of 221 and 289 bp; homozygotes are: a band of 289 bp.

[0118] Table 3: PCR identification conditions and primers.

[0119]

[0120] In addition, Cre activity can be verified at the DNA level. Usually, a small piece of tissue expressing Cre is taken, genomic DNA is extracted, and the flox region is amplified by PCR. The presence or absence of the flox region can be used to qualitatively determine whether Cre is working. The PCR identification conditions and primers are shown below. Cre activity is a 1386 bp band; no Cre activity is a 3638 bp band; the wild type is a 3505 bp band.

[0121] Table 4: PCR identification conditions and primers.

[0122]

[0123] 3.5 Experimental Procedure

[0124] After the animal model was obtained, i.e., 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.

[0125] 3.6 Experimental Results

[0126] The results are as follows Figure 8 First, the diastolic function of mice was tested at 5 weeks, and the diastolic function parameters E / E' and E / A increased significantly, proving that the diastolic function was impaired, indicating that the model described in the above literature was successfully obtained. Furthermore, in the continuous testing for 5 to 15 weeks, the model treatment group (using tamoxifen for Jun The occurrence and development of HFpEF in the gene knockout group were well suppressed, as shown in Jun After being knocked out, the diastolic function of mice treated with a high-fat diet combined with L-NAME (HFD + 0.5g / L L-NAME) was significantly improved and lasted until the fifteenth week; Jun In the model control group mice that were not knocked out, a continuous deterioration of diastolic function was observed; at the same time, the expression of Jun in the model treatment group was downregulated compared with the model control group. This suggests that by inhibiting or knocking out Jun The expression of TNF-α can prevent and treat HFpEF in the mouse HFpEF model.

[0127] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Able to knock out Jun Use of a gene reagent in the preparation of a drug for preventing and / or treating heart failure with preserved ejection fraction; wherein, The reagents are CRISPR / Cas9 and Cre recombinase.

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