Use of Ago2 in preparing drugs for treating heart failure or diabetic cardiomyopathy, and its protein, gene, and transformant

Abstract

The present invention "the use of Ago2 in the preparation of drugs for treating heart failure or diabetic cardiomyopathy and its protein, gene, and transformant" belongs to the field of biomedicine. The present invention provides the use of Ago2 in the preparation of drugs for treating heart failure or diabetic cardiomyopathy, and based on the new use of Ago2, provides a drug for treating heart failure or diabetic cardiomyopathy and a preparation method, as well as proteins, genes, recombinant expression vectors and transformants that have the effect of treating heart failure or diabetic cardiomyopathy. The present invention has confirmed through animal experiments that the anti-heart failure drug of the present invention based on Ago2 can significantly improve the cardiac function of animals with heart failure, and play an effective role in preventing heart failure and diabetic cardiomyopathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to use of Ago2 in preparing drugs for treating heart failure or diabetic cardiomyopathy, and its protein, gene, transformant and drug. Background Art

[0002] Cardiovascular disease has become the leading cause of death in China. Heart failure is a clinical syndrome of different cardiovascular diseases developing to the terminal stage. Although some new drugs have been used in clinical practice in recent years, about 50% of heart failure patients still die within 5 years after diagnosis. 。 Heart failure has a poor prognosis and high mortality rate. It is one of the main causes that threaten human health and increase the medical burden.

[0003] Diabetes is one of the major diseases that threaten human health. It is estimated that by 2025, the number of people with diabetes worldwide will reach 300 million. Among them, 80% of diabetic patients eventually die of cardiovascular complications, and diabetic cardiomyopathy is the leading cause of death in diabetic patients. The pathogenesis of diabetic cardiomyopathy involves glucose toxicity, lipotoxicity, cell apoptosis and necrosis, impaired calcium balance, mitochondrial dysfunction, changes in myocardial insulin signaling, and oxidative stress. Among them, hyperglycemia is considered to be the most important factor in the occurrence and development of diabetic cardiomyopathy. However, many large-scale clinical studies have shown that for patients with diabetes and heart failure, strict blood sugar control does not improve the prognosis of patients with diabetic heart failure. For example, large-scale randomized controlled clinical trials (ACCORD, ADVANCE, VADT, UKPDS) aimed at studying the effect of blood sugar control on cardiovascular event outcomes have shown that intensive blood sugar control can reduce the incidence of myocardial infarction, but it does not reduce the readmission rate and mortality rate of heart failure. This phenomenon indicates that diabetic patients have been in a state of hyperglycemia, and even after hypoglycemic treatment, they are still prone to diabetes-related cardiovascular complications, which is called the "hyperglycaemic memory" phenomenon. Clinically, there is currently no effective treatment for diabetic cardiomyopathy, so it is urgent to discover new treatments.

[0004] Currently, adenovirus and lentivirus are mostly used as expression vectors for gene therapy, but lentivirus is mostly modified from leukemia virus or HIV, and adenovirus vectors have a short expression time and are immunogenic to the body, which are not suitable for future clinical applications. Recombinant adeno-associated virus vector (rAAV) overcomes the shortcomings that other gene expression vectors are difficult to overcome, has no immunogenicity, and can drive the target gene to be expressed in the body for a long time, thus becoming the most promising vector for gene therapy.

[0005] MicroRNA (miRNAs) are a newly discovered class of non-coding small RNAs that play an important role in regulating gene expression. Mature miRNAs generally bind to the 3' untranslated region (3'UTR) sequence of their target mRNAs completely or incompletely through the principle of base complementary pairing. Studies have found that miRNAs are involved in heart failure caused by various causes. In recent years, studies have found that the drug CDR132L designed for miR-132 has good safety and tolerability in humans, without obvious toxic reactions (EurHeart J. 2021; 42(2): 178). At the same time, CDR132L can significantly reduce the patient's heart failure marker NT-proBNP, significantly narrow the QRS wave of the electrocardiogram, and also show a downward trend in myocardial fibrosis markers. However, CDR132L does not significantly improve the EF value of patients with heart failure. Therefore, it is necessary to find more important miRNAs in heart failure and key regulatory factors for miRNA function. Ago2 has been reported to be a core protein that regulates miRNAs (Science. 2014; 346(6209): 608-13; Cell. 2014; 158(3): 607). However, there have been no reports on the association between Ago2 and heart failure, or Ago2 and diabetic cardiomyopathy. There is even less research on the therapeutic effects of Ago2 in heart failure, the therapeutic effects of Ago2 in diabetic cardiomyopathy, and the use of related therapeutic drugs. Summary of the invention

[0006] Based on the above-mentioned problems and deficiencies that objectively exist in this field, the inventors accidentally discovered the relationship between mitochondrial Ago2 and the pathogenesis of heart failure and diabetic cardiomyopathy, and further conducted molecular experiments to develop an anti-heart failure drug and a drug for treating diabetic cardiomyopathy with mitochondrial Ago2 as the drug target. At the same time, animal experiments were conducted to verify the effects, and it was found that high expression of mitochondrial-targeted Ago2 can significantly improve the heart function of mice and treat heart failure and diabetic cardiomyopathy.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions:

[0008] Use of Ago2 in preparing medicine for treating heart failure or diabetic cardiomyopathy.

[0009] The target of the drug for treating heart failure or diabetic cardiomyopathy is Ago2;

[0010] Preferably, the Ago2 is mitochondrial-targeted Ago2;

[0011] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, and mouse Ago2.

[0012] A drug for treating heart failure or diabetic cardiomyopathy, comprising: a pharmacologically active ingredient; characterized in that the pharmacologically active ingredient comprises: a substance that highly expresses mitochondrial-targeted Ago2.

[0013] The substance highly expressing mitochondrial-targeted Ago2 is selected from: an expression vector connected to a leader peptide sequence capable of locating mitochondria and an Ago2 gene sequence, a transformant transformed with an expression vector connected to a leader peptide sequence capable of locating mitochondria and an Ago2 gene sequence, an expression vector connected in sequence to a myocardial-specific promoter, a leader peptide sequence capable of locating mitochondria and an Ago2 gene sequence, and a transformant transformed with an expression vector connected in sequence to a myocardial-specific promoter, a leader peptide sequence capable of locating mitochondria and an Ago2 gene sequence;

[0014] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0015] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0016] Preferably, the leader peptide sequence capable of localizing mitochondria is selected from the group consisting of: Cox8, Su9, Sod2;

[0017] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0018] Preferably, the expression vector connected to the leader peptide sequence that can locate mitochondria and the Ago2 gene sequence is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected to the myocardial specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence in sequence;

[0019] Preferably, the host cell of the transformant is selected from: 293 cells;

[0020] Preferably, the transformant transformed with an expression vector connected with a leader peptide sequence capable of localizing mitochondria and an Ago2 gene sequence is selected from: a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or a recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2;

[0021] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0022] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0023] A protein having the effect of treating heart failure or diabetic cardiomyopathy, characterized by comprising: a leader peptide sequence and an Ago2 sequence that can locate mitochondria.

[0024] The leader peptide that can localize to mitochondria is selected from: Cox8, Su9, Sod2;

[0025] Preferably, the protein having the effect of treating heart failure or diabetic cardiomyopathy is a fusion protein composed of Cox8 and Ago2.

[0026] A gene having the effect of treating heart failure or diabetic cardiomyopathy, characterized by comprising: a leader peptide gene sequence and an Ago2 gene sequence that can locate mitochondria.

[0027] The gene having the effect of treating heart failure or diabetic cardiomyopathy further comprises: a myocardium-specific promoter;

[0028] Preferably, the leader peptide that can localize to mitochondria is selected from: Cox8, Su9, Sod2;

[0029] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0030] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0031] Preferably, the gene having the effect of treating heart failure or diabetic cardiomyopathy is selected from: a gene consisting of a Cox8 gene sequence and an Ago2 gene sequence, or a gene consisting of a myocardial-specific promoter tnt sequence, a Cox8 gene sequence and an Ago2 gene sequence;

[0032] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0033] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0034] A recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy, characterized in that it is an expression vector connected to the sequence of the gene having the effect of treating heart failure or diabetic cardiomyopathy.

[0035] The expression vector is selected from: an adeno-associated virus expression vector pAAV-D(+);

[0036] Preferably, the recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected in sequence to the myocardial-specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence.

[0037] A transformant having the effect of treating heart failure or diabetic cardiomyopathy, characterized in that it is a host cell transformed with the recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy.

[0038] The host cell is selected from: 293 cells;

[0039] Preferably, the transformant having the effect of treating heart failure or diabetic cardiomyopathy is selected from: recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2.

[0040] A method for preparing a drug for treating heart failure or diabetic cardiomyopathy, characterized in that a fusion protein consisting of a leader peptide capable of localizing to mitochondria and Ago2 is expressed.

[0041] Connecting the gene sequence of the leader peptide that can locate mitochondria and the gene sequence of Ago2 to an expression vector to obtain a recombinant expression vector;

[0042] Preferably, the myocardial-specific promoter, the gene sequence of the leader peptide that can locate mitochondria, and the gene sequence of Ago2 are sequentially connected to an expression vector to obtain a recombinant expression vector;

[0043] Preferably, the recombinant expression vector is transformed into a host cell to obtain a transformant;

[0044] Preferably, the recombinant expression vector is transformed into a host cell using a calcium-phosphate co-transfection method to obtain a transformant;

[0045] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0046] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0047] Preferably, the leader peptide sequence capable of localizing mitochondria is selected from the group consisting of: Cox8, Su9, Sod2;

[0048] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0049] Preferably, the recombinant expression vector is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected in sequence to the myocardial specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence;

[0050] Preferably, the transformant is selected from: a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or a recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2;

[0051] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0052] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0053] Under the premise that the patent laws of some countries or regions allow, the present invention also requests protection for substances that highly express mitochondrial-targeted Ago2, or, a fusion protein consisting of a leader peptide sequence and an Ago2 sequence that can locate mitochondria, or, a fusion protein consisting of Cox8 and Ago2, or, a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to a Cox8 gene sequence and an Ago2 gene sequence, or, a myocardial-specific promoter tnt, a Cox8 gene sequence, and an Ago2 gene sequence connected in sequence. The present invention relates to the use of the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2, or the recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or the recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 in the treatment of heart failure or diabetic cardiomyopathy.

[0054] The present invention provides a drug for treating heart failure or diabetic cardiomyopathy, characterized in that the active ingredient of the drug includes a substance that takes mitochondrial-targeted Ago2 as a drug target and can increase the expression of mitochondrial-targeted Ago2.

[0055] The active ingredients of the drug include substances that highly express mitochondrial-targeted Ago2.

[0056] The mitochondrial-targeted Ago2 substance comprises a mitochondrial targeting signal sequence fragment and an Ago2 sequence fragment, and the sequence fragment of only cox8-Ago2 is shown in, for example, SEQ ID NO.1 and SEQ ID NO.2.

[0057] The medicament also includes pharmaceutically acceptable excipients, and / or reagents for buffering, synthesizing, and / or purifying the sequence fragments.

[0058] A method for preparing a drug for treating heart failure or diabetic cardiomyopathy, characterized by comprising: using a substance that can highly express the mitochondrial-targeted Ago2 as an active ingredient of the drug for treating heart failure or diabetic cardiomyopathy.

[0059] The sequence fragment capable of expressing Cox8-Ago2 is inserted into the expression vector, thereby preparing a recombinant plasmid capable of stably expressing the granulocyte-targeted Ago2. The sequence fragment of Cox8-Ago2 is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0060] One object of the present invention is to provide a drug for treating heart failure or diabetic cardiomyopathy, wherein the active ingredient of the drug includes mitochondrial-targeted Ago2 as a drug target, and the drug effect of treating heart failure or diabetic cardiomyopathy is achieved by a substance that highly expresses the mitochondrial-targeted Ago2.

[0061] High expression: Ago2 containing mitochondrial localization sequences can be highly expressed in mitochondria through various vectors. Ago2 can be highly expressed in mitochondria through leading signals such as mitochondrial localization sequences such as Cox8, Su9, Sod2, etc.; or Ago2 can be delivered into mitochondria through mitochondrial targeted nanoparticles;

[0062] Any of the above methods can play a role in increasing the expression of mitochondrial-targeted Ago2. Any behavior of packaging the above substances into a product package marked with anti-heart failure use for commercial purposes on any scale falls within the scope of protection claimed by the present invention.

[0063] Furthermore, the active ingredients of the drug include substances that highly express mitochondrial-targeted Ago2.

[0064] Furthermore, the substance that highly expresses mitochondrial-targeted Ago2 comprises a leader peptide sequence fragment of mitochondrial-localized cox8 and a sequence fragment of Ago2 as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0065] Furthermore, the drug also includes pharmaceutically acceptable adjuvants and / or reagents for buffering, synthesizing, and / or purifying the sequence fragments. Those skilled in the art can add various pharmaceutically acceptable adjuvants / adjuvants to the anti-heart failure drug of the present invention according to objective needs to prepare various dosage forms for easy sale or promotion.

[0066] Another object of the present invention is to provide a method for preparing a drug for treating heart failure or diabetic cardiomyopathy, comprising: using a substance that highly expresses mitochondrial-targeted Ago2 as an active ingredient of the drug for treating heart failure or diabetic cardiomyopathy.

[0067] In a further embodiment, the preparation method comprises: inserting a sequence fragment containing a leader peptide sequence of mitochondrial-targeted cox8 and Ago2 into an expression vector, thereby preparing a recombinant plasmid of mitochondrial-targeted Ago2 that can be stably expressed.

[0068] In a specific embodiment, the expression vector comprising the leader peptide sequence of mitochondrial localized cox8 and the sequence fragment of Ago2 is an adeno-associated virus expression vector pAAV-D(+).

[0069] In order to achieve the purpose of gene therapy for heart failure or diabetic cardiomyopathy, the present invention recombined the mitochondrial targeted cox8-Ago2 with a recombinant adeno-associated virus vector containing the myocardial specific promoter tnt, and obtained a high titer that meets the treatment requirements after detection, and confirmed in animal experiments that it can effectively improve the cardiac function of mice with heart failure or diabetic cardiomyopathy. Therefore, based on the above findings and results, the present invention provides a drug for the treatment of clinical heart failure or diabetic cardiomyopathy with mitochondrial Ago2 as a therapeutic target.

[0070] The present invention relates to a drug for treating heart failure or diabetic cardiomyopathy. The anti-heart failure / anti-diabetic cardiomyopathy drug relates to the construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-cox8-Ago2) that can specifically target mitochondrial Ago2, and the drug effect of treating heart failure or anti-diabetic cardiomyopathy is achieved by highly expressing the mitochondrial-targeted rAAV-Ago2. The present invention utilizes a chemical synthesis method to construct a pAAV-D(+)-tnt-cox8-Ago2 expression plasmid, and utilizes a three-plasmid calcium phosphate co-transfection method to package and prepare a recombinant adeno-associated virus containing a target fragment and purify it. Animal experiments have confirmed that the anti-heart failure and anti-diabetic cardiomyopathy drugs provided by the present invention can significantly improve the cardiac function of animals with heart failure, and play an effective role in preventing heart failure or diabetic cardiomyopathy.

[0071] Based on the mitochondrial targeting cox8 leading base sequence and Ago2 base sequence, the present invention designs and synthesizes the mitochondrial targeting Ago2 sequence, and successfully inserts it into the eukaryotic expression vector pAAV-D(+) to form the recombinant plasmid pAAV-D(+)-cox8-Ago2. Afterwards, the following three plasmids: 1) pXX9 plasmid, 2) phelper plasmid, 3) pAAV-D(+)-cox8-Ago2 plasmid, are respectively transferred into 293 cells by calcium-phosphate co-transfection method to package and prepare recombinant adeno-associated virus (rAAV9) capable of expressing mitochondrial targeting cox8-Ago2, and after purification, the titer is determined by real-time PCR method. In the next step, the recombinant adeno-associated virus (rAAV-cox8-Ago2) of the same serotype was packaged and prepared and injected into the heart failure model mice induced by aortic ligation (TAC) via the tail vein. It was found that the long-term expression of mitochondrial Ago2 in the mouse heart changed significantly, indicating that the recombinant adeno-associated virus can mediate the long-term effective expression of mitochondrial Ago2. Ultrasound and catheter results showed that the expression of mitochondrial-targeted Ago2 mediated by the recombinant adeno-associated virus can significantly improve the cardiac function of TAC mice, further supporting the anti-heart failure effect of mitochondrial-targeted Ago2.

[0072] At the same time, the recombinant adeno-associated virus (rAAV-cox8-Ago2) of the same serotype was packaged and prepared and injected into the heart function damage model mice caused by type 1 diabetes (streptozotocin STZ) and type 2 diabetes (leptin receptor gene mutation db / db) through the tail vein. It was found that the long-term expression of mitochondrial Ago2 in the mouse heart changed significantly, indicating that the recombinant adeno-associated virus can mediate the long-term effective expression of mitochondrial Ago2. Ultrasound and catheter results show that the expression of mitochondrial-targeted Ago2 mediated by recombinant adeno-associated virus can significantly improve the cardiac function of diabetic mice, further supporting the anti-diabetic cardiomyopathy effect of mitochondrial-targeted Ago2. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0074] Figure 1 It shows that the Ago2 content localized in the heart mitochondria of mice with heart failure is significantly increased; A is the western blot result of Ago2 content in sham mice and TAC mice; B is a bar graph of Ago2 content in sham mice and TAC mice; the meanings of the marks in the figure are as follows: VDAC1 represents the internal reference protein of mitochondria; sham represents the sham operation control group; TAC represents the heart failure model mice caused by TAC.

[0075] Figure 2 The figure shows the plasmid structure of pAAV-D(+)-tnt-cox8-Ago2, wherein tnt promoter represents a cardiomyocyte-specific promoter, and m-Ago2 represents mouse-derived Ago2.

[0076] Figure 3 The Western blotting of Experimental Example 2 detected the expression of Ago2 in the hearts of TAC mice with different treatments; the meanings of the marks in the figure are listed as follows: rAAV-tnt-GFP represents TAC mice injected with a recombinant adeno-associated virus vector expressing GFP that specifically targets cardiomyocytes, rAAV-tnt-cox8-Ago2 represents TAC mice injected with a recombinant adeno-associated virus vector that specifically targets cardiomyocytes and highly expresses Ago2 in mitochondria, and VDAC1 is represented as an internal reference protein of mitochondria; the test results show that rAAV-tnt-cox8-Ago2 can significantly increase the expression of Ago2 in the heart mitochondria of TAC mice.

[0077] Figure 4 This is the effect of rAAV-tnt-cox8-Ago2 treatment on the cardiac function of TAC mice monitored by cardiac ultrasound and catheter in Experimental Example 2, wherein A is a bar graph of ejection fraction, B is a bar graph of shortening fraction, C is a bar graph of the maximum rate of increase of left ventricular pressure during isovolumetric contraction, and D is a bar graph of the minimum rate of increase of left ventricular pressure during isovolumetric contraction; the results showed that rAAV-tnt-cox8-Ago2 can significantly increase the cardiac contractile and diastolic functions of TAC mice.

[0078] Figure 1-Figure 4The meanings of the markers are listed as follows: rAAV-tnt-GFP represents TAC mice injected with a recombinant adeno-associated virus vector expressing GFP that specifically targets cardiomyocytes; rAAV-tnt-cox8-Ago2 represents TAC mice injected with a recombinant adeno-associated virus vector that specifically targets cardiomyocytes and highly expresses Ago2 in mitochondria; control represents TAC mice injected with normal saline as a blank control; sham represents sham-operated control mice; and TAC represents TAC-induced heart failure model mice.

[0079] Figure 5 The western blot results of Experimental Examples 3 and 4 for the Ago2 content localized in the heart mitochondria of diabetic cardiomyopathy mice show that the Ago2 content localized in the heart mitochondria of diabetic cardiomyopathy mice is significantly reduced; wherein, A is a western blot result of the Ago2 content in the heart mitochondria of control mice and STZ-induced type 1 diabetic mice; B is a bar graph of the Ago2 content in the control mice and STZ mice; C is a western blot result of the Ago2 content in the heart mitochondria of BKS control mice and db / db type 2 diabetic mice; D is a bar graph of the Ago2 content in the BKS control mice and db / db mice; the meanings of the marks in the figure are as follows: VDAC1 represents the internal reference protein of mitochondria; Control represents the control group; STZ represents the type 1 diabetic model mice caused by STZ; BKS represents the db / db littermate control normal mice, and db / db represents the type 2 diabetic model mice caused by gene mutation.

[0080] Figure 6 The Western blotting of Experimental Example 3 detected the expression of Ago2 in the hearts of STZ mice with different treatments; the meanings of the marks in the figure are listed as follows: rAAV-tnt-GFP represents STZ mice injected with recombinant adeno-associated virus vectors expressing GFP that specifically targets cardiomyocytes, rAAV-tnt-cox8-Ago2 represents mice injected with recombinant adeno-associated virus vectors that specifically target cardiomyocytes and highly express Ago2 in mitochondria, and VDAC1 is represented as the internal reference protein of mitochondria; the test results show that rAAV-tnt-cox8-Ago2 can significantly increase the expression of Ago2 in the heart mitochondria of STZ mice.

[0081] Figure 7This is a bar graph of the effect of rAAV-tnt-cox8-Ago2 treatment on the cardiac function of STZ-induced type 1 diabetic mice monitored by cardiac ultrasound and catheter in Experimental Example 3, wherein A is a bar graph of ejection fraction, B is a bar graph of shortening fraction, C is a bar graph of the maximum rate of rise of left ventricular pressure during isovolumetric contraction, and D is a bar graph of the minimum rate of rise of left ventricular pressure during isovolumetric contraction; the results show that rAAV-tnt-cox8-Ago2 can significantly increase the cardiac systolic and diastolic functions of type 1 diabetic mice. The meanings of the marks in the figure are listed as follows: rAAV-tnt-GFP represents STZ mice injected with a recombinant adeno-associated virus vector expressing GFP that specifically targets myocardial cells, rAAV-tnt-cox8-Ago2 represents diabetic mice injected with a recombinant adeno-associated virus vector that specifically targets myocardial cells and highly expresses Ago2 in mitochondria; control represents mice injected with saline as a blank control; STZ represents type 1 diabetic model mice caused by STZ.

[0082] Figure 8 This is a graph of Western blotting results for detecting Ago2 in the hearts of db / db mice with different treatments in Experimental Example 4; the meanings of the markers in the figure are listed as follows: rAAV-tnt-GFP represents db / db mice injected with a recombinant adeno-associated virus vector expressing GFP that specifically targets cardiomyocytes, rAAV-tnt-cox8-Ago2 represents db / db mice injected with a recombinant adeno-associated virus vector that specifically targets cardiomyocytes and highly expresses Ago2 in mitochondria, and VDAC1 is represented as an internal reference protein of mitochondria; the test results show that rAAV-tnt-cox8-Ago2 can significantly increase the expression of Ago2 in the heart mitochondria of db / db mice.

[0083] Fig. 9 This is a bar graph of the effect of rAAV-tnt-cox8-Ago2 treatment on cardiac function of db / db type 2 diabetic mice monitored by cardiac ultrasound and catheter in Experimental Example 4, wherein A is a bar graph of ejection fraction, B is a bar graph of shortening fraction, C is a bar graph of the maximum rate of increase of left ventricular pressure during isovolumetric contraction, and D is a bar graph of the minimum rate of increase of left ventricular pressure during isovolumetric contraction; the results show that rAAV-tnt-cox8-Ago2 can significantly increase the cardiac systolic and diastolic functions of type 2 diabetic mice.

[0084] Figure 5-Figure 9The meanings of the markers are listed as follows: rAAV-tnt-GFP represents db / db mice injected with a recombinant adeno-associated virus vector expressing GFP that specifically targets cardiomyocytes; rAAV-tnt-cox8-Ago2 represents db / db mice injected with a recombinant adeno-associated virus vector that specifically targets cardiomyocytes and highly expresses Ago2 in mitochondria; BKS represents littermate control mice; db / db represents type 2 diabetes model mice caused by gene mutation. DETAILED DESCRIPTION

[0085] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0086] The sources of the instruments, equipment, reagents, consumables and biological materials involved in the following embodiments or experimental examples are as follows:

[0087] 1) Instruments and equipment

[0088] Nanodrop® ND-1000 nucleic acid analyzer, ABI 9700 PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge;

[0089] 2) Reagents and consumables

[0090] Streptozotocin STZ (purchased from Sigma), RNasey Mini Kit (purchased from Qiagen), TRIZOL (purchased from Invitrogen), TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0 (agarose gel DNA recovery kit, purchased from TaKaRa), EZNA® Endo-Free Plasmid Maxi Kit (plasmid extraction kit, purchased from OMEGA), EasyPure Plasmid MiniPrep Kit (plasmid extraction kit, purchased from Beijing Quanshijin Company); the eukaryotic expression vector pAAV-D(+) was constructed and donated by the collaborating Professor Xiao Xiao, and the cox8-Ago2DNA sequence fragment was synthesized by Wuhan Corester Biotechnology Co., Ltd.

[0091] Sources of biological materials:

[0092] C57 background mice were purchased from Beijing Huafukang Company;

[0093] BKS mice and db / db mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.;

[0094] 293T cells were from ATCC, USA.

[0095] Group 1 Example: Pharmaceutical Use of Ago2

[0096] This group of embodiments provides the use of Ago2 in preparing a drug for treating heart failure or diabetic cardiomyopathy.

[0097] In a specific embodiment, the target of the drug for treating heart failure or diabetic cardiomyopathy is Ago2;

[0098] Preferably, the Ago2 is mitochondrial-targeted Ago2;

[0099] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, and mouse Ago2.

[0100] Group 2 Examples: Drugs for treating heart failure or diabetic cardiomyopathy according to the present invention

[0101] This group of embodiments provides a drug for treating heart failure or diabetic cardiomyopathy. All embodiments in this group have the following common features: the drug comprises: a pharmacologically active ingredient; the pharmacologically active ingredient comprises: a substance that highly expresses mitochondrial-targeted Ago2.

[0102] In some embodiments, the substance that highly expresses mitochondrial-targeted Ago2 is selected from: an expression vector connected to a leader peptide sequence that can locate mitochondria and an Ago2 gene sequence, a transformant transformed with an expression vector connected to a leader peptide sequence that can locate mitochondria and an Ago2 gene sequence, an expression vector sequentially connected to a myocardial-specific promoter, a leader peptide sequence that can locate mitochondria, and an Ago2 gene sequence, and a transformant transformed with an expression vector sequentially connected to a myocardial-specific promoter, a leader peptide sequence that can locate mitochondria, and an Ago2 gene sequence;

[0103] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0104] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0105] Preferably, the leader peptide sequence capable of localizing mitochondria is selected from the group consisting of: Cox8, Su9, Sod2;

[0106] The leader peptide Cox8 is the Cox8 described in the article “Double deletion of PINK1 and Parkin impairs hepaticmitophagy and exacerbatesacetaminophen-induced liver injury in mice”;

[0107] The leader peptide Su9 is the Su9 described in the article “MicroRNA directly enhances mitochondrial translation during muscle differentiation”;

[0108] The leader peptide Sod2 is the Sod2 described in the paper “Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs”.

[0109] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0110] tnt is the tnt recorded in the article "Nuclear miR-320 Mediates Diabetes-Induced Cardiac Dysfunction by Activating Transcription of Fatty AcidMetabolic Genes to CauseLipotoxicity in the Heart";

[0111] α-MHC is the α-MHC recorded in the article “Deletion of BCATm increases insulin-stimulated glucoseoxidation in the heart”;

[0112] MLC-2v is the MLC-2v described in the article “Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System”;

[0113] Desmin is the Desmin described in the article “Muscle-Specific Promoters for Gene Therapy”.

[0114] Herein, heart failure and heart failure both have the conventional technical meanings well known to those skilled in the art, and are the common technical meanings expressed by the term "heart failure".

[0115] Both diabetic cardiomyopathy and diabetic cardiomyopathy have conventional technical meanings well known to those skilled in the art, and are the common technical meanings expressed by the term "diabetic cardiomyopathy".

[0116] Preferably, the expression vector connected to the leader peptide sequence that can locate mitochondria and the Ago2 gene sequence is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected to the myocardial specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence in sequence;

[0117] Preferably, the host cell of the transformant is selected from: 293 cells;

[0118] Preferably, the transformant transformed with an expression vector connected with a leader peptide sequence capable of localizing mitochondria and an Ago2 gene sequence is selected from: a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or a recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2;

[0119] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0120] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0121] This group of embodiments provides a drug for treating heart failure, wherein the active ingredient of the drug for treating heart failure uses mitochondrial Ago2 as a drug target, and the drug effect of treating heart failure is achieved by overexpressing the mitochondrial targeted Ago2. The present invention has found through some experiments that the content of Ago2 localized in the heart mitochondria of mice with heart failure is significantly increased ( Figure 1), which suggests that Ago2 in mitochondria may play a regulatory role in the pathophysiological process of heart failure. Further experiments on mice showed that increasing the content of mitochondrial Ago2 significantly improved the cardiac ejection fraction of TAC mice ( Figure 4 A), thereby enhancing cardiac function and achieving the goal of treating heart failure.

[0122] The present invention also found through other experiments that the Ago2 content in the heart mitochondria of STZ type 1 diabetes and db / db type 2 mice was significantly reduced ( Figure 5 ), which suggests that Ago2 in mitochondria may play a regulatory role in the pathophysiological process of diabetic cardiomyopathy. Further mouse experiments showed that increasing the content of mitochondrial Ago2 can significantly improve the cardiac ejection fraction of STZ and db / db diabetic mice ( Fig. 9 A, 7A), thereby enhancing cardiac function and achieving the goal of treating diabetic cardiomyopathy.

[0123] Based on many years of research experience in the clinical treatment of heart failure / diabetic cardiomyopathy, the inventors speculated that Ago2 is very likely to play a crucial role in the occurrence and development of heart failure or diabetic cardiomyopathy, and conducted relevant experiments.

[0124] The present invention has experimentally found that the level of mitochondrial Ago2 in TAC heart failure mice is significantly high, and overexpression of mitochondrial Ago2 in TAC heart failure mice can resist heart failure. Based on the inventor's many years of experience in clinical research and development, it is speculated that the logical relationship between the two may be as follows: In heart failure, the increase in mitochondrial Ago2 protein indicates that mitochondrial Ago2 may play an important role in heart failure. However, whether this increase is a compensatory protective effect or a destructive effect requires subsequent functional studies. In the functional study, the present invention found that overexpression of mitochondrial Ago2 can treat heart failure, indicating that the elevated mitochondrial Ago2 in heart failure plays a compensatory protective role, but the strength is not strong enough, and a larger amount of overexpression is needed to amplify the protective effect.

[0125] The present invention experimentally discovered that in the hearts of two types of diabetic cardiomyopathy mice, the level of mitochondrial Ago2 was reduced. Overexpression of mitochondrial Ago2 in diabetic cardiomyopathy mice can resist diabetic cardiomyopathy. Based on the inventor's many years of experience in clinical research and development, it is speculated that the logical relationship between the two may be as follows: In diabetes, the reduction of mitochondrial Ago2 protein indicates that mitochondrial Ago2 may play an important role in diabetes. However, whether this reduction is a compensatory protective effect or a destructive effect requires subsequent functional studies. In the functional study, the present invention found that overexpression of mitochondrial Ago2 can resist diabetic cardiomyopathy, indicating that the reduced mitochondrial Ago2 in anti-diabetic cardiomyopathy may be the cause of the progression of diabetic cardiomyopathy, and exogenous supplementation of Ago2 can protect the heart.

[0126] Therefore, in mice with heart failure and diabetic cardiomyopathy, although the change trends of mitochondrial Ago2 are inconsistent (the reason is unclear and needs further study), overexpression of mitochondrial Ago2 can improve cardiac function. This shows that the overexpression of mitochondrial Ago2 discovered in the present invention can play a role in treating heart failure and diabetic cardiomyopathy.

[0127] Furthermore, the active ingredients of the drug include substances that can increase mitochondrial-targeted Ago2. It is well known to those skilled in the art that a mitochondrial localization signal can be added before the Ago2 sequence to specifically increase the expression of mitochondrial Ago2 and thus play a therapeutic role. Other means can also be used, such as certain mitochondrial-targeted nanoparticles, to directly deliver Ago2 to mitochondria, which can also achieve the purpose of increasing the mitochondrial level of Ago2.

[0128] Preferably, the active ingredient of the drug comprises a mitochondrial-targeted Ago2 sequence fragment. The most common method in molecular biology is to add a mitochondrial localization signal sequence in front of the target sequence fragment to transport Ago2 to the mitochondria for therapeutic purposes. Cox8 mitochondrial signal peptide is a commonly used mitochondrial localization signal, and the sequence of co8-Ago2 is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0129] In some alternative embodiments of the present invention, some other mitochondrial localization signal sequences, such as Su9, Sod2 and other leading sequences, can also deliver Ago2 specifically to mitochondria. The recombinant plasmid of Ago2 containing these leading sequences has an anti-heart failure effect similar to or equivalent to that in the following Experimental Example 2. Due to limited space, they will not be described one by one in this article.

[0130] In some alternative embodiments of the present invention, some Ago2 sequence fragments from other species, such as human Ago2, rat Ago2 and other sequences, which are highly homologous to the mouse Ago2 gene sequence, and the recombinant plasmids containing Ago2 from these species have anti-heart failure effects similar to or equivalent to those in the following Experimental Example 2. Due to limited space, they will not be described one by one in this article.

[0131] In some alternative embodiments of the present invention, some other promoters of the recombinant adeno-associated virus plasmid pAAV-D(+), such as α-MHC, MLC-2v, Desmin and other sequences, like tnt, can achieve the effect of specifically targeting the recombinant adeno-associated virus vector to the myocardium. The recombinant plasmid pAAV-D(+)-cox8-Ago2 containing these promoter sequences has an anti-heart failure effect similar to or equivalent to that in the following Experimental Example 2. Due to limited space, they will not be described one by one in this article.

[0132] In the most specific embodiment of the present invention, the active ingredient of the drug is a recombinant adeno-associated virus plasmid pAAV-D(+)-tnt-cox8-Ago2 that expresses mitochondrial-targeted mouse Ago2; the recombinant adeno-associated virus vector (rAAV) used in the present invention overcomes the shortcomings that are difficult to overcome by other gene expression vectors. It can carry the target gene to transfect dividing and non-dividing cells (i.e., it has a wide range of transgenic genes), has no side effects (non-immunogenic), has high infection efficiency, can drive the target gene to be expressed in vivo for a long time, and has successfully solved the problem of large-scale replication in vitro without adenovirus contamination, thus becoming the most promising vector for gene therapy.

[0133] More specifically, the cox8 mitochondrial guide sequence and mus-Ago2 sequence as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted into the adenovirus expression vector pAAV-D(+) to construct the pAAV-D(+)-tnt-cox-Ago2 plasmid for expression. The above double-stranded nucleotide sequence was synthesized by Wuhan Corester Biotechnology Co., Ltd. and inserted into the vector pAAV-D(+).

[0134] Furthermore, the drug also includes pharmaceutically acceptable excipients, and / or reagents for buffering, culturing, and / or propagating the recombinant adeno-associated virus plasmid pAAV-D(+)-cox8-Ago2; those skilled in the art can add various pharmaceutically acceptable excipients / excipients to the anti-heart failure drug of the present invention according to objective needs to prepare various dosage forms for easy sales or promotion.

[0135] Group 3 Examples, Proteins of the Present Invention

[0136] This group of embodiments provides a protein having the effect of treating heart failure or diabetic cardiomyopathy. All embodiments of this group have the following common features: the protein having the effect of treating heart failure or diabetic cardiomyopathy comprises: a leader peptide sequence and an Ago2 sequence that can locate mitochondria.

[0137] In some embodiments, the leader peptide that can localize to mitochondria is selected from the group consisting of: Cox8, Su9, Sod2;

[0138] Preferably, the protein having the effect of treating heart failure or diabetic cardiomyopathy is a fusion protein composed of Cox8 and Ago2.

[0139] The fourth group of examples, genes of the present invention

[0140] This group of embodiments provides a gene that has the effect of treating heart failure or diabetic cardiomyopathy. All embodiments in this group have the following common features: the gene comprises: a leader peptide gene sequence that can locate mitochondria and an Ago2 gene sequence.

[0141] In a further embodiment, the gene having the effect of treating heart failure or diabetic cardiomyopathy further comprises: a myocardium-specific promoter;

[0142] Preferably, the leader peptide that can localize to mitochondria is selected from: Cox8, Su9, Sod2;

[0143] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0144] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0145] The m-Ago2 and mus-Ago2 in this article both represent mouse-derived Ago2.

[0146] Preferably, the gene having the effect of treating heart failure or diabetic cardiomyopathy is selected from: a gene consisting of a Cox8 gene sequence and an Ago2 gene sequence, or a gene consisting of a myocardial-specific promoter tnt sequence, a Cox8 gene sequence and an Ago2 gene sequence;

[0147] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0148] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0149] The fifth group of embodiments, the recombinant expression vector of the present invention

[0150] This group of embodiments provides a recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy. All embodiments in this group have the following common features: the recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy is an expression vector connected to the sequence of a gene having the effect of treating heart failure or diabetic cardiomyopathy provided by any one of the fourth group of embodiments.

[0151] In a specific embodiment, the expression vector is selected from: an adeno-associated virus expression vector pAAV-D(+);

[0152] Preferably, the recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected in sequence to the myocardial-specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence.

[0153] Group 6 Example, transformant of the present invention

[0154] This group of embodiments provides a transformant having the effect of treating heart failure or diabetic cardiomyopathy. All embodiments in this group have the following common features: the transformant having the effect of treating heart failure or diabetic cardiomyopathy is a host cell transformed with a recombinant expression vector having the effect of treating heart failure or diabetic cardiomyopathy provided by any one of the fifth group of embodiments.

[0155] In some embodiments, the host cell is selected from: 293 cells;

[0156] Preferably, the transformant having the effect of treating heart failure or diabetic cardiomyopathy is selected from: recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2.

[0157] Group 7 Example, Preparation Method of the Drug of the Present Invention

[0158] This group of embodiments provides a method for preparing a drug for treating heart failure or diabetic cardiomyopathy. All embodiments in this group have the following common features: expressing a fusion protein consisting of a leader peptide that can locate mitochondria and Ago2.

[0159] In some embodiments, the gene sequence of the leader peptide that can be localized to mitochondria and the gene sequence of Ago2 are connected to an expression vector to obtain a recombinant expression vector;

[0160] Preferably, the myocardial-specific promoter, the gene sequence of the leader peptide that can locate mitochondria, and the gene sequence of Ago2 are sequentially connected to an expression vector to obtain a recombinant expression vector;

[0161] Preferably, the recombinant expression vector is transformed into a host cell to obtain a transformant;

[0162] Preferably, the recombinant expression vector is transformed into a host cell using a calcium-phosphate co-transfection method to obtain a transformant;

[0163] Preferably, the Ago2 is selected from: human Ago2, rat Ago2, mouse Ago2;

[0164] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0165] Preferably, the leader peptide sequence capable of localizing mitochondria is selected from the group consisting of: Cox8, Su9, Sod2;

[0166] Preferably, the myocardium-specific promoter is selected from the group consisting of: tnt, α-MHC, MLC-2v, Desmin;

[0167] Preferably, the recombinant expression vector is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2 connected to the Cox8 gene sequence and the Ago2 gene sequence, or a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2 connected in sequence to the myocardial specific promoter tnt, the Cox8 gene sequence, and the Ago2 gene sequence;

[0168] Preferably, the transformant is selected from: a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or a recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2;

[0169] Preferably, the Cox8 gene sequence is shown as SEQ ID NO.1;

[0170] Preferably, the Ago2 gene sequence is shown as SEQ ID NO.2.

[0171] In some specific embodiments of this group, the recombinant adeno-associated virus plasmid pAAV-D(+)-cox8-Ago2 which can highly express the mitochondrial targeted substance - the sequence fragment of Ago2 containing the cox8 mitochondrial localization signal is used as the active ingredient of the anti-heart failure drug.

[0172] Furthermore, the mus-cox8-Ago2 double-stranded nucleotide was synthesized by full gene synthesis technology and cloned into the pAAV-D(+) vector, thereby preparing a recombinant plasmid that can stably express mitochondrial-targeted Ago2. In a further specific scheme, the cox8-Ago2 sequence fragment that can express mitochondrial-targeted is shown in SEQ ID NO.1 and SEQ ID NO.2; the expression vector is an adeno-associated virus expression vector pAAV-D(+).

[0173] The specific experimental operation steps of this group of embodiments can be found in Experimental Example 1.

[0174] The technical effects of the present invention are further verified and illustrated by experimental examples below.

[0175] Experimental Example 1: Construction of recombinant adeno-associated virus

[0176] 1. Construction of pAAV-D(+) vector

[0177] Based on the base sequences of the mitochondrial guide sequence Cox8 and mus-Ago2 (mouse-derived Ago2), the cox8-Ago2 double-stranded nucleotide was synthesized using full gene synthesis technology and cloned into the pAAV-D(+) vector ( Figure 2 ), and the double-stranded nucleotides were synthesized by Wuhan Krust Biotechnology Co., Ltd. The sequences of cox8 and Ago2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0178] 2. Plasmid Transformation

[0179] Add pAAV-D(+)-cox8-Ago2 plasmid to 100 μl DH5α competent cells and place in ice for 30 min; heat at 42°C for 45 sec, and place in ice for 1 min; add 500 μl antibiotic-free LB medium, shake and culture at 37°C and 100 rpm for 60 min; culture on Amp+ LB plate medium, and select white monoclonal colonies for identification.

[0180] 3. Plasmid extraction

[0181] Pick a single clone colony and add it to 3 ml of LB liquid medium containing Amp+, and culture it at 37°C with shaking at 280 rpm overnight. Kit was used to extract plasmid. The specific steps are as follows: 1. Take 1.5 ml of overnight cultured bacteria and centrifuge at 10,000g for 1 min, and try to remove the supernatant; 2. Add 250μl of colorless solution RB (containing RNaseA) and oscillate to suspend the bacterial precipitate; 3. Add 250μl of blue solution LB, gently invert and mix 4-6 times to fully lyse the bacteria and form a blue transparent solution; 4. Add 350μl of yellow solution NB, gently mix 5-6 times until a compact yellow agglomerate is formed, and let stand at room temperature for 2 min; 5. Centrifuge at 15,000g for 5 min, carefully aspirate the supernatant and add it to the adsorption column; 6. Centrifuge at 15,000g for 1 min, and discard the effluent; 7. Add 650μl of solution WB, centrifuge at 15,000g for 1 min, and discard the effluent; 8. Centrifuge at 15,000g for 2 min to completely remove the residual WB; 9. Place the adsorption column in a new Ep tube and add 20μl Preheat EB at 70°C and let stand at room temperature for 1 min; centrifuge at 10,000 g for 1 min to elute DNA, and store the eluted DNA at -20°C.

[0182] 4. Plasmid extraction

[0183] Prepare a 1L sterile conical flask, add 300ml sterile LB medium, and add ampicillin solution to a final concentration of 100μg / ml. Add 50μl of the required plasmids (pXX9, phelper, pAAV-D(+)-GFP, pAAV-D(+)-cox8-Ago2) respectively, and culture at 280rpm and 37℃ overnight. According to the instructions of OMEGA's Endo-Free Plasmid Maxi Kit, extract the plasmid. The specific steps are as follows: 1. Centrifuge at 5000g for 10min at room temperature to collect bacteria; 2. Discard the culture medium, add 10ml Solution I / RNase A mixture, vortex and shake to completely resuspend; 3. Add 10ml Solution II to the resuspended mixture, gently invert and mix 10-15 times, and place at room temperature.

[0184] 5. rAAV-mediated viral packaging

[0185] When 293T cells grow to 90%, 1-2 hours before calcium-phosphorus transfection, replace each culture dish with 12-15 ml of fresh culture medium (containing serum), add calcium chloride (CaCl2) first into a 50 ml centrifuge tube, then add plasmid to form a Ca-DNA mixed solution, mix thoroughly, slowly add 2XHEBS BUFFER to the Ca-DNA mixed solution to form a Ca-DNA-P mixed solution, add 2XHEBS while shaking the centrifuge tube, mix thoroughly to form calcium-phosphorus particles. After 8-12 hours, replace with 18-20 ml of serum-free culture medium, after 72 hours, aspirate and discard the culture medium, wash 3 times with PBS, add 1 ml of Tris+NaCl (pH 8.5) to each culture dish, scrape the cells with a scraper, collect them in a clean centrifuge tube, and freeze at -80°C.

[0186] 6. Virus Purification

[0187] Take out the cells frozen at -80℃, thaw and dissolve at 37℃, repeat freezing and thawing 4 times, centrifuge at 8,000g for 15min, put the supernatant into a clean centrifuge tube, and discard the cell pellet. Mix the rAAV with -20℃ pre-cooled anhydrous ethanol at a volume ratio of 3:1, place in a -20℃ refrigerator for 2hr, centrifuge at 4℃, 13,000rpm for 15min, and discard the supernatant; after the ethanol evaporates, add the corresponding volume of Tris+NaCl (pH 8.5) to dissolve the precipitate. Filter with a Millipore small filter (0.22μm).

[0188] 7. Virus Titer Determination

[0189] Sample processing:

[0190] rAAV virus solution 40μl

[0191] Proteinase K (20 mg / ml) 5 μl

[0192] 55°C, reaction for 1 hr;

[0193] Phenol:chloroform:isoamyl alcohol 45 μl

[0194] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C;

[0195] Chloroform 45 μl

[0196] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C.

[0197] Real-time PCR:

[0198] Primer 1 (10 μm) 0.4 μl

[0199] Primer 2 (10 μm) 0.4 μl

[0200] SYBR Green I Mix 10 μl

[0201] ddH2O 8.2μl

[0202] Template 1μl

[0203] 95℃30sec---(95℃5sec---60℃5sec---72℃20sec)×40 cycles---MeltingCurve

[0204] Experimental Example 2: Using rAAV9-type recombinant adeno-associated virus expressing cox8-Ago2 as an example to detect its therapeutic effect on heart failure

[0205] 1. Detection of Ago2 expression in cardiac mitochondria of TAC mice:

[0206] Eight-week-old C57 mice were subjected to TAC surgery: transverse aortic constriction (TAC) and sham operation control group. At the end of the experiment (4 weeks later), myocardial mitochondria were extracted using the Bio-Tian tissue mitochondrial extraction kit and the expression of mitochondrial Ago2 in the heart of TAC mice was detected by Western blot. The results showed that the content of mitochondrial Ago2 in the heart of TAC mice was significantly increased ( Figure 1 ).

[0207] Eight-week-old C57 mice were injected with rAAV-tnt-cox8-Ago2 and control rAAV-tnt-GFP viruses through the tail vein, with a virus titer of 1×10 11 PFU / mouse. Two weeks later, TAC surgery was performed: transverse aortic constriction (TAC). At the end of the experiment (four weeks later), myocardial mitochondria were extracted and Western blot was used to detect the expression of mitochondrial Ago2 in the heart of TAC mice. The results showed that rAAV-tnt-cox8-Ago2 treatment could significantly increase the expression of mitochondrial Ago2 in the heart ( Figure 3 ).

[0208] 2. TAC mouse cardiac function detection: At the end of the experiment, cardiac ultrasound was used to detect the cardiac function of TAC mice. The method is as follows:

[0209] The instrument used was an ultrasound machine equipped with a 30 MHz high-frequency probe. After the mice were anesthetized with isoflurane, they were placed supine on the detection platform. Two-dimensional images of the left ventricle were collected along the short-axis and long-axis sections at the level of the left ventricular papillary muscle beside the sternum of the mice. At the same time, more than 5 continuous cardiac cycle M-mode ultrasound images were obtained under the guidance of two-dimensional images. According to the results of the collected images, the cardiac hemodynamic indicators of cardiac ultrasound detection were obtained. The following indicators were calculated after analysis by relevant software: including heart rate (HR), left ventricular internal dimension (diastole, LVIDd), left ventricular internal dimension (systole, LVIDs), left ventricular posterior wall diastole thickness (diastole, LVPWd), left ventricular posterior wall systole thickness (LVPWs), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (systole, LVPWs), left ventricular ventricular posterior wall diastole thickness (LVPWs), left ventricular septal diastole thickness (LVSd), left ventricular septal systolic thickness (LVSd), left ventricular septal diastole ... thickness, systole, IVSs), ejection fraction (EF) and fractional shortening (FS). The results showed that rAAV-cox8-Ago2 treatment could significantly improve the cardiac function of TAC mice ( Figure 4 ).

[0210] Experimental Example 3: Using rAAV9 type recombinant adeno-associated virus expressing cox8-Ago2 as an example to detect its therapeutic effect on cardiac function damage caused by STZ-induced type 1 diabetes

[0211] 1. Detection of Ago2 expression in heart mitochondria of STZ mice:

[0212] Preparation of STZ solution: STZ was dissolved in sodium citrate buffer and freshly prepared into 8 mg / mL STZ solution. Eight-week-old C57 mice were used. The model group mice were fasted for 12 hours before surgery and intraperitoneally injected with 40 mg / kg STZ for 5 consecutive days. The control group was given the same dose of sodium citrate buffer. Three days after the STZ injection, fasting blood glucose was measured, and those with blood glucose higher than 16.7 mmol / L were included in the formal experiment. Myocardial mitochondria were extracted using the Bio-Time Tissue Mitochondrial Extraction Kit, and the expression of mitochondrial Ago2 in the heart of STZ mice was detected by Western blot. The results showed that the content of mitochondrial Ago2 in the heart of STZ mice was significantly reduced ( Figure 5 A, B).

[0213] Eight-week-old C57 mice were used to establish STZ model. After successful establishment of the model, rAAV-tnt-cox8-Ago2 and control rAAV-tnt-GFP viruses were injected through the tail vein, with a virus titer of 1×10 11 PFU / each. At the end of the experiment (16 weeks later), myocardial mitochondria were extracted and Western blot was used to detect the expression of mitochondrial Ago2 in the heart of STZ mice. The results showed that rAAV-tnt-cox8-Ago2 treatment could significantly increase the expression of cardiac mitochondrial Ago2 ( Figure 6 ).

[0214] 2. Cardiac function test of STZ mice: At the end of the experiment, cardiac ultrasound was used to test the cardiac function of STZ mice. The method is as follows:

[0215] The instrument used was an ultrasound machine equipped with a 30 MHz high-frequency probe. After the mice were anesthetized with isoflurane, they were placed supine on the detection platform. Two-dimensional images of the left ventricle were collected along the short-axis and long-axis sections at the level of the left ventricular papillary muscle beside the sternum of the mice. At the same time, more than 5 continuous cardiac cycle M-mode ultrasound images were obtained under the guidance of two-dimensional images. According to the results of the collected images, the cardiac hemodynamic indicators of cardiac ultrasound detection were obtained. The following indicators were calculated after analysis by relevant software: including heart rate (HR), left ventricular internal dimension (diastole, LVIDd), left ventricular internal dimension (systole, LVIDs), left ventricular posterior wall diastole thickness (diastole, LVPWd), left ventricular posterior wall systole thickness (LVPWs), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (systole, LVPWs), left ventricular ventricular posterior wall diastole thickness (LVPWs), left ventricular septal diastole thickness (LVSd), left ventricular septal systolic thickness (LVSd), left ventricular septal diastole ... thickness, systole, IVSs), ejection fraction (EF) and fractional shortening (FS). The results showed that rAAV-cox8-Ago2 treatment could significantly improve the cardiac function of STZ mice ( Figure 7 ).

[0216] Experimental Example 4: Using rAAV9-type recombinant adeno-associated virus expressing cox8-Ago2 as an example to detect its therapeutic effect on cardiac function damage caused by type 2 diabetes

[0217] 1. Detection of Ago2 expression in db / db mouse heart mitochondria:

[0218] Myocardial mitochondria were extracted using the Bio-Tech Mitochondrial Extraction Kit and Western blot was used to detect the expression of mitochondrial Ago2 in the hearts of db / db mice and their littermate control BKS mice. The results showed that the content of mitochondrial Ago2 in the hearts of db / db mice was significantly reduced ( Figure 5 C, D).

[0219] Eight-week-old BKS and db / db mice were injected with rAAV-tnt-cox8-Ago2 and control rAAV-tnt-GFP viruses through the tail vein, with a virus titer of 1×10 11 PFU / each. At the end of the experiment (24 weeks later), myocardial mitochondria were extracted and Western blot was used to detect the expression of mitochondrial Ago2 in the heart of db / db mice. The results showed that rAAV-tnt-cox8-Ago2 treatment could significantly increase the expression of cardiac mitochondrial Ago2 ( Figure 8 ).

[0220] 2. Detection of cardiac function of db / db mice: At the end of the experiment, cardiac ultrasound was used to detect the cardiac function of db / db mice. The method was as follows: The instrument used was an ultrasound machine equipped with a 30MHz high-frequency probe. After the mice were anesthetized with isoflurane, they were placed supine on the detection platform. Two-dimensional images of the left ventricle were collected along the short-axis and long-axis sections at the level of the left ventricular papillary muscle beside the sternum of the mice. At the same time, more than 5 continuous cardiac cycle M-mode ultrasound images were obtained under the guidance of two-dimensional images. According to the results of the collected images, the cardiac hemodynamic indicators of cardiac ultrasound detection were obtained. The following indicators were calculated after analysis by relevant software: including heart rate (HR), left ventricular internal dimension (diastole, LVIDd), left ventricular internal dimension (systole, LVIDs), left ventricular posterior wall diastole thickness (diastole, LVPWd), left ventricular posterior wall systole thickness (LVPWs), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (diastole, IVSd), interventricular septal thickness (systole, LVPWs), left ventricular ventricular posterior wall diastole thickness (LVPWs), left ventricular septal diastole thickness (LVSd), left ventricular septal systolic thickness (LVSd), left ventricular septal diastole ... thickness, systole, IVSs), ejection fraction (EF) and fractional shortening (FS). The results showed that rAAV-cox8-Ago2 treatment could significantly improve the cardiac function of db / db mice ( Fig. 9 ).

Claims

1. Use of a transformant transformed with an expression vector connected with a leader peptide sequence capable of localizing mitochondria and an Ago2 gene sequence in the preparation of a drug for treating heart failure or diabetic cardiomyopathy, characterized in that: The transformant transformed with an expression vector connected to a leader peptide sequence that can locate mitochondria and an Ago2 gene sequence is selected from: a recombinant adeno-associated virus rAAV-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-cox8-Ago2, or a recombinant adeno-associated virus rAAV-tnt-cox8-Ago2 transformed with a recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-cox8-Ago2.

2. The use according to claim 1, characterized in that The target of the drug for treating heart failure or diabetic cardiomyopathy is Ago2.

3. The use according to claim 1, characterized in that The Ago2 is mitochondria-targeted Ago2.

4. The use according to claim 2, characterized in that: The Ago2 is mitochondria-targeted Ago2.

5. The use according to any one of claims 1 to 3, characterized in that: The Ago2 is selected from: human Ago2, rat Ago2, and mouse Ago2.

Images