Application of SACS in preparation of medicine for resisting diabetic cardiomyopathy, product of SACS and preparation method of SACS
By constructing SACS-shRNA and recombinant adeno-associated viral vector pAAV-D(+), antidiabetic cardiomyopathy drugs were prepared, which solved the problem of lack of effective treatment methods in the prior art and achieved significant improvement effects on diabetic cardiomyopathy.
Patent Information
- Application Number
- CN202510394502.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-07-04
AI Technical Summary
Currently, effective treatment measures are lacking to deal with diabetic cardiomyopathy. Existing gene therapy vectors such as adenovirus and lentiviruses have immunogenic problems, while recombinant adeno-associated viral vectors (rAAVs) are considered the most promising due to their long-term expression ability. However, the application and therapeutic drugs of SACS in diabetic cardiomyopathy have not been reported.
SACS-shRNA and recombinant adeno-associated viral vector pAAV-D(+) were used to construct a recombinant expression vector. By knocking out or knocking down SACS gene expression, a cardiomyocyte-specific promoter was used to prepare anti-diabetic cardiomyopathy drugs, which were used to significantly improve cardiac function abnormalities in animal models.
It significantly improves blood glucose abnormalities and cardiac function in diabetic cardiomyopathy animals, reduces cardiomyopathy hypertrophy, and achieves effective treatment of diabetic cardiomyopathy.
Smart Images
Figure CN120242018A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of SACS in the preparation of anti-diabetic cardiomyopathy drugs, as well as products and preparation methods thereof. Background Art
[0002] Diabetes is one of the major diseases threatening human health. It is estimated that by 2025, the number of diabetic patients worldwide will reach 300 million. 1 . The prevalence of diabetes in Chinese adults is 11.6% (about 110 million), and the prevalence of pre-diabetes is as high as 50%. Among them, 80% of diabetic patients ultimately die from cardiovascular complications, with diabetic cardiomyopathy being the leading cause of death. The pathogenesis of diabetic cardiomyopathy involves glucose toxicity, lipotoxicity, apoptosis and necrosis, impaired calcium homeostasis, mitochondrial dysfunction, altered myocardial insulin signaling, and oxidative stress, etc. Among them, hyperglycemia is considered to be the most important risk factor in the occurrence and development of diabetic cardiomyopathy. However, many large-scale clinical trials have shown that strict blood glucose control does not improve the prognosis of diabetic patients with heart failure. For example, large-scale randomized controlled clinical trials (ACCORD, ADVANCE, VADT, UKPDS) aimed at studying the impact of blood glucose control on cardiovascular event outcomes have shown that intensive blood glucose control can reduce the incidence of myocardial infarction, but does not reduce the re-hospitalization rate and mortality of patients with heart failure. This phenomenon indicates that as long as diabetic patients have ever been in a hyperglycemic state, even after hypoglycemic treatment, they are still prone to diabetes-related cardiovascular complications. This phenomenon is called the "hyperglycaemic memory" phenomenon. Clinically, there is currently no effective treatment for diabetic cardiomyopathy. Therefore, it is urgent to explore new treatment measures.
[0003] Currently, adenoviruses and lentiviruses are mostly used as expression vectors for gene therapy. However, lentiviruses are mostly modified from leukemia viruses or HIV, and adenovirus vectors have a short expression time and are immunogenic to the body, both of which are not suitable for future clinical applications. Recombinant adeno-associated virus vectors (rAAV) overcome the disadvantages that are difficult to overcome by other gene expression vectors. They are non-immunogenic and can drive the long-term expression of target genes in vivo, thus becoming the most promising vector for gene therapy.
[0004] The SACS gene encodes the sacsin protein, which includes an N-terminal UbL domain, a DnaJ domain, and a C-terminal HEPN domain. The SACS gene is highly expressed in the central nervous system, and mutations in this gene lead to autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). ARSACS is a neurodegenerative disease characterized by early-onset cerebellar ataxia with spasticity and peripheral neuropathy. However, so far, there have been no reports on the association between SACS and diabetic cardiomyopathy, and there is also a complete lack of reports on the therapeutic effect of SACS in diabetic cardiomyopathy and the use of related therapeutic drugs. Summary of the Invention
[0005] To solve the above-mentioned problems and deficiencies objectively existing in the prior art in this field, the present invention provides the application of SACS in the preparation of drugs for treating diabetic cardiomyopathy, as well as its products and preparation methods.
[0006] The above technical objectives of the present invention are achieved through the following technical solutions:
[0007] The application of SACS in the preparation of drugs for treating diabetic cardiomyopathy.
[0008] The drug for treating diabetic cardiomyopathy uses SACS as a drug target.
[0009] Preferably, the drug for treating diabetic cardiomyopathy includes a pharmacologically active ingredient; the pharmacologically active ingredient includes: a substance that knocks out or knocks down or silences or downregulates the expression of SACS.
[0010] Preferably, the substance that knocks out or knocks down or silences or downregulates the expression of SACS includes: SACS-shRNA, an expression vector linked with SACS-shRNA, and a host transformed with the expression vector linked with SACS-shRNA.
[0011] Preferably, the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0012] Preferably, the expression vector is selected from: the adeno-associated virus expression vector pAAV D(+).
[0013] Preferably, the host is selected from: viruses and / or cells.
[0014] Preferably, the virus is selected from adeno-associated viruses.
[0015] Preferably, the cell is selected from 293 cells.
[0016] A SACS-shRNA, the corresponding DNA sequence of which is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0017] A recombinant expression vector, which is an expression vector ligated with the above-mentioned SACS-shRNA.
[0018] The promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0019] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0020] A transformant, characterized in that it is a host transformed with the above-mentioned recombinant expression vector.
[0021] The host is selected from: virus and / or cell;
[0022] Preferably, the virus is selected from adeno-associated virus;
[0023] Preferably, the cell is selected from 293 cells;
[0024] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0025] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0026] An anti-diabetic cardiomyopathy drug, comprising a pharmaceutically active ingredient, and the pharmaceutically active ingredient is selected from the group consisting of the above-mentioned SACS-shRNA, the above-mentioned recombinant expression vector, and the above-mentioned transformant;
[0027] A preparation method of an anti-diabetic cardiomyopathy drug, characterized in that substances capable of knocking out, knocking down, silencing, or down-regulating the expression of SACS are designed, screened, synthesized, prepared, and produced.
[0028] In some specific embodiments, the preparation method comprises the following steps: preparing SACS-shRNA; the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0029] The preparation is selected from the group consisting of synthesis, amplification, expression, cloning, secretion, enrichment, and propagation;
[0030] Preferably, the synthesis refers to the whole gene synthesis technology;
[0031] Preferably, the cloning refers to ligating SACS-shRNA to an expression vector;
[0032] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0033] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0034] Use of the SACS-shRNA, and / or the recombinant expression vector, and / or the transformant in the preparation of a medicament for treating diabetic cardiomyopathy.
[0035] The present invention provides a use of SACS-shRNA in the preparation of a medicament for treating diabetic cardiomyopathy.
[0036] Preferably, the expression vector is the adeno-associated virus expression vector pAAV-D(+);
[0037] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0038] Preferably, the expression vector ligated with SACS-shRNA is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-SACS-shRNA ligated with the SACS-shRNA sequence;
[0039] Preferably, the host cell of the transformant is selected from: 293 cells;
[0040] Preferably, the transformant transformed with the SACS-shRNA expression vector is selected from: the recombinant adeno-associated virus rAAV-U6-SACS-shRNA transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-SACS-shRNA;
[0041] Preferably, the SACS-shRNA sequence is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4;
[0042] A recombinant expression vector having an effect of treating diabetic cardiomyopathy, characterized in that it is an expression vector ligated with a sequence of a gene having an effect of treating diabetic cardiomyopathy.
[0043] The expression vector is selected from: the adeno-associated virus expression vector pAAV-D(+);
[0044] Preferably, the recombinant expression vector having the effect of treating diabetic cardiomyopathy is selected from: the recombinant adeno-associated virus expression vector pAAV-D(+)-SACS-shRNA ligated with the SACS-shRNA gene sequence.
[0045] 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.
[0046] The host cell is selected from: 293 cells;
[0047] Preferably, the transformant having the effect of treating diabetic cardiomyopathy is selected from: the recombinant adeno-associated virus rAAV-SACS-shRNA transformed with the recombinant adeno-associated virus expression vector pAAV-D(+)-SACS-shRNA;
[0048] Furthermore, the drug further comprises a pharmaceutically acceptable excipient, and / or a reagent for buffering, synthesizing, and / or purifying the sequence fragment. Those skilled in the art can add various pharmaceutically acceptable adjuvants / 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.
[0049] In a further embodiment, the preparation method includes: inserting the SACS-shRNA sequence fragment into an expression vector to prepare a recombinant plasmid capable of stably expressing SACS-shRNA.
[0050] In a specific embodiment, the expression vector containing the SACS-shRNA sequence fragment is the adeno-associated virus expression vector pAAV-D(+).
[0051] In order to achieve the purpose of gene therapy for diabetic cardiomyopathy, the present invention recombinantly constructs the SACS-shRNA sequence fragment with a recombinant adeno-associated virus vector, and after detection, a high titer meeting the treatment requirements is obtained, and it is confirmed in animal experiments that it can effectively improve the cardiac function of diabetic cardiomyopathy mice. Therefore, based on the above findings and results, the present invention provides a small RNA drug represented by SACS-shRNA for the treatment of clinical diabetic cardiomyopathy.
[0052] When performing high-throughput mass spectrometry detection on the hearts of diabetic cardiomyopathy mice in the present invention, it was found that SACS was significantly elevated in diabetic cardiomyopathy. Based on this, an anti-diabetic cardiomyopathy drug targeting SACS was developed, and its effect was verified on an animal model. It was found that knocking down SACS using shRNA could significantly improve the cardiac function of mice and treat diabetic cardiomyopathy.
[0053] The present invention relates to a drug for treating diabetic cardiomyopathy. The anti-diabetic cardiomyopathy drug relates to the construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-SACS-shRNA) of SACS-shRNA, and exerts the efficacy of treating diabetic cardiomyopathy by highly expressing the rAAV-SACS-shRNA. The present invention constructs a pAAV-D(+)-SACS-shRNA expression plasmid using chemical synthesis, and packages and purifies a recombinant adeno-associated virus containing the target fragment by the calcium phosphate co-transfection method of three plasmids. Animal experiments have confirmed that the anti-diabetic cardiomyopathy drug provided by the present invention can significantly improve the blood glucose abnormality and cardiac function abnormality of diabetic cardiomyopathy animals, and play an effective role in anti-diabetic cardiomyopathy.
[0054] The present invention designed and synthesized the sequence of SACS-shRNA, and successfully inserted it into the eukaryotic expression vector pAAV-D(+) to form the recombinant plasmid pAAV-D(+)-SACS-shRNA. Then, the following three plasmids: 1) pXX9 plasmid, 2) phelper plasmid, 3) pAAV-D(+)-SACS-shRNA plasmid, were transfected into 293 cells by calcium phosphate co-transfection method to package and prepare a recombinant adeno-associated virus (rAAV9) capable of expressing SACS-shRNA. After purification, the titer was measured by real-time PCR method. Next, the packaged recombinant adeno-associated virus (rAAV-SACS-shRNA) of the same serotype was injected into diabetic model mice caused by diabetes (streptozotocin STZ) via the tail vein. Ultrasonic and catheter results showed that SACS-shRNA mediated by recombinant adeno-associated virus could significantly improve the blood glucose abnormality and cardiac function abnormality of diabetic mice. Brief Description of the Drawings
[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0056] Figure 1It is a schematic structural diagram of the pAAV-D(+)-SACS-shRNA plasmid prepared in Experimental Example 1 of the present invention; wherein, U6 and H1 represent promoters; sh1 represents SEQ ID NO.1, sh2 represents SEQ ID NO.2, sh3 represents SEQ ID NO.3, sh4 represents SEQ ID NO.4; ITR represents an inverted repeat sequence; Amp represents an ampicillin resistance gene sequence; From Figure 1 looking, the promoter of sh3 is H1, the promoter of sh4 is U6, the promoter of sh1 is U6, the promoter of sh2 is H1. Those skilled in the art know that swapping the promoters among sh1, sh2, sh3, sh4 or replacing them with other known promoters in the art such as CMV will also have the same effect.
[0057] Figure 2 It shows that the content of SACS in the hearts of diabetic cardiomyopathy mice in Experimental Example 2 of the present invention is significantly increased; wherein, A is a column chart of the SACS content detected by mass spectrometry in the hearts of control mice and STZ-induced diabetic mice; B is a column chart of the SACS content detected by mass spectrometry in human cardiomyocytes AC16 intervened with high glucose; Control represents control group mice; STZ represents model group mice; Highglucose represents the cell group intervened with high glucose;
[0058] Figure 3 It is the detection by cardiac ultrasound in Experimental Example 2 of the effect of rAAV-SACS-shRNA treatment on the cardiac function of STZ-induced diabetic mice. Among them, A is a column chart of ejection fraction, B is a column chart of shortening fraction, C is a column chart of random blood glucose; D is the protein knockdown effect diagram of cardiac SACS; E is the cardiomyocyte area diagram. The results show that rAAV-SACS-shRNA can significantly increase the cardiac contractile function of diabetic mice while reducing hyperglycemia; rAAV-SACS-shRNA can reduce the protein content of cardiac SACS and myocardial hypertrophy. The meanings of each label in the figure are listed as follows: rAAV-Control-shRNA represents STZ mice injected with the negative control rAAV-Control-shRNA virus, rAAV-SACS-shRNA represents STZ mice injected with the rAAV-SACS-shRNA virus, and Control represents control group mice. Detailed implementation manners
[0059] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0060] I. Instruments and Equipment
[0061] ND-1000 Nucleic Acid Analyzer, ABI 9700 PCR Instrument, ABI 7900HT Fluorescent Real-Time Quantitative PCR Instrument, Beckman X-15R Low-Temperature High-Speed Centrifuge;
[0062] II. Reagents and Consumables
[0063] RNasey Mini Kit 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 TransGen Biotech Co., Ltd.); The eukaryotic expression vector pAAV-D(+) is from Vigene Biosciences Co., Ltd., and the core sequence of SACS-shRNA was designed by the applicant and synthesized by Vigene Biosciences Co., Ltd.
[0064] III. Sources of Biological Materials
[0065] (I) The DH5α competent cells used in Experimental Example 1 are commercially available; 293T cells are from the American Type Culture Collection (ATCC).
[0066] (II) The C57 mice used in Experimental Example 2 were purchased from Beijing Huafukang Bioscience Co., Ltd.; human cardiomyocytes AC16 were purchased from the American Type Culture Collection (ATCC); the negative control rAAV-Control-shRNA virus was purchased from Shandong Vigene Biosciences Co., Ltd.
[0067] Group 1 of Examples, Pharmaceutical Use of SACS of the Present Invention
[0068] This group of examples provides the application of SACS in the preparation of anti-diabetic cardiomyopathy drugs.
[0069] In some specific examples, the anti-diabetic cardiomyopathy drug targets SACS;
[0070] Preferably, the anti-diabetic cardiomyopathy drug comprises a pharmacologically active ingredient; the pharmacologically active ingredient comprises: a substance that knocks out or knocks down or silences or down-regulates the expression of SACS;
[0071] Preferably, the substance that knocks out or knocks down or silences or down-regulates the expression of SACS comprises: SACS-shRNA, an expression vector linked with SACS-shRNA, and a host transformed with the expression vector linked with SACS-shRNA;
[0072] Preferably, the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;
[0073] Preferably, the expression vector is selected from: the adeno-associated virus expression vector pAAV D(+);
[0074] Preferably, the host is selected from: virus and / or cell;
[0075] Preferably, the virus is selected from adeno-associated virus;
[0076] Preferably, the cell is selected from 293 cells.
[0077] Preferably, the SACS is selected from: human SACS, rat SACS.
[0078] Group 2 Examples, SACS-shRNA of the present invention
[0079] This group of examples provides a SACS-shRNA. All examples in this group have the following common characteristics: the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0080] The SACS-shRNAs of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 have been verified by animal experiments to have an anti-diabetic cardiomyopathy effect. The corresponding DNA sequence refers to the DNA sequence in which the U base in the shRNA is replaced by T base and the other bases are the same as those in the shRNA.
[0081] Preferably, the SACS is selected from: human SACS, rat SACS.
[0082] The above SACS-shRNAs are all prepared for the first time in the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, propagating, enriching, ligating, transforming, cloning, or expressing the above SACS-shRNAs, and / or any act of using the above SACS-shRNAs for pharmaceutical purposes or as a drug ingredient, and / or any act of using the above recombinant expression vector for treatment falls within the protection scope of the present invention.
[0083] Group 3 Examples, the recombinant expression vector of the present invention
[0084] This group of examples provides a recombinant expression vector. All examples in this group have the following common feature: the recombinant expression vector is an expression vector ligated with the SACS-shRNA described in any item of Group 2 Examples.
[0085] In some examples, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0086] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0087] The above recombinant expression vectors are all prepared for the first time in the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering for sale, using, importing, exporting, secreting, propagating, enriching, ligating, transforming, cloning, or expressing the above recombinant expression vectors, and / or any act of using the above recombinant expression vectors for pharmaceutical purposes or as a drug ingredient, and / or any act of using the above recombinant expression vectors for treatment falls within the protection scope of the present invention.
[0088] Group 4 Examples, the transformant of the present invention
[0089] This group of examples provides a transformant. All examples in this group have the following common feature: the transformant is a host transformed with the recombinant expression vector described in any item of Group 3 Examples.
[0090] In specific examples, the host is selected from: virus and / or cell;
[0091] Preferably, the virus is selected from adeno-associated virus;
[0092] Preferably, the cell is selected from 293 cells;
[0093] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0094] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0095] The above-mentioned transformants are all prepared for the first time in the present invention. Any act of amplifying, synthesizing, producing, manufacturing, selling, offering to sell, using, importing, exporting, secreting, propagating, enriching, ligating, transforming, cloning, or expressing the above-mentioned transformants, and / or any act of using the above-mentioned transformants for pharmaceutical purposes or as drug ingredients, and / or any act of using the above-mentioned transformants for treatment falls within the protection scope of the present invention.
[0096] Group 5 Examples, the drug of the present invention
[0097] This group of examples provides an anti-diabetic cardiomyopathy drug. All examples in this group have the following common characteristics: the anti-diabetic cardiomyopathy drug includes a pharmaceutically active ingredient, and the pharmaceutically active ingredient is selected from the group consisting of SACS-shRNA described in any one of Group 2 examples, the recombinant expression vector described in any one of Group 3 examples, and the transformant described in any one of Group 4 examples;
[0098] In a further embodiment, the anti-diabetic cardiomyopathy drug further includes: pharmaceutical excipients; and / or reagents for amplifying, synthesizing, producing, manufacturing, selling, offering to sell, using, importing, exporting, secreting, propagating, enriching, ligating, transforming, cloning, or expressing SACS-shRNA described in any one of Group 2 examples, and / or the recombinant expression vector described in any one of Group 3 examples, and / or the transformant described in any one of Group 4 examples;
[0099] 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 make various dosage forms for easy sales or promotion.
[0100] In a specific embodiment, the pharmaceutical excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants.
[0101] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0102] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0103] Group 6 Examples, Preparation Method of the Drug of the Present Invention
[0104] This group of examples provides a preparation method of an anti-diabetic cardiomyopathy drug. All examples in this group have the following common features: designing, screening, synthesizing, preparing, and producing substances that can knockout or knockdown or silence or down-regulate the expression of SACS.
[0105] In some specific examples, the preparation method includes the following steps: preparing SACS-shRNA; the DNA sequences corresponding to SACS-shRNA are selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0106] The preparation is selected from the group consisting of: synthesis, amplification, expression, cloning, secretion, enrichment, and propagation;
[0107] Preferably, the synthesis refers to the whole gene synthesis technology;
[0108] Preferably, the cloning refers to ligating SACS-shRNA to an expression vector;
[0109] Preferably, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0110] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
[0111] In a further example, the preparation method further includes: formulating or mixing the pharmaceutically active ingredient with a pharmaceutical excipient.
[0112] 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 sales or promotion.
[0113] In a specific embodiment, the pharmaceutical excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants.
[0114] Group 7 of the embodiments, the pharmaceutical use of SACS-shRNA of the present invention
[0115] This group of embodiments provides the use of SACS-shRNA in the preparation of anti-diabetic cardiomyopathy drugs. All embodiments in this group have the following characteristics: the DNA sequence corresponding to SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0116] In some embodiments, the anti-diabetic cardiomyopathy drug uses SACS-shRNA or a recombinant expression vector or a transformant as the pharmaceutically active ingredient;
[0117] In a further embodiment, the anti-diabetic cardiomyopathy drug further includes pharmaceutical excipients;
[0118] In a specific embodiment, the pharmaceutical excipients are selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants.
[0119] In a specific embodiment, the transformant is a host containing a recombinant expression vector; the recombinant expression vector is an expression vector linked to the shRNA of the sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and can play a role in treating diabetic cardiomyopathy;
[0120] In a preferred embodiment, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin;
[0121] Preferably, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+);
[0122] Preferably, the host is selected from: virus and / or cell;
[0123] Preferably, the virus is selected from adeno-associated virus;
[0124] Preferably, the cell is selected from 293 cells.
[0125] The present invention will be further described below and in the examples:
[0126] Experimental Example 1. Construction of Recombinant Adeno-Associated Virus
[0127] 1. Design of SACS-shRNA Sequences
[0128] The applicant designed several potential shRNAs of mouse SACS using the biological website (Invitrogen Block-iT RNAi Designer). The SACS-shRNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The applicant selected the sequences composed of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 for animal experiments as representatives of the examples. The constructions described below are only some examples of the present invention. For those of ordinary skill in the art, without creative efforts, similar example results can also be obtained according to other shRNA sequences of SACS.
[0129] 2. Construction of pAAV-D(+) Vector
[0130] The rAAV-SACS-shRNA double-stranded nucleotides were synthesized by full gene synthesis technology and cloned onto the pAAV-D(+) vector ( Figure 1 ), and the double-stranded nucleotides were synthesized by Vigene Biosciences Co., Ltd. The SACS-shRNA sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.
[0131] 3. Plasmid Transformation
[0132] The pAAV-D(+)-SACS-shRNA plasmid was added to 100 μl of DH5α competent cells and placed on ice for 30 min; after heating at 42 °C for 45 sec, it was then placed on ice for 1 min; 500 μl of antibiotic-free LB medium was added, and the mixture was cultured with shaking at 37 °C and 100 rpm for 60 min; it was cultured on an Amp+ LB plate medium, and white monoclonal colonies were selected for identification.
[0133] 4. Plasmid miniprep
[0134] Pick a single colony and add it to 3 ml of LB liquid medium with Amp+, and culture it overnight at 37 °C with shaking at 280 rpm. Use the EasyPure Plasmid MiniPrep Kit from Beijing TransGen Biotech Co., Ltd. to extract the plasmid. The specific operation steps are as follows: 1. Centrifuge 1.5 ml of the overnight cultured bacteria at 10,000 g for 1 min, and aspirate as much supernatant as possible; 2. Add 250 μl of colorless solution RB (containing RNase A) and vortex to suspend the bacterial pellet; 3. Add 250 μl of blue solution LB and gently invert the mixture up and down 4 - 6 times to fully lyse the bacteria and form a blue and transparent solution; 4. Add 350 μl of yellow solution NB and mix gently 5 - 6 times until a firm yellow agglomerate is formed, and let it stand at room temperature for 2 min; 5. Centrifuge at 15,000 g for 5 min, carefully aspirate the supernatant and add it to the adsorption column; 6. Centrifuge at 15,000 g for 1 min and discard the effluent; 7. Add 650 μl of solution WB, centrifuge at 15,000 g for 1 min and discard the effluent; 8. Centrifuge at 15,000 g for 2 min to completely remove the residual WB; 9. Place the adsorption column in a new Ep tube, add 20 μl of EB preheated at 70 °C to the center of the column, and let it stand at room temperature for 1 min; 10. Centrifuge at 10,000 g for 1 min to elute the DNA, and store the eluted DNA at -20 °C.
[0135] 5. Plasmid maxiprep
[0136] Prepare a 1 L sterile conical flask, add 300 ml of sterile LB medium, and add ampicillin solution to a final concentration of 100 μg / ml. Add 50 μl of the required plasmids (pXX9, phelper, pAAV-D(+)-Control-shRNA, pAAV-D(+)-SACS-shRNA) respectively, and culture overnight at 37 °C with shaking at 280 rpm. Operate according to the instructions of the Endo-Free Plasmid Maxi Kit from OMEGA company to extract the plasmid. The specific steps are as follows: 1. Centrifuge the bacteria at 5000 g for 10 min at room temperature to collect them; 2. Discard the medium, add 10 ml of Solution I / RNase A mixture, and vortex to resuspend completely; 3. Add 10 ml of Solution II to the resuspended mixture, gently invert and mix 10 - 15 times, and then let it stand at room temperature
[0137] 6. rAAV-mediated virus packaging
[0138] The 293T cells were grown to 90%. 1-2 hours before calcium phosphate transfection, 12-15 ml of fresh medium (containing serum) was changed for each culture dish. First, calcium chloride (CaCl2) was added to a 50 ml centrifuge tube, and then the plasmid was added to form a Ca-DNA mixture, which was thoroughly mixed. 2X HEBS BUFFER was slowly added dropwise to the Ca-DNA mixture to form a Ca-DNA-P mixture. While adding 2X HEBS, the centrifuge tube was shaken to thoroughly mix and form calcium phosphate particles. After 8-12 hours, 18-20 ml of serum-free medium was changed. After 72 hours, the medium was aspirated, and the cells were washed 3 times with PBS. 1 ml of Tris + NaCl (pH 8.5) was added to each culture dish, and the cells were scraped with a spatula and collected in a clean centrifuge tube and stored at -80 °C.
[0139] 7. Virus purification
[0140] The cells stored at -80 °C were taken out, thawed and dissolved at 37 °C, and repeatedly frozen and thawed 4 times, centrifuged at 8,000 g for 15 min. The supernatant was transferred to a clean centrifuge tube, and the cell pellet was discarded. Anhydrous ethanol pre-cooled at -20 °C and rAAV were thoroughly mixed at a volume ratio of 3:1, placed in a -20 °C refrigerator for 2 hours, then centrifuged at 4 °C and 13,000 rpm for 15 min, and the supernatant was discarded; after the ethanol evaporated, an appropriate volume of Tris + NaCl (pH 8.5) was added to dissolve the precipitate. It was filtered through a Millipore small filter (0.22 μm).
[0141] 8. Virus titer determination
[0142] Sample treatment:
[0143] 40 μl of rAAV virus solution
[0144] 5 μl of proteinase K (20 mg / ml)
[0145] React at 55 °C for 1 hour;
[0146] 45 μl of phenol:chloroform:isoamyl alcohol
[0147] Centrifuge at 4 °C and 12,000 g for 5 min to recover the aqueous phase;
[0148] 45 μl of chloroform
[0149] Centrifuge at 4 °C and 12,000 g for 5 min to recover the aqueous phase.
[0150] Real-time PCR:
[0151] 0.4 μl of Primer 1 (10 μm)
[0152] 0.4 μl of Primer 2 (10 μm)
[0153] 10 μl of SYBR Green I Mix
[0154] 8.2 μl of ddH2O
[0155] 1 μl of Template
[0156] 95°C for 30 sec --- (95°C for 5 sec --- 60°C for 5 sec --- 72°C for 20 sec) × 40 cycles --- Melting Curve Example 2. Taking the recombinant adeno-associated virus expressing SACS-shRNA of rAAV9 type as an example, its therapeutic effect on diabetic cardiomyopathy was detected
[0157] 1. Detection of SACS expression in the hearts of STZ mice:
[0158] Preparation of STZ (streptozotocin) solution: Dissolve STZ in sodium citrate buffer to freshly prepare an STZ solution with a concentration of 8 mg / mL. Use 8-week-old C57 mice, fast them for 12 h before the operation. Mice in the model group were intraperitoneally injected with STZ at a dose of 40 mg / kg for 5 consecutive days; the control group was given the same dose of sodium citrate buffer to intervene in the cultured cells; the high-glucose intervention cell group: the cultured cells were intervened with a 33.3 mM glucose solution; the normal-glucose cell group: the cultured cells were intervened with a 5.5 mM glucose solution. Three days after the completion of STZ injection, measure the fasting blood glucose, and select those with blood glucose higher than 16.7 mmol / L for the formal experiment. Detect the expression of SACS in the hearts of STZ mice and in the human cardiomyocyte AC16 intervened with high glucose by mass spectrometry. The results showed that the content of SACS in the hearts of STZ mice and in the human cardiomyocytes intervened with high glucose was significantly increased ( Figure 2 ).
[0159] 2. Detection of cardiac function in STZ mice: At the end of the experiment, the cardiac function of STZ mice was detected by echocardiography. The method is as follows:
[0160] Use 8-week-old C57 mice to establish an STZ model. After the model was successfully established, the transformant rAAV-SACS-shRNA virus transformed with the pAAV-D(+)-SACS-shRNA plasmid of Experimental Example 1 and the negative control rAAV-Control-shRNA virus were respectively injected through the tail vein. The virus titer was 1×10 11PFU / mouse. At the end of the experiment (after 16 weeks), cardiac function indexes and blood glucose levels were detected. The instrument used was an ultrasound machine equipped with a 30 MHz high-frequency probe. After anesthetizing the mice with isoflurane, the mice were placed supine on the detection platform, and two-dimensional left ventricular images 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 consecutive M-mode ultrasound images were obtained under the guidance of the two-dimensional images. According to the collected images, the software was used to analyze the results, and the cardiac hemodynamic indexes detected by echocardiography were obtained. After analysis by relevant software, the following indexes were calculated: including heart rate (HR), left ventricular internal dimension in diastole (LVIDd), left ventricular internal dimension in systole (LVIDs), left ventricular posterior wall thickness in diastole (LVPWd), left ventricular posterior wall thickness in systole (LVPWs), interventricular septal thickness in diastole (IVSd), interventricular septal thickness in systole (IVSs), ejection fraction (EF), and fractional shortening (FS), etc. The results showed that rAAV-SACS-shRNA treatment could significantly improve the cardiac function abnormalities in STZ mice ( Figure 3 A - B) and blood glucose abnormalities ( Figure 3 C). Western Blot detection showed that rAAV-SACS-shRNA treatment could significantly reduce the expression of SACS protein in the heart ( Figure 3 D). Cell area detection found that rAAV-SACS-shRNA treatment could significantly reduce myocardial hypertrophy ( Figure 3 E).
Claims
1. Use of SACS in the preparation of anti-diabetic cardiomyopathy drugs.
2. Use of SACS according to claim 1 in the preparation of anti-diabetic cardiomyopathy drugs, characterized in that, The anti-diabetic cardiomyopathy drug targets SACS; and / or, the anti-diabetic cardiomyopathy drug comprises a pharmaceutically active ingredient; the pharmaceutically active ingredient comprises: a substance that knocks out or knocks down or silences or down-regulates the expression of SACS; and / or, the substance that knocks out or knocks down or silences or down-regulates the expression of SACS comprises: SACS-shRNA, an expression vector linked with SACS-shRNA, a host transformed with an expression vector linked with SACS-shRNA; and / or, the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4; and / or, the expression vector is selected from: adeno-associated virus expression vector pAAV D(+); and / or, the host is selected from: virus, and / or, cell; and / or, the virus is selected from adeno-associated virus; and / or, the cell is selected from 293 cells.
3. A SACS-shRNA, characterized in that, Its corresponding DNA sequence is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.
4.
4. A recombinant expression vector, characterized in that, Is an expression vector linked with the SACS-shRNA described in claim 3.
5. The recombinant expression vector according to claim 4, wherein The promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin; and / or, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).
6. A transformant, characterized in that, Is a host transformed with the recombinant expression vector described in claim 4 or 5.
7. A transformant according to claim 6, characterized in that, The host is selected from: virus, and / or, cell; and / or, the virus is selected from adeno-associated virus; and / or, the cell is selected from 293 cells; and / or, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin; and / or, the expression vector is selected from adeno-associated virus expression vector pAAV D(+).
8. An anti-diabetic cardiomyopathy drug, comprising a pharmacodynamic active ingredient, characterized in that, The pharmaceutically active ingredient is selected from the group consisting of the SACS-shRNA described in claim 3, the recombinant expression vector described in claim 4 or 5, and the transformant described in claim 6 or 7.
9. A preparation method of an anti-diabetic cardiomyopathy drug, characterized in that, Design, screen, synthesize, prepare, produce a substance that can knock out or knock down or silence or down-regulate the expression of SACS. In some specific embodiments, the preparation method comprises the following steps: preparing SACS-shRNA; the DNA sequence corresponding to the SACS-shRNA is selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.
4. The preparation is selected from the group consisting of: synthesis, amplification, expression, cloning, secretion, enrichment, propagation; and / or, the synthesis refers to total gene synthesis technology; And / or, the cloning refers to ligating SACS-shRNA to an expression vector; And / or, the promoter of the expression vector is selected from: non-specific promoters H1, U6, CMV, or cardiomyocyte-specific promoters tnt, α-MHC, MLC-2v, Desmin; And / or, the expression vector is selected from the adeno-associated virus expression vector pAAV D(+).
10. Use of the SACS-shRNA according to claim 3, and / or a recombinant expression vector according to claim 4 or 5, and / or a transformant according to claim 6 or 7 in the preparation of a medicament for treating diabetic cardiomyopathy.