ShRNA (short hairpin ribonucleic acid), recombinant expression vector, transformant, anti-heart failure medicine, method and pharmaceutical application thereof

Abstract

The invention relates to shRNA (short hairpin Ribonucleic Acid), a recombinant expression vector, a transformant, an anti-heart failure medicine, a preparation method and pharmaceutical application thereof, and belongs to the field of biological medicine. The invention provides shRNA (short hairpin ribonucleic acid), which is selected from shRNA-AHFD-HF (short hairpin ribonucleic acid)-1 or shRNA-AHFD-HF (short hairpin ribonucleic acid)-2. The DNA (deoxyribonucleic acid) sequence corresponding to the shRNA-AHFD-HF-1 is as shown in SEQ ID NO. 1; dNA (deoxyribonucleic acid) sequences corresponding to the shRNA-AHFD-HF-2 comprise SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. The invention also provides a recombinant expression vector based on the shRNA, a transformant, an anti-heart failure drug and a pharmaceutical application thereof, and provides a preparation method of the anti-heart failure drug. Animal experiments prove that the anti-heart-failure medicine provided by the invention can obviously improve the heart function of animals with heart failure and has an effective anti-heart-failure effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to shRNA, its recombinant expression vector, transformant, anti-heart failure drug, preparation method and pharmaceutical use. Background Art

[0002] The number of people suffering from cardiovascular diseases in my country is as high as 330 million, and cardiovascular diseases have become the leading cause of death among Chinese people. Heart failure is the common outcome of all cardiovascular diseases in the terminal stage. According to current research, metabolic disorders and population aging are closely related to the occurrence and development of heart failure. In recent years, various new drugs for the treatment of heart failure have been used in clinical practice, but about 50% of patients still die within 5 years after diagnosis. Therefore, it is urgent to explore more effective strategies for the treatment of heart failure.

[0003] 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.

[0004] Small nucleic acid drugs refer to drugs that can use small nucleic acid molecules such as siRNA, miRNA and antisense nucleic acid ASO to specifically silence the expression of disease genes in order to cure specific diseases. Among them, siRNA refers to double-stranded RNA or hairpin structure RNA (shRNA) that is cut by nuclease to form siRNA, which generates sense strands and antisense strands under the action of helicase, and forms RNA-induced silencing complexes to cut and degrade the target gene, thereby inhibiting the expression of the target gene. Currently, there are many new small RNA drugs on the market (such as siRNA drug Inclisiran for the treatment of hyperlipidemia, ASO drug casimersen for the treatment of Duchenne muscular dystrophy, etc.), which have great clinical application prospects. However, the clinical use of small RNA drugs for the treatment of heart failure is quite limited.

[0005] Therefore, there is an urgent need to develop more small RNA drugs for anti-heart failure in this field to provide more options for doctors and patients. Summary of the invention

[0006] In order to solve the technical problem of providing more anti-heart failure small RNA drugs to increase more choices for doctors and patients, the present invention has developed a small RNA drug rAAV-shRNA-AHFD-HF (AHFD-HF, anti-high-fat-diet induced-heart-failure) that delivers short hairpin RNA (shRNA) via rAAV, and conducted animal experiments to verify its effect. It was found that rAAV-shRNA-AHFD-HF-1 and rAAV-shRNA-AHFD-HF-2 can significantly improve the cardiac function of mice and treat heart failure induced by lipid metabolism disorders.

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

[0008] A shRNA, which is selected from shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0009] A recombinant expression vector is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0010] The expression vector is pAAV D(+);

[0011] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0012] Preferably, the recombinant expression vector is selected from: pAAV-D(+)-cTNT-shRNA-AHFD-HF-1, pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2.

[0013] A transformant, which is a host transformed with a recombinant expression vector; the recombinant expression vector is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4 and SEQ ID NO.5.

[0014] The host is selected from: viruses, and / or cells;

[0015] Preferably, the virus is selected from adeno-associated virus;

[0016] Preferably, the cells are selected from 293T cells;

[0017] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0018] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0019] Preferably, the transformant is selected from: rAAV-cTNT-shRNA-AHFD-HF-1, rAAV-H1-U6-shRNA-AHFD-HF-2.

[0020] An anti-heart failure drug comprises a pharmacologically active ingredient, wherein the pharmacologically active ingredient comprises: the shRNA, and / or the recombinant expression vector, and / or the transformant.

[0021] The pharmacologically active ingredient is selected from the group consisting of: the shRNA according to claim 1, the recombinant expression vector according to claim 2 or 3, and the transformant according to claim 4 or 5;

[0022] Preferably, the anti-heart failure drug further comprises: pharmaceutical excipients.

[0023] Use of the shRNA, and / or the recombinant expression vector, and / or the transformant in preparing anti-heart failure drugs.

[0024] Preferably, the anti-heart failure means: significantly reducing the expression of AHFD-HF and improving heart function.

[0025] A method for preparing an anti-heart failure drug, comprising preparing the shRNA, and / or the recombinant expression vector, and / or the transformant.

[0026] The preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and expansion;

[0027] Preferably, the synthesis refers to whole gene synthesis technology;

[0028] Preferably, the cloning refers to connecting the shRNA to an expression vector;

[0029] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0030] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0031] The present invention designs and develops a small RNA drug that delivers short hairpin RNA (shRNA) via rAAV, which can significantly improve cardiac function and has the potential to treat heart failure.

[0032] The present invention provides use of shRNA in preparing medicine for treating heart failure.

[0033] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0034] Preferably, the expression vector promoter is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0035] Preferably, the expression vector connected to shRNA-AHFD-HF is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-shRNA-AHFD-HF connected to the shRNA-AHFD-HF sequence; or, a recombinant adeno-associated virus expression vector pAAV-D(+)-cTNT / H1-U6-shRNA-AHFD-HF connected in sequence to the myocardial specific promoter cTNT / non-specific promoter H1-U6 and the shRNA-AHFD-HF sequence; Preferably, the host cell of the transformant is selected from: 293T cells;

[0036] Preferably, the transformant transformed with the shRNA-AHFD-HF expression vector is selected from: recombinant adeno-associated virus rAAV-shRNA-AHFD-HF transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-shRNA-AHFD-HF; or, recombinant adeno-associated virus rAAV-cTNT / H1-U6-shRNA-AHFD-HF transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cTNT / H1-U6-shRNA-AHFD-HF; preferably, the sequence of the shRNA-AHFD-HF-1 is as shown in SEQ ID NO.1; the sequence of shRNA-AHFD-HF-2 is as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; A recombinant expression vector having the effect of treating heart failure, characterized in that it is an expression vector connected to the sequence of a gene having the effect of treating heart failure.

[0037] The expression vector is selected from: an adeno-associated virus expression vector pAAV-D(+);

[0038] Preferably, the recombinant expression vector having the effect of treating heart failure is selected from: a recombinant adeno-associated virus expression vector pAAV-D(+)-shRNA-AHFD-HF connected to the shRNA-AHFD-HF gene sequence.

[0039] A transformant with the effect of treating heart failure, characterized in that it is a host cell transformed with the recombinant expression vector with the effect of treating heart failure.

[0040] The host cell is selected from: 293T cells;

[0041] Preferably, the transformant having the effect of treating heart failure is selected from: recombinant adeno-associated virus rAAV-shRNA-AHFD-HF transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-shRNA-AHFD-HF. Or, recombinant adeno-associated virus rAAV-cTNT-shRNA-AHFD-HF-1, rAAV-H1-U6-shRNA-AHFD-HF-2 transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-cTNT-shRNA-AHFD-HF-1, pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2.

[0042] 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.

[0043] In a further embodiment, the preparation method comprises: inserting the shRNA-AHFD-HF sequence fragment into an expression vector, thereby preparing a recombinant plasmid that can stably express shRNA-AHFD-HF.

[0044] In a specific embodiment, the expression vector containing the shRNA-AHFD-HF sequence fragment is an adeno-associated virus expression vector pAAV-D(+).

[0045] In order to achieve the purpose of gene therapy for heart failure, the present invention recombined the shRNA-AHFD-HF sequence fragment with a recombinant adeno-associated virus vector, 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. Therefore, based on the above findings and results, the present invention provides a small RNA drug represented by shRNA-AHFD-HF for the treatment of clinical heart failure.

[0046] The present invention relates to a drug for treating heart failure. The anti-heart failure drug relates to the construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-shRNA-AHFD-HF) of shRNA-AHFD-HF, and the drug effect of treating heart failure is achieved by high expression of the rAAV-shRNA-AHFD-HF. The present invention utilizes a chemical synthesis method to construct a pAAV-D(+)-shRNA-AHFD-HF 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 drug provided by the present invention can significantly improve the cardiac function of animals with heart failure and play an effective anti-heart failure role.

[0047] The present invention designs and synthesizes the sequence of shRNA-AHFD-HF, and successfully inserts it into the eukaryotic expression vector pAAV-D(+) to form a recombinant plasmid pAAV-D(+)-shRNA-AHFD-HF. Afterwards, the following three plasmids: 1) pXX9 plasmid, 2) phelper plasmid, 3) pAAV-D(+)-shRNA-AHFD-HF plasmid, are respectively transferred into 293T cells by calcium-phosphorus co-transfection method to package and prepare recombinant adeno-associated virus (rAAV9) that can express shRNA-AHFD-HF. After purification, the titer is determined by real-time PCR. In the next step, the recombinant adeno-associated virus (rAAV-shRNA-AHFD-HF) of the same serotype prepared by packaging is injected into the heart failure model mice caused by high-fat feeding (HFD) through the tail vein. Ultrasound and catheter results show that shRNA-AHFD-HF mediated by recombinant adeno-associated virus can significantly improve the cardiac function of HFD mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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.

[0049] Figure 1 The plasmid composition is pAAV-D(+)-cTNT-shRNA-AHFD-HF of Experimental Example 1 of the present invention; Figure 1 The meanings of the marks are listed as follows: cTNT promoter represents a cardiomyocyte-specific promoter sequence, ITR represents a nucleotide sequence repeated at both ends of the adeno-associated virus (AAV) genome in opposite directions (Inverted Terminal Repeat), shRNA-AHFD-HF refers to the shRNA corresponding to SEQ ID NO.1 of the present invention, and Amp represents an ampicillin resistance gene sequence.

[0050] Figure 2 The plasmid composition is pAAV-D(+)-H1-U6-shRNA-AHFD-HF of Experimental Example 1 of the present invention; Figure 1The meanings of the marks are listed as follows: H1-U6 promoter represents the small RNA promoter sequence, ITR represents the nucleotide sequence repeated at both ends of the adeno-associated virus (AAV) genome in opposite directions (Inverted Terminal Repeat), shRNA-AHFD-HF refers to the shRNA corresponding to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 of the present invention, and Amp represents the ampicillin resistance gene sequence.

[0051] Figure 3 Experimental Example 2 of the present invention verifies the knockdown efficiency of the recombinant adeno-associated virus rAAV-shRNA-AHFD-HF-1 containing sh-AHFD-HF-1 (SEQ ID NO.1) in the high-fat-induced mouse heart.

[0052] Figure 4 Experimental Example 2 of the present invention verifies the knockdown efficiency of the recombinant adeno-associated virus rAAV-shRNA-AHFD-HF-2 containing sh-AHFD-HF-2 (SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5) in the high-fat-induced mouse heart.

[0053] Figure 5 The effect of the treatment of recombinant adeno-associated virus rAAV-shRNA-AHFD-HF-1 containing sh-AHFD-HF-1 (SEQ ID NO.1) or recombinant adeno-associated virus rAAV-shRNA-AHFD-HF-2 containing sh-AHFD-HF-2 (SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5) on the cardiac function of high-fat fed mice was monitored by cardiac ultrasound and catheter in Experimental Example 3 of the present invention, 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-shRNA-AHFD-HF can significantly improve the cardiac systolic and diastolic function of high-fat fed mice.

[0054] Figure 2-Figure 5The meanings of the markers are listed as follows: Control represents high-fat-fed mice injected with the control solvent PBS, GFP represents high-fat-fed mice injected with the recombinant adeno-associated virus vector rAAV-cTNT-GFP expressing the control shRNA specifically targeting cardiomyocytes, sh-AHFD-HF-1 represents high-fat-fed mice injected with the recombinant adeno-associated virus vector rAAV-cTNT-shRNA-AHFD-HF specifically targeting cardiomyocytes, which corresponds to SEQ ID NO.1, sh-AHFD-HF-2 represents high-fat-fed mice injected with the small RNA non-specific promoter rAAV-H1-U6-shRNA-AHFD-HF, which corresponds to the group consisting of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5); Chow represents normal diet-fed control mice, and HFD represents heart failure model mice caused by high-fat feeding. DETAILED DESCRIPTION

[0055] 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.

[0056] 1. Sources of biological materials

[0057] (I) The DH5α competent cells used in Experimental Example 1 were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; 293T cells were from ATCC, USA; rAAV was purchased from Shandong Weizhen Biotechnology Co., Ltd.;

[0058] (ii) The C57 mice used in Experimental Example 3 were purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[0059] 2. The sources of the instruments, equipment, reagents, consumables and biological materials involved in the following embodiments or experimental examples are as follows:

[0060] (I) Instruments and equipment

[0061] ND-1000 nucleic acid analyzer, ABI 9700PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge;

[0062] (II) Reagents and consumables

[0063] 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), 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 rAAV-shRNA-AHFD-HF sequence fragment was synthesized by Wuhan Ruibo Biotechnology Co., Ltd.

[0064] Group 1 Example, shRNA of the present invention

[0065] This group of embodiments provides a shRNA. All embodiments in this group have the following common features: the shRNA is selected from shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4 and SEQ ID NO.5.

[0066] The above shRNA can play a role in treating heart failure induced by lipid metabolism disorder. The corresponding DNA sequence refers to a DNA sequence in which the U base in the shRNA corresponds to the T base, and the other bases are consistent with the shRNA.

[0067] The above-mentioned shRNAs are all designed and synthesized for the first time by the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, and expressing any of the above-mentioned shRNAs, and / or any act of using any of the above-mentioned shRNAs for pharmaceutical preparation or as a drug ingredient, and / or any act of using any of the above-mentioned shRNAs for treatment falls within the scope of protection of the present invention.

[0068] According to actual production needs, those skilled in the art can design specific amplification primers for the sequence of the shRNA to obtain its gene sequence and connect it with an expression vector to obtain a recombinant expression vector that can express the shRNA, or further transform the recombinant expression vector into competent cells to obtain a transformant that can express the shRNA, and multiply and culture the transformant under conditions suitable for its growth to efficiently produce the shRNA. This has no technical barriers for those skilled in the art and is possible and easy to do.

[0069] The second group of embodiments, the recombinant expression vector of the present invention

[0070] This group of embodiments provides a recombinant expression vector. All embodiments in this group have the following common features: the recombinant expression vector is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0071] In a specific embodiment, the expression vector is pAAV D(+);

[0072] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0073] Preferably, the recombinant expression vector is selected from: pAAV-D(+)-cTNT-shRNA-AHFD-HF-1, pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2.

[0074] The above-mentioned recombinant expression vectors are all prepared for the first time by the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, and expressing the above-mentioned recombinant expression vectors, and / or any act of using the above-mentioned recombinant expression vectors for pharmaceutical preparation or as drug ingredients, and / or any act of using the above-mentioned recombinant expression vectors for treatment falls within the scope of protection of the present invention.

[0075] The third group of examples, transformants of the present invention

[0076] This group of embodiments provides a transformant. All embodiments in this group have the following common features: the transformant is a host transformed with a recombinant expression vector; the recombinant expression vector is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0077] In some embodiments, the host is selected from: a virus, and / or a cell;

[0078] Preferably, the virus is selected from adeno-associated virus;

[0079] Preferably, the cells are selected from 293T cells;

[0080] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0081] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0082] Preferably, the transformant is selected from: rAAV-cTNT-shRNA-AHFD-HF-1, rAAV-H1-U6-shRNA-AHFD-HF-2.

[0083] The above-mentioned transformants are all prepared for the first time by the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, expressing the above-mentioned transformants, and / or any act of using the above-mentioned transformants for pharmaceutical preparation or as a drug ingredient, and / or any act of using the above-mentioned transformants for treatment falls within the scope of protection of the present invention.

[0084] The fourth group of embodiments, the anti-heart failure drug of the present invention

[0085] This group of embodiments provides an anti-heart failure drug. All embodiments in this group have the following common features: the anti-heart failure drug includes a pharmacologically active ingredient, and the pharmacologically active ingredient includes: the shRNA described in any one of the first group of embodiments, and / or the recombinant expression vector described in any one of the second group of embodiments, and / or the transformant described in any one of the third group of embodiments.

[0086] In a specific embodiment, the pharmacologically active ingredient is selected from the group consisting of: the shRNA described in any one of the first group of embodiments, the recombinant expression vector described in any one of the second group of embodiments, and the transformant described in any one of the third group of embodiments;

[0087] Preferably, the anti-heart failure drug further comprises: pharmaceutical excipients, and / or reagents for amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, expressing the shRNA described in any one of the first group of embodiments, and / or the recombinant expression vector described in any one of the second group of embodiments, and / or the transformant described in any one of the third group of embodiments;

[0088] Based on the teachings of the present invention, those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-heart failure drugs of the present invention according to production needs and practical requirements to prepare various dosage forms for easy sale or promotion.

[0089] In a specific embodiment, the pharmaceutical excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, and release retardants.

[0090] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0091] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0092] The fifth group of embodiments, pharmaceutical use of the present invention

[0093] This group of embodiments provides the use of the shRNA described in any one of the first group of embodiments, and / or the recombinant expression vector described in any one of the second group of embodiments, and / or the transformant described in any one of the third group of embodiments in the preparation of anti-heart failure drugs.

[0094] Preferably, the anti-heart failure means: significantly reducing the expression of AHFD-HF and improving heart function.

[0095] In a specific embodiment, the preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and expansion;

[0096] Preferably, the synthesis refers to whole gene synthesis technology;

[0097] Preferably, the cloning refers to connecting the shRNA to an expression vector;

[0098] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0099] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0100] In some embodiments, the anti-heart failure drug uses shRNA or a recombinant expression vector or transformant as a pharmacologically active ingredient;

[0101] In a further embodiment, the anti-heart failure drug further comprises a pharmaceutical excipient;

[0102] In a specific embodiment, the pharmaceutical excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, and release retardants.

[0103] In a specific embodiment, the transformant is a host containing a recombinant expression vector; the recombinant expression vector is an expression vector connected to the shRNA sequence shown in SEQ ID NO.1, which can play a role in treating heart failure;

[0104] In a preferred embodiment, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0105] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0106] Preferably, the host is selected from: viruses, and / or cells;

[0107] Preferably, the virus is selected from adeno-associated virus;

[0108] Preferably, the cells are selected from 293T cells.

[0109] Group 6 Example, method for preparing anti-heart failure drug of the present invention

[0110] This group of embodiments provides a method for preparing an anti-heart failure drug. All embodiments in this group have the following common features: preparing the shRNA described in any one of the first group of embodiments, and / or the recombinant expression vector described in any one of the second group of embodiments, and / or the transformant described in any one of the third group of embodiments.

[0111] In a specific embodiment, the preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and expansion;

[0112] Preferably, the synthesis refers to whole gene synthesis technology;

[0113] Preferably, the cloning refers to connecting the shRNA to an expression vector;

[0114] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0115] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0116] In a further embodiment, the preparation method further comprises: combining or mixing the pharmaceutically active ingredient with a pharmaceutical excipient.

[0117] Based on the teachings of the present invention, those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-heart failure drugs of the present invention according to production needs and practical requirements to prepare various dosage forms for easy sale or promotion.

[0118] In a specific embodiment, the pharmaceutical excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, and release retardants.

[0119] The present invention is further described below with examples:

[0120] Experimental Example 1: Construction of recombinant adeno-associated virus

[0121] 1. Construction of pAAV-D(+) vector

[0122] The rAAV-shRNA-AHFD-HF-1 or rAAV-shRNA-AHFD-HF-2 double-stranded nucleotides (one of the double strands of rAAV-shRNA-AHFD-HF-1 is SEQ ID NO.1 and the other is the reverse complementary sequence of SEQ ID NO.1) were synthesized by full gene synthesis technology and cloned into the pAAV-D(+) vector ( Figure 1-2 ), and the double-stranded nucleotides were synthesized by Ruibo Biotechnology Co., Ltd. The sequence of shRNA-AHFD-HF-1 is SEQ ID NO.1; the sequence of shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0123] 2. Plasmid Transformation

[0124] Add pAAV-D(+)-cTNT-shRNA-AHFD-HF-1 and pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2 plasmids to 100 μl DH5α competent cells and place on ice for 30 min; heat at 42°C for 45 sec, and place on 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.

[0125] 3. Plasmid extraction

[0126] 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.

[0127] 4. Plasmid extraction

[0128] 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(+)-cTNT-shRNA-AHFD-HF-1, pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2) 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, and vortex to completely resuspend; 3. Add 10ml Solution II to the resuspended mixture, gently invert and mix 10-15 times, and place at room temperature.

[0129] 5. rAAV-mediated viral packaging

[0130] 293T cells grow to 90%, 1-2 hours before calcium-phosphorus transfection, each culture dish is replaced with 12-15 ml of fresh culture medium (containing serum), calcium chloride (CaCl2) is first added to a 50 ml centrifuge tube, and then plasmid is added to form a Ca-DNA mixed solution, which is fully mixed, and 2XHEBS BUFFER is slowly added to the Ca-DNA mixed solution to form a Ca-DNA-P mixed solution, while adding 2X HEBS, the centrifuge tube is shaken, and fully mixed to form calcium-phosphorus particles. After 8-12 hours, 18-20 ml of serum-free culture medium is replaced, and after 72 hours, the culture medium is aspirated and washed 3 times with PBS, 1 ml of Tris+NaCl (pH 8.5) is added to each culture dish, and cells are scraped with a scraper, collected in a clean centrifuge tube, and frozen at -80°C.

[0131] 6. Virus Purification

[0132] 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).

[0133] 7. Virus Titer Determination

[0134] Sample processing:

[0135] rAAV virus solution 40μl

[0136] Proteinase K (20 mg / ml) 5 μl

[0137] 55°C, reaction for 1 hr;

[0138] Phenol:chloroform:isoamyl alcohol 45 μl

[0139] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C;

[0140] Chloroform 45 μl

[0141] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C.

[0142] Real-time PCR:

[0143] Primer 1 (10 μm) 0.4 μl

[0144] Primer 2 (10 μm) 0.4 μl

[0145] SYBR Green I Mix 10 μl

[0146] ddH2O 8.2μl

[0147] Template 1μl

[0148] 95℃30sec---(95℃5sec---60℃5sec---72℃20sec)×40 cycles---MeltingCurve Experimental Example 2. Western blotting to verify the knockdown efficiency of recombinant adeno-associated virus of shRNA-AHFD-HF 1. Protein extraction:

[0149] a. Sample preparation: Tissue sample: 20 mg of heart tissue was cut into a 2 mL enzyme-free EP tube, 2 3 mm grinding beads were added, and IP lysis buffer was added at a ratio of 300 μL lysis buffer per 20 mg of heart tissue. The mixture was ground using an automated tissue grinder for 2 minutes. After grinding was complete, the mixture was centrifuged at 4°C, 12,000 g for 10 minutes, and the supernatant was transferred to another clean EP tube.

[0150] b. Prepare standard curve: dilute 2 mg / mL standard with IP lysis buffer to 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL by serial dilution method.

[0151] c. Determination of protein concentration by BCA method: Calculate the total number of samples (including standard curve) for which protein concentration needs to be determined, prepare AB mixed solution according to the ratio of 100μL A solution and 2μL LB solution for each sample, oscillate and mix well, and place on ice. Add 5μL of protein stock solution to the ELISA tube, and then add 95μL of AB mixed solution. Avoid bubbles when adding, otherwise it will affect the accuracy of the multifunctional ELISA reader reading. Gently shake the wall of the ELISA tube to mix, incubate at 37℃ for 15 minutes, and use the multifunctional ELISA reader to detect the absorbance value at 562nm.

[0152] d. Sample preparation: Calculate the protein concentration, standardize the remaining samples according to the total amount of protein contained in the sample with the lowest concentration, and dilute each sample in a new EP tube with IP lysis buffer to ensure the same total concentration and total volume. Add 5× loading buffer in proportion. Note that 5× loading buffer is viscous and easily adheres to the wall of the gun tip tube. Ensure that the amount added is accurate, otherwise the amount of loading buffer added during the subsequent spotting process will be insufficient, which will make it difficult for the protein to precipitate. Shake the added sample and heat it at 95°C in a constant temperature metal bath for 10 minutes. After heating, place the sample on ice for 5 minutes.

[0153] 2. Glue making:

[0154] a. Wash the prepared glass plate with deionized water to ensure that there are no impurities on the glass plate, otherwise it will cause abnormal protein electrophoresis bands in the subsequent electrophoresis. After washing, place the glass plate in a 37°C oven to dry it or use filter paper to absorb the residual water on the glass plate to avoid excessive water residue diluting the concentration of the gel;

[0155] b. Select the required concentration of separation gel according to the molecular weight of the target band, prepare separation gel and stacking gel for use. Note that the coagulant should be temporarily added before the gel is poured into the glass plate. Align the bottoms of the thin and thick glass plates and fix them on the gel holder, pour in the separation gel with the coagulant added, and then flatten the separation gel with deionized water;

[0156] c. When the separation gel is flattened to form a horizontal straight line, discard the water used to press the line and absorb it with filter paper. Pour the stacking gel with the coagulant added, and then gently insert the comb to avoid bubbles that may cause leaks, and let it stand and wait for solidification.

[0157] 3. Electrophoresis:

[0158] a. Clamp the prepared gel and place it in the electrophoresis tank, check whether it is well sealed to avoid leakage. Prepare electrophoresis solution according to the specification of 1L electrophoresis solution for 4 gels, and pour the prepared electrophoresis solution into the electrophoresis tank;

[0159] b. Gently pull out the comb, shake the protein sample again and add it to the loading well. When loading, be careful to avoid protein overflow, which may cause contamination between wells and inaccurate loading amount. To better identify the loading order, add more protein marker before the first loading well and less protein marker after the last well;

[0160] c. After loading, first perform electrophoresis at 80V constant voltage for 30 minutes, then at 110V constant voltage. The specific time of electrophoresis depends on the molecular weight of the target protein. If the molecular weight is small, the electrophoresis time should be shortened accordingly to avoid the electrophoresis time of small molecule protein being too long, the band being dispersed and skewed;

[0161] If the molecular weight is large, the electrophoresis time should be appropriately extended to separate the large protein bands from other bands.

[0162] 4. Transfer

[0163] a. Cut the PVDF membrane of the required size according to the size of the gel, and mark it with a ballpoint pen, and then soak the prepared PVDF membrane in methanol solution to activate it. Prepare the transfer solution according to the specification of 1.5L transfer solution for 4 gels.

[0164] b. According to the wet transfer sandwich method, place the transfer chuck with the black side facing down, and put in two layers of flat filter paper, gel, PVDF membrane, and two layers of flat filter paper in sequence. Be careful not to make a mistake in the corresponding directions of the gel and PVDF membrane, and do not leave bubbles in the gap;

[0165] c. Place the properly placed transfer clip into the transfer tank, confirm the forward and reverse directions of the transfer clip again, connect the transfer tank power supply, and transfer the membrane at a constant current of 200mA per transfer tank. The transfer time depends on the molecular weight of the target protein.

[0166] 5. Closure

[0167] After the transfer, place the membrane in a flat antibody incubation box and rinse twice with TBST to remove the residual transfer solution. Submerge the PVDF membrane with 5% BSA blocking solution and incubate on a shaker at room temperature for 2 hours to block non-specific protein binding sites.

[0168] 6. Primary Antibody Incubation

[0169] The blocking solution was recovered, 10 mL of the corresponding diluted antibody was added, and the cells were incubated overnight on a shaker at 4°C.

[0170] 7. Secondary Antibody Incubation and Membrane Washing

[0171] Recover the primary antibody and wash it 4 times with TBST on a shaker, 10 minutes each time. After the primary antibody is washed, add 15mL of the corresponding diluted secondary antibody according to the species of the primary antibody and incubate at room temperature for 2 hours. After incubation, wash it 4 times and then expose it.

[0172] 8. Exposure

[0173] Mix ECLA solution and B solution and evenly coat them on the PVDF membrane for exposure. Select the specific exposure time and intensity according to the strip. After the exposure is completed, reselect the antibodies required for incubation according to the results and detect the remaining indicators.

[0174] Experimental Example 3: Using rAAV9-type recombinant adeno-associated virus expressing shRNA-AHFD-HF as an example to detect its therapeutic effect on heart failure

[0175] Eight-week-old C57 mice were used. After high-fat diet for 16 weeks, rAAV-cTNT-shRNA-AHFD-HF-1 (sh-AHFD-HF-1 group, corresponding to SEQ ID NO.1), rAAV-H1-U6-shRNA-AHFD-HF-2 (sh-AHFD-HF-2 group, corresponding to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 (promoters H1-U6, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 were sequentially connected to the expression vector)), control rAAV-cTNT-GFP virus (GFP group) and control solvent PBS (Control group) were injected through the tail vein. Control mice fed with normal diet (Chow group) and heart failure model mice induced by high-fat diet without any treatment (HFD group) were set up. The virus titer was 1×10 11PFU / each, at the end of the experiment (8 weeks later), the cardiac function of mice was tested in the following way: 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 acquired 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), The results showed that rAAV-shRNA-AHFD-HF treatment could significantly reduce the expression of AHFD-HF (representing HMGCS2 gene) Figure 3-4 ), and significantly improved the cardiac function of HFD mice ( Figure 5 ).

Claims

1. A shRNA, characterized in that Selected from shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

2. A recombinant expression vector, characterized in that: It is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

3. A recombinant expression vector according to claim 1, characterized in that: The expression vector is pAAV D(+); And / or, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV; And / or, the recombinant expression vector is selected from: pAAV-D(+)-cTNT-shRNA-AHFD-HF-1, pAAV-D(+)-H1-U6-shRNA-AHFD-HF-2.

4. A transformant, characterized in that The invention relates to a host transformed with a recombinant expression vector; the recombinant expression vector is an expression vector connected with shRNA-AHFD-HF-1 or shRNA-AHFD-HF-2; the DNA sequence corresponding to the shRNA-AHFD-HF-1 is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-AHFD-HF-2 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

5. A transformant according to claim 4, characterized in that The host is selected from: viruses, and / or cells; and / or, the virus is selected from adeno-associated viruses; And / or, the cells are selected from 293T cells; And / or, the expression vector is selected from adeno-associated virus expression vector pAAV D(+); And / or, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV; And / or, the transformant is selected from: rAAV-cTNT-shRNA-AHFD-HF-1, rAAV-H1-U6-shRNA-AHFD-HF-2.

6. An anti-heart failure drug, comprising a pharmacologically active ingredient, characterized in that: The pharmacologically active ingredients include: the shRNA according to claim 1, and / or the recombinant expression vector according to claim 2 or 3, and / or the transformant according to claim 4 or 5.

7. An anti-heart failure drug according to claim 6, characterized in that: The pharmacologically active ingredient is selected from the group consisting of: the shRNA according to claim 1, the recombinant expression vector according to claim 2 or 3, and the transformant according to claim 4 or 5; And / or, the anti-heart failure drug further comprises: pharmaceutical excipients.

8. Use of the shRNA according to claim 1, and / or the recombinant expression vector according to claim 2 or 3, and / or the transformant according to claim 4 or 5 in the preparation of an anti-heart failure drug. And / or, the anti-heart failure means: significantly reducing the expression of AHFD-HF and improving heart function.

9. A method for preparing an anti-heart failure drug, characterized in that: Prepare the shRNA according to claim 1, and / or the recombinant expression vector according to claim 2 or 3, and / or the transformant according to claim 4 or 5.

10. The method for preparing an anti-heart failure drug according to claim 9, characterized in that: The preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and expansion; And / or, the synthesis refers to whole gene synthesis technology; And / or, the cloning refers to connecting the shRNA to an expression vector; And / or, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters cTNT, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV; And / or, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

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