Application of UBAP1 as diabetic cardiomyopathy drug target and product of UBAP1

Abstract

The invention relates to application of UBAP1 as a diabetic cardiomyopathy drug target and a product of the UBAP1, belonging to the field of biological medicines. The invention provides application of UBAP1 as a diabetic cardiomyopathy drug target and application of a substance for inhibiting UBAP1 expression in preparation of a diabetic cardiomyopathy drug, and provides shRNA and a recombinant expression vector, a transformant and an anti-diabetic cardiomyopathy drug thereof. The shRNA comprises the following components: shRNA-ADCM (Amplified Dendritic Cell Module) and shRNA-4in1; a DNA (deoxyribonucleic acid) sequence corresponding to the shRNA-ADCM is shown as SEQ ID NO. 1; dNA (deoxyribonucleic acid) sequences corresponding to the shRNA-4in1 comprise SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. The results of animal experiments such as ultrasound, catheters and the like show that the recombinant adeno-associated virus mediated shRNA-ADCM and shRNA-4in1 can obviously improve the heart function of a db / db mouse.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of UBAP1 as a drug target for diabetic cardiomyopathy and a product thereof. Background Art

[0002] Diabetes is a chronic metabolic disease with an increasing incidence rate year by year, with a total number of patients worldwide exceeding 500 million. More than one-third of patients will experience left ventricular hypertrophy, abnormal myocardial contraction and relaxation, and progress to diabetic cardiomyopathy, which will eventually lead to heart failure. The pathogenesis of diabetic cardiomyopathy includes 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 change the prognosis of patients with diabetic heart failure. For example, large-scale randomized controlled clinical trials (ACCORD, ADVANCE, VADT, UKPDS) aimed at studying the effects of blood sugar control on cardiovascular event outcomes showed that intensive blood sugar control can reduce the incidence of myocardial infarction, but does not reduce the re-admission rate and mortality rate of heart failure. This shows that diabetic patients have been in a state of hyperglycemia and are still prone to diabetes-related cardiovascular complications even after hypoglycemic treatment. In recent years, although some drugs have been used in preclinical studies or clinical trials, these drugs have not been transformed into clinical applications or have been proven to fail to reduce the incidence and mortality.

[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, many new small RNA drugs have been launched 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 prospects for clinical application.

[0005] However, the clinical small RNA drugs for treating diabetic cardiomyopathy are quite limited, and there is not much room for selection. At the same time, the targets of diabetic cardiomyopathy are also very rare. This field urgently needs to develop more therapeutic targets for diabetic cardiomyopathy and more small RNAs targeting the targets for targeted treatment of diabetic cardiomyopathy. Summary of the invention

[0006] In order to solve the technical problem that small RNA drugs for treating diabetic cardiomyopathy are quite limited and there is not much room for selection, and to provide more therapeutic targets for diabetic cardiomyopathy and more small RNAs targeting the targets for targeted treatment, the present invention provides the use of UBAP1 as a drug target for diabetic cardiomyopathy and its products. The products involve small RNA drugs rAAV-shRNA-ADCM (ADCM, anti-diabetic-cardiomyopathy) and rAAV-shRNA-4in1. Animal experiments are carried out to verify the effects, and it is found that rAAV-ADCM and shRNA-4in1 can significantly improve the cardiac function of mice and treat diabetic cardiomyopathy.

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

[0008] Use of UBAP1 as a drug target for diabetic cardiomyopathy.

[0009] The diabetic cardiomyopathy drug has a substance that inhibits UBAP1 expression as a pharmacologically active ingredient;

[0010] Preferably, the substance that inhibits UBAP1 expression includes: shRNA-ADCM, shRNA-4in1, a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1, and a transformant transformed with a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0011] Use of a substance that inhibits UBAP1 expression in preparing a drug for diabetic cardiomyopathy.

[0012] The substance inhibiting UBAP1 expression includes: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0013] A shRNA, comprising: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 comprises: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0014] A recombinant expression vector is an expression vector connected with the shRNA.

[0015] The expression vector is selected from: the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0016] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV.

[0017] A transformant is a host transformed with the recombinant expression vector.

[0018] The host is selected from: viruses, and / or cells;

[0019] Preferably, the virus is selected from adeno-associated virus;

[0020] Preferably, the cells are selected from 293 cells.

[0021] An anti-diabetic cardiomyopathy drug comprises a pharmacologically active ingredient, wherein the pharmacologically active ingredient is selected from: the aforementioned shRNA, and / or the aforementioned recombinant expression vector, and / or the aforementioned transformant.

[0022] The present invention pioneered the discovery that UBAP1 is a target for diabetic cardiomyopathy, and based on this, provides the use of UBAP1 as a drug target for diabetic cardiomyopathy, and the use of a substance that inhibits the expression of UBAP1 to prepare a drug for diabetic cardiomyopathy. At the same time, based on this gene, the present invention designed and developed a small RNA drug that delivers short hairpin RNA (shRNA) via rAAV, which can target UBAP1, a target for diabetic cardiomyopathy, and significantly improve cardiac function, and has the potential to treat diabetic cardiomyopathy.

[0023] The present invention provides use of shRNA in preparing medicine for treating diabetic cardiomyopathy.

[0024] Preferably, the expression vector is an adeno-associated virus expression vector pAAV-D(+);

[0025] Preferably, the expression vector promoter is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0026] Preferably, the expression vector connected to shRNA-ADCM and shRNA-4in1 is selected from: recombinant adeno-associated virus expression vectors pAAV-D(+)-shRNA-ADCM and pAAV-D(+)-shRNA-4in1 connected to shRNA-ADCM and shRNA-4in1 sequences; or, recombinant adeno-associated virus expression vectors pAAV-D(+)-tnt-shRNA-ADCM and pAAV-D(+)-shRNA-4in1 connected to myocardial specific promoter tnt, shRNA-ADCM or shRNA-4in1 sequences in sequence;

[0027] Preferably, the host cell of the transformant is selected from: 293 cells;

[0028] Preferably, the transformant transformed with the shRNA-ADCM and shRNA-4in1 expression vectors is selected from: recombinant adeno-associated virus rAAV-shRNA-ADCM and rAAV-U6-shRNA-4in1 transformed with the recombinant adeno-associated virus expression vectors pAAV-D(+)-shRNA-ADCM and pAAV-D(+)-shRNA-4in1; or, recombinant adeno-associated virus rAAV-tnt-shRNA-ADCM and rAAV-U6-shRNA-4in1 transformed with the recombinant adeno-associated virus expression vectors pAAV-D(+)-tnt-shRNA-ADCM and pAAV-D(+)-shRNA-4in1;

[0029] Preferably, the shRNA-ADCM sequence is as shown in SEQ ID NO.1; the shRNA-4in1 sequence includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0030] A recombinant expression vector having the effect of treating diabetic cardiomyopathy, characterized in that it is an expression vector connected to the sequence of the gene having the effect of treating diabetic cardiomyopathy.

[0031] The expression vector is selected from: an adeno-associated virus expression vector pAAV-D(+);

[0032] Preferably, the recombinant expression vector having the effect of treating diabetic cardiomyopathy is selected from: recombinant adeno-associated virus expression vectors pAAV-D(+)-shRNA-ADCM and pAAV-D(+)-shRNA-4in1 connected with shRNA-ADCM and shRNA-4in1 gene sequences.

[0033] A transformant having the effect of treating diabetic cardiomyopathy, characterized in that it is a host cell transformed with the recombinant expression vector having the effect of treating diabetic cardiomyopathy.

[0034] The host cell is selected from: 293 cells;

[0035] Preferably, the transformant having the effect of treating diabetic cardiomyopathy is selected from: recombinant adeno-associated virus rAAV-shRNA-ADCM, rAAV-U6-shRNA-4in1 transformed with recombinant adeno-associated virus expression vector pAAV-D(+)-shRNA-ADCM, pAAV-D(+)-shRNA-4in1. Or, recombinant adeno-associated virus rAAV-tnt-shRNA-ADCM, rAAV-U6-shRNA-4in1 transformed with recombinant adeno-associated virus expression vector pAAV-D(+)-tnt-shRNA-ADCM;

[0036] Furthermore, the drug also includes pharmaceutically acceptable excipients and / or reagents for buffering, synthesizing, and / or purifying the sequence fragments. Those skilled in the art can add various pharmaceutically acceptable excipients / excipients to the anti-diabetic cardiomyopathy drug of the present invention according to objective needs to prepare various dosage forms for easy sales or promotion.

[0037] In a further embodiment, the preparation method comprises: inserting the shRNA-ADCM and shRNA-4in1 sequence fragments into an expression vector, thereby preparing a recombinant plasmid that can stably express shRNA-ADCM and shRNA-4in1.

[0038] In a specific embodiment, the expression vector containing the shRNA-ADCM and shRNA-4in1 sequence fragments is an adeno-associated virus expression vector pAAV-D(+).

[0039] In order to achieve the purpose of gene therapy for heart failure, the present invention recombined shRNA-ADCM and shRNA-4in1 sequence fragments with recombinant adeno-associated virus vectors, and obtained high titers that meet the treatment requirements after detection, and confirmed in animal experiments that it can effectively improve the cardiac function of diabetic mice. Therefore, based on the above findings and results, the present invention provides a small RNA drug represented by shRNA-ADCM and shRNA-4in1 for the treatment of clinical diabetic cardiomyopathy.

[0040] The present invention relates to a drug for treating diabetic cardiomyopathy. The anti-diabetic cardiomyopathy drug involves the construction and preparation method of recombinant adeno-associated virus recombinants (rAAV-shRNA-ADCM) of shRNA-ADCM and shRNA-4in1, and the drug effect of treating heart failure is achieved by high expression of the rAAV-shRNA-ADCM. The present invention utilizes a chemical synthesis method to construct a pAAV-D(+)-shRNA-ADCM 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-diabetic cardiomyopathy drug provided by the present invention can significantly improve the cardiac function of diabetic mice and play an effective role in treating diabetic cardiomyopathy.

[0041] The present invention designs and synthesizes the sequences of shRNA-ADCM and shRNA-4in1, and successfully inserts them into the eukaryotic expression vector pAAV-D(+) to form a recombinant plasmid pAAV-D(+)-shRNA-ADCM. Afterwards, the following five plasmids: pXX9, phelper, pAAV-D(+)-shCON, pAAV-D(+)-shRNA-ADCM, and pAAV-D(+)-shRNA-4in1, are respectively transferred into 293 cells by calcium-phosphate co-transfection to package and prepare recombinant adeno-associated viruses (rAAV9) that can express shRNA-ADCM and shRNA-4in1. After purification, the titer is determined by real-time PCR. In the next step, the recombinant adeno-associated viruses (rAAV-shRNA-ADCM, rAAV-U6-shRNA-4in1) of the same serotype prepared by packaging are injected into leptin receptor homozygous mutation (db / db) type 2 diabetic mice through the tail vein. Ultrasound and catheter results showed that recombinant adeno-associated virus-mediated shRNA-ADCM and shRNA-4in1 could significantly improve the cardiac function of db / db mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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.

[0043] Figure 1 The figure shows the plasmid structure of pAAV-D(+)-shRNA-ADCM, wherein the tnt promoter represents a cardiomyocyte-specific promoter.

[0044] Figure 2 This is an amplification curve diagram of the virus titer determination containing pAAV-D(+)-shRNA-ADCM plasmid in Experimental Example 1 of the present invention.

[0045] Figure 3 This is an amplification curve diagram of the virus titer determination containing the pAAV-D(+)-shRNA-4in1 plasmid in Experimental Example 1 of the present invention.

[0046] Figure 4 This is a graph showing the amplification curve of the rAAV-tnt-shCON virus titer determination in Experimental Example 1 of the present invention.

[0047] Figure 5 This is the effect of rAAV-shRNA-ADCM treatment on the cardiac function of db / db mice monitored by cardiac ultrasound and catheter in Experimental Example 2 of the present invention, wherein A is a bar graph of ejection fraction, B is a bar graph of left ventricular 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 decrease of left ventricular pressure during isovolumetric contraction; the results show that rAAV-shRNA-ADCM can significantly increase the cardiac contractile and diastolic functions of db / db mice.

[0048] Figure 6This is a western blot result diagram of the effects of rAAV-shRNA-ADCM, rAAV-U6-shRNA-4in1, and control rAAV-tnt-shCON viruses of Experimental Example 2 of the present invention on the expression of UBAP1 and GAPDH genes in bks and db / db mice, respectively. Among them, the + sign indicates that the corresponding recombinant line-related virus rAAV on the left has been added, and the - sign indicates that it has not been added. This figure shows that from left to right, bks mice were intervened with the first group of rAAV-tnt-shCON, the second group of rAAV-tnt-shRNA-ADCM, and the third group of rAAV-U6-shRNA-4in1; db / db mice were intervened with the fourth group of rAAV-tnt-shCON, the fifth group of rAAV-tnt-shRNA-ADCM, and the sixth group of rAAV-U6-shRNA-4in1; and Figure 5 The order of grouping in is consistent.

[0049] The meanings of the symbols in the figure are listed as follows: rAAV-tnt-shCON represents db / db mice or bks mice injected with the control rAAV-tnt-shCON virus transformed with the pAAV-tnt empty plasmid that specifically targets cardiomyocytes; rAAV-tnt-shRNA-ADCM represents db / db mice or bks mice injected with the recombinant adeno-associated virus rAAV-tnt-shRNA-ADCM that specifically targets cardiomyocytes shRNA-ADCM that is transformed with the recombinant expression vector pAAV-D(+)-tnt-shRNA-ADCM; rAAV-U6-shRNA-4in1 represents db / db mice or bks mice injected with the recombinant adeno-associated virus rAAV-U6-shRNA-4in1 that is transformed with the recombinant expression vector pAAV-D(+)-shRNA-4in1 that specifically targets cardiomyocytes shRNA-4in1; bks represents healthy control mice (C57BLKS / J strain mice); and db / db represents type 2 diabetes model mice. DETAILED DESCRIPTION

[0050] 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.

[0051] The sources of the instruments, equipment, reagents, consumables and biological materials involved in the following embodiments or experimental examples are as follows:

[0052] 1. Instruments and Equipment

[0053] ND-1000 nucleic acid analyzer, ABI 9700PCR instrument, ABI 7900HT fluorescence real-time quantitative PCR instrument, Beckman X-15R low-temperature high-speed centrifuge;

[0054] 2. Reagents and consumables

[0055] 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 cooperating Professor Xiao Xiao, and the rAAV-shRNA-ADCM sequence fragment was synthesized by Wuhan Corester Biotechnology Co., Ltd.

[0056] 3. Sources of biological materials

[0057] (I) The DH5α competent cells used in Experimental Example 1 are commercially available; 293T cells are from ATCC, USA.

[0058] (ii) The bks and db / db mice used in Experimental Example 2 were purchased from Gempharmatech (https: / / cn.gempharmatech.com / ).

[0059] The first group of examples, use of UBAP1 of the present invention

[0060] This group of embodiments provides the use of UBAP1 as a drug target for diabetic cardiomyopathy.

[0061] In a specific embodiment, the diabetic cardiomyopathy drug has a substance that inhibits UBAP1 expression as a pharmacologically active ingredient;

[0062] Preferably, the substance that inhibits UBAP1 expression includes: shRNA-ADCM, shRNA-4in1, a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1, and a transformant transformed with a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0063] The above shRNA can play a role in treating diabetic cardiomyopathy. 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.

[0064] The use of UBAP1 as a drug target for diabetic cardiomyopathy is proposed for the first time in the present invention. Any act of developing, designing, screening, amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, expressing, inhibiting, knocking down, silencing, and knocking out UBAP1 based on UBAP1 and using it for pharmaceutical manufacturing falls within the scope of protection of the present invention.

[0065] Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, expressing any of the above shRNAs, and / or any act of using any of the above shRNAs for pharmaceutical preparation or as a drug ingredient, and / or any act of using any of the above shRNAs for treatment falls within the scope of protection of the present invention.

[0066] 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.

[0067] The second group of embodiments, pharmaceutical use of the present invention

[0068] This group of embodiments provides the use of a substance that inhibits UBAP1 expression in preparing a drug for diabetic cardiomyopathy.

[0069] In some embodiments, the substance that inhibits UBAP1 expression includes: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0070] Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, multiplying, enriching, connecting, transforming, cloning, expressing any of the above shRNAs, and / or any act of using any of the above shRNAs for pharmaceutical preparation or as a drug ingredient, and / or any act of using any of the above shRNAs for treatment falls within the scope of protection of the present invention.

[0071] 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.

[0072] Group 3 Example, shRNA of the present invention

[0073] This group of embodiments provides a shRNA. All embodiments in this group have the following common features: the shRNA includes: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0074] 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.

[0075] 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.

[0076] The fourth group of embodiments, the recombinant expression vector of the present invention

[0077] 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 the shRNA described in any one of the third group of embodiments.

[0078] In some embodiments, the expression vector is selected from: the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0079] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV.

[0080] 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.

[0081] Group 5 Example, transformant of the present invention

[0082] This group of embodiments provides a transformant. All embodiments in this group have the following common features: the transformant is a host transformed with the recombinant expression vector described in any one of the fourth group of embodiments.

[0083] In some embodiments, the host is selected from: a virus, and / or a cell;

[0084] Preferably, the virus is selected from adeno-associated virus;

[0085] Preferably, the cells are selected from 293 cells.

[0086] 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.

[0087] Group 6 Examples, Drugs of the Present Invention

[0088] This group of embodiments provides an anti-diabetic cardiomyopathy drug. All embodiments in this group have the following common features: the anti-diabetic cardiomyopathy drug comprises a pharmacologically active ingredient, and the pharmacologically active ingredient is selected from: an shRNA described in any one of the third group of embodiments, and / or a recombinant expression vector described in any one of the fourth group of embodiments, and / or a transformant described in any one of the fifth group of embodiments.

[0089] In a further embodiment, the anti-diabetic cardiomyopathy 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 third group of embodiments, and / or, the recombinant expression vector described in any one of the fourth group of embodiments, and / or, the transformant described in any one of the fifth group of embodiments;

[0090] Based on the teachings of the present invention, those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-diabetic cardiomyopathy drug of the present invention according to production needs and practical requirements to prepare various dosage forms for easy sale or promotion.

[0091] 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.

[0092] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0093] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0094] Group 7 Example: Preparation method of the anti-diabetic cardiomyopathy drug of the present invention

[0095] This group of embodiments provides a method for preparing an anti-diabetic cardiomyopathy drug. All embodiments in this group have the following common features: preparing shRNA; and the DNA sequence corresponding to the shRNA is selected from SEQ ID NO.1.

[0096] In a specific embodiment, the preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and expansion;

[0097] Preferably, the synthesis refers to whole gene synthesis technology;

[0098] Preferably, the cloning refers to connecting the shRNA to an expression vector;

[0099] Preferably, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0100] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).

[0101] In a further embodiment, the preparation method further comprises: combining or mixing the pharmaceutically active ingredient with a pharmaceutical excipient.

[0102] Based on the teachings of the present invention, those skilled in the art can add various pharmaceutically acceptable adjuvants / excipients to the anti-diabetic cardiomyopathy drug of the present invention according to production needs and practical requirements to prepare various dosage forms for easy sale or promotion.

[0103] 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.

[0104] Group 5 Example: Pharmaceutical Use of shRNA of the Present Invention

[0105] This group of embodiments provides the use of shRNA in preparing anti-diabetic cardiomyopathy drugs. All embodiments in this group have the following common features: the shRNA includes: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5.

[0106] In some embodiments, the anti-diabetic cardiomyopathy drug uses shRNA or a recombinant expression vector or transformant as a pharmacologically active ingredient;

[0107] In a further embodiment, the anti-diabetic cardiomyopathy drug further comprises a pharmaceutical excipient;

[0108] 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.

[0109] 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 diabetic cardiomyopathy;

[0110] In a preferred embodiment, the promoter of the expression vector is selected from: cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin, or non-specific promoters H1, U6, CMV;

[0111] Preferably, the expression vector is selected from adeno-associated virus expression vector pAAV D(+);

[0112] Preferably, the host is selected from: viruses, and / or cells;

[0113] Preferably, the virus is selected from adeno-associated virus;

[0114] Preferably, the cells are selected from 293 cells.

[0115] The present invention will be further described below by experimental examples:

[0116] Experimental Example 1: Construction of recombinant adeno-associated virus

[0117] 1. Construction of pAAV-D(+) vector

[0118] The double-stranded nucleotides of rAAV-shRNA-ADCM (SEQ ID NO.1) and rAAV-U6-shRNA-4in1 (composed of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5) were synthesized by full gene synthesis technology and cloned into pAAV-D(+) vector ( Figure 1 ), and double-stranded nucleotides were synthesized by Wuhan Crester Biotechnology Co., Ltd.

[0119] 2. Plasmid Transformation

[0120] Add pAAV-D(+)-shRNA-ADCM 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.

[0121] 3. Plasmid extraction

[0122] Pick a single clone colony, add it to 3ml Amp+ LB liquid culture medium, and shake and culture it at 37℃280rpm overnight. Use Beijing Quanshijin Company EasyPure Plasmid MiniPrep Kit to extract plasmids. The specific steps are as follows: 1. Take 1.5ml of overnight cultured bacteria and centrifuge at 10,000g for 1min, and try to remove the supernatant; 2. Add 250μl colorless solution RB (containing RNaseA) and oscillate to suspend the bacterial precipitate; 3. Add 250μl blue solution LB, gently turn it upside down and mix it 4-6 times to fully lyse the bacteria and form a blue transparent solution; 4. Add 350μl yellow solution NB, gently mix it 5-6 times until a compact yellow agglomerate is formed, and let it stand at room temperature for 2min; 5.1 1. Centrifuge at 5,000g for 5 minutes, carefully aspirate the supernatant and add it to the adsorption column; 2. Centrifuge at 15,000g for 1 minute, and discard the effluent; 3. Add 650μl WB solution, centrifuge at 15,000g for 1 minute, and discard the effluent; 4. Centrifuge at 15,000g for 2 minutes to completely remove the residual WB; 5. Place the adsorption column in a new Eppendorf tube, add 20μl EB preheated at 70℃ in the center of the column, and let it stand at room temperature for 1 minute; 6. Centrifuge at 10,000g for 1 minute to elute the DNA, and store the eluted DNA at -20℃.

[0123] 4. Plasmid extraction

[0124] 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(+)-shCON, pAAV-D(+)-shRNA-ADCM, pAAV-D(+)-shRNA-4in1) respectively, and culture at 280rpm and 37℃ overnight. According to the instructions of OMEGA's Endo-Free PlasmidMaxi Kit, extract the plasmid. The specific steps are as follows: 1. Centrifuge at 5000g for 10 minutes 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.

[0125] 6. rAAV-mediated viral packaging

[0126] 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 2×HEBS 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.

[0127] 7. Virus Purification

[0128] 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).

[0129] 8. Virus Titer Determination

[0130] Sample processing:

[0131] rAAV virus solution 40μl

[0132] Proteinase K (20 mg / ml) 5 μl

[0133] 55°C, reaction for 1 hr;

[0134] Phenol:chloroform:isoamyl alcohol 45 μl

[0135] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C;

[0136] Chloroform 45 μl

[0137] The aqueous phase was recovered by centrifugation at 12,000 g for 5 min at 4°C.

[0138] Real-time PCR:

[0139] Primer 1 (10 μm) 0.4 μl

[0140] Primer 2 (10 μm) 0.4 μl

[0141] SYBR Green I Mix 10 μl

[0142] ddH2O 8.2μl

[0143] Template 1μl

[0144] 95℃30sec---(95℃5sec---60℃5sec---72℃20sec)×40 cycles---MeltingCurve

[0145] The titer of the virus (rAAV-shRNA-ADCM) containing the pAAV-D(+)-shRNA-ADCM plasmid is as follows Figure 2 As shown, the titer value is 3.47×10 13 vg / ml.

[0146] The titer of the virus (rAAV-U6-shRNA-4in1) containing the pAAV-D(+)-shRNA-4in1 plasmid is as follows Figure 3 As shown, the titer value is 1.32×10 13 vg / ml.

[0147] The results of the control rAAV-tnt-shCON virus titer determination are as follows Figure 4 As shown, the titer value is 7.86×10 13 vg / ml.

[0148] Experimental Example 2: Using rAAV9-type recombinant adeno-associated virus expressing shRNA-ADCM as an example to detect its therapeutic effect on diabetic cardiomyopathy

[0149] Ten-week-old bks and db / db mice were injected with rAAV-tnt-shRNA-ADCM, rAAV-U6-shRNA-4in1 (composed of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5), and control rAAV-tnt-CON virus (transformed with pAAV-tnt empty vector plasmid) through the tail vein, respectively. The virus titer was 1×10 11PFU / each, at the end of the experiment (14 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 (syst ... The results showed that rAAV-shRNA-ADCM and rAAV-U6-shRNA-4in1 treatment could significantly improve the cardiac function of db / db mice ( Figure 5 ).

[0150] The Western blot results of the effects of rAAV-shRNA-ADCM, rAAV-U6-shRNA-4in1, and control rAAV-tnt-shCON viruses on the expression of UBAP1 and GAPDH genes in bks and db / db mice, respectively, are shown in the figure below. Figure 6 The specific steps of Western blot are as follows:

[0151] Weigh 20 mg of heart tissue, place it in a 2.0 ml centrifuge tube, and add tissue grinding beads and lysis buffer. Grind for 180 seconds at 60 Hz in a 4°C grinder. Centrifuge the obtained tissue grinding solution at 4°C, 12000g for 10 minutes. Transfer the supernatant to a new centrifuge tube, determine the protein concentration using the BCA method, and use the lysis buffer to dilute the sample to the same concentration, then add 5×SDS loading buffer to the diluted protein solution and shake to mix. Heat the sample at 95°C for 10 minutes and immediately place it on ice for denaturation. Use 10% polyacrylamide gel for electrophoresis.

[0152] Electrophoresis fluid formula:

[0153] Add the protein sample and protein marker to the gel loading well, connect the power supply, and use 80V voltage for electrophoresis. After the protein marker is separated, adjust the voltage to 110V and continue electrophoresis. When bromophenol blue is electrophoresed to the bottom edge of the gel, stop electrophoresis and transfer to the membrane.

[0154] Transfer solution formula:

[0155] Activate the PVDF membrane with methanol and transfer it using the sandwich method. The transfer current is 200 mA and the transfer time is 2 hours. After the transfer is completed, incubate the membrane with 5% BSA solution at room temperature on a shaker for 1.5 hours to block the membrane. After blocking, add the primary antibody solution and incubate overnight at 4°C.

Claims

1. Use of UBAP1 as a drug target for diabetic cardiomyopathy.

2. The use of UBAP1 as a drug target for diabetic cardiomyopathy according to claim 1, characterized in that: The diabetic cardiomyopathy drug has a substance that inhibits UBAP1 expression as a pharmacologically active ingredient; And / or, the substance that inhibits UBAP1 expression includes: shRNA-ADCM, shRNA-4in1, a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1, and a transformant transformed with a recombinant expression vector connected to shRNA-ADCM or shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.

5.

3. Use of substances that inhibit UBAP1 expression in the preparation of drugs for diabetic cardiomyopathy.

4. Use of the substance for inhibiting UBAP1 expression according to claim 2 for preparing a drug for diabetic cardiomyopathy, characterized in that: The substance inhibiting UBAP1 expression includes: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; the DNA sequence corresponding to the shRNA-4in1 includes: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.

5.

5. A shRNA, characterized in that include: shRNA-ADCM, shRNA-4in1; the DNA sequence corresponding to the shRNA-ADCM is SEQ ID NO.1; The DNA sequences corresponding to the shRNA-4in1 include: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.

5.

6. A recombinant expression vector, characterized in that: An expression vector connected with the shRNA according to claim 5.

7. A recombinant expression vector according to claim 6, characterized in that: 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 promoter tnt, α-MHC, MLC-2v, Desmin, or non-specific promoter H1, U6, CMV.

8. A transformant, characterized in that A host transformed with the recombinant expression vector according to claim 6.

9. A transformant according to claim 8, 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 293 cells.

10. An anti-diabetic cardiomyopathy drug, comprising a pharmacologically active ingredient, characterized in that: The pharmacologically active ingredient is selected from: a shRNA according to claim 5, and / or a recombinant expression vector according to claim 6 or 7, and / or a transformant according to claim 8 or 9.

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