Patent Application
20240263178
This application claims priority to and the benefit of Chinese patent Application No. 202310099726.4 filed Feb. 6, 2023. The entire contents of Chinese patent Application No. 202310099726.4 and the English translation of Chinese patent Application No. 202310099726.4 are incorporated herein by reference.
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing .xml file entitled “000005 us_SequenceListing.xml”, file size 7,712 bytes, created on Jan. 21, 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
The present invention belongs to the pharmaceutical field of cardiovascular diseases, and specifically relates to use of LncRNA DCRT as drug and its drug for treating cardiovascular diseases, screening method, and preparation method thereof.
Heart failure is a complex syndrome caused by impaired ventricular filling and/or ejection capacity led by various structural or functional diseases of heart, which significantly increases risk of hospitalization and death worldwide. Despite advances in scientific research and treatment strategies, treating heart failure remains a challenge. Dilated cardiomyopathy is one of the most common causes of heart failure, it's defined as left ventricular systolic dysfunction and dilation under abnormal load conditions proportional to degree of left ventricular damage or without coronary artery disease. Heart failure is a clinical syndrome that progresses to the terminal stage of different cardiovascular diseases, with poor prognosis and high mortality rate, which is one of the main causes threatening human health and increasing medical burden. Despite progress in recent treatment, a large number of patients with dilated cardiomyopathy still experience arrhythmias, heart failure, and sudden cardiac death. Dilated cardiomyopathy ranks third among various cardiovascular diseases that lead to heart failure and is the leading cause of heart transplant surgery.
For pathogenesis of dilated cardiomyopathy, genetic mutations involving genes account for 35% of cases. Endocrine abnormalities, drugs and toxins, as well as infectious factors, are all causes of acquired dilated cardiomyopathy. Importantly, mitochondrial dysfunction plays a crucial role in pathogenic process of dilated cardiomyopathy. Mitochondria are “power source” of eukaryotic cells, a highly dynamic organelle that is particularly important for cells that require high energy, such as cardiomyocytes. Mitochondria provide a large amount of adenosine triphosphate every day, which maintains heart function through oxidative phosphorylation. Mutations in genes encoding mitochondrial proteins often lead to abnormal oxidative phosphorylation, which not only results in ATP deficiency but also increases ROS production and changes in antioxidant defense system, ultimately promoting development of dilated cardiomyopathy. Therefore, maintaining normal mitochondrial function in myocardial cells can serve as a new target for treating heart failure.
Long chain non coding RNAs are a newly discovered class of non coding RNAs with nucleotide sequences greater than 200 nt, which play an important role in regulating gene expression. Recently, an increasing number of studies have found that lncRNA plays an important role in various cardiovascular diseases, including myocardial metabolism, myocardial hypertrophy, cardiac conduction system, and cardiac development. Clarifying molecular mechanisms underlying function of lncRNA is a major challenge in this field. In recent years, more and more studies have shown that lncRNA is involved in various processes of gene expression in disease process, including transcriptional regulation, post transcriptional regulation, epigenetic regulation and etc. The function of lncRNA can be regulated by cis- or trans-action to regulate expression level of downstream target genes. Mechanism of lncRNAs located in different subcellular structures is different. Localization in cytoplasm and nucleus of lncRNA helps us determine the action mode of lncRNA. The lncRNA located in the nucleus may affect gene transcription through various mechanisms, including epigenetic modifications, interactions with transcription factors, and affecting mRNA processing or output. Action modes of lncRNAs distributed in the cytoplasm mainly include: affecting stability of mRNA, affecting translation initiation, acting as competitive endogenous RNAs, or affecting post translation modifications, etc. Due to lncRNAs lack conservation among genuses, although many studies have reported the function and mechanism of long-chain non coding RNAs in mouse models, little is known about their roles in human heart. There is currently no report of an association between lncRNA DCRT and cardiovascular disease.
Based on the objective problems and shortcomings in this field, the present invention accidentally discovered relationship between lncRNA DCRT and mechanism of dilated cardiomyopathy, and further conducted molecular experiments to develop an anti-dilated cardiomyopathy drug with lncRNA DCRT as the drug target. At the same time, animal experiments were conducted to verify its effectiveness, and it was found that molecules targeting lncRNA DCRT and upregulating expression of DCRT can significantly improve cardiac function impairment and pathological increase in myocardial cell area in dilated cardiomyopathy.
Technical solution of the present invention is as follows:
The cardiovascular disease is dilated cardiomyopathy.
A drug for treating cardiovascular disease, characterized in that, its active pharmaceutical ingredient is taking lncRNA DCRT with a sequence shown as SEQ ID NO.1 as target.
Preferably, the substance regulating expression of lncRNA DCRT is selected from a group consisting of primers upregulating or overexpressing lncRNA DCRT, a recombinant expression vector upregulating or overexpressing lncRNA DCRT, and a transformant upregulating or overexpressing lncRNA DCRT;
A method of preparing a drug for treating cardiovascular disease, characterized in that, comprising the following step: a substance upregulating or overexpressing lncRNA DCRT is used as the active pharmaceutical ingredient of the drug; sequence of lncRNA DCRT is shown as SEQ ID NO.1 active pharmaceutical ingredient.
The method of preparing a drug for treating cardiovascular disease also comprising the following step: a substance upregulating or overexpressing lncRNA DCRT is mixed with a medicinal excipient to produce the drug;
A screening method of a drug for treating cardiovascular disease, characterized in that, comprising the following step: screening candidate substance with lncRNA DCRT as target; sequence of lncRNA DCRT is shown as SEQ ID NO.1. The screening method of a drug for treating cardiovascular disease further comprising the following step: further screening substance regulating lncRNA DCRT expression from the candidate substance as active pharmaceutical ingredient of the drug active pharmaceutical ingredient;
The present invention provides a drug for treating dilated cardiomyopathy, wherein a active pharmaceutical ingredient of the drug targets lncRNA DCRT and exerts the therapeutic effect of treating dilated cardiomyopathy by upregulating expression of lncRNA DCRT.
Further configuration of the present invention is that the active pharmaceutical ingredient comprises a substance containing upregulated or overexpressed lncRNA DCRT, and sequence of the lncRNA DCRT is shown as SEQ ID NO.1.
By adopting above technical solution, the active pharmaceutical ingredient of the drug includes substance that upregulate or overexpress lncRNA DCRT, thereby increasing the expression level of lncRNA DCRT and further treating the disease.
Further configuration of the present invention is to further include pharmaceutically acceptable excipient.
By adopting above technical solution, a person skilled in the art can add various pharmaceutically acceptable excipient/excipient to the anti dilated cardiomyopathy drug of the present invention according to objective needs, and make various dosage forms for easy sales or promotion.
Further embodiments of the present invention include reagent for buffering, and/or synthesizing, and/or purifying the lncRNA DCRT.
The present invention also provides a drug for treating dilated cardiomyopathy, wherein the active pharmaceutical ingredient of the drug have degradation or inhibitory effect on substance that downregulate the expression of lncRNA DCRT sequence.
The present invention also provides a screening method for anti dilated cardiomyopathy drug, detecting whether candidate substance can upregulate the expression of lncRNA DCRT.
The present invention also provides a substance that can upregulate the expression of lncRNA DCRT for preparing anti dilated cardiomyopathy drug.
The present invention also provides a use of a full-length sequence fragment of lncRNA DCRT in preparing drug for treating dilated cardiomyopathy. Full-length sequence fragment of lncRNA DCRT is shown as SEQ ID NO.1.
Another aspect of the present invention provides a use of a substance upregulating the expression of lncRNA DCRT in preparing anti dilated cardiomyopathy drug. Any act of packaging above substance in commercial packaging labeled with anti dilated cardiomyopathy use for any scale falls within protection scope of the present invention.
The beneficial effects of the present invention are as follows:
The present invention innovatively discovered highly conserved lncRNA DCRT in the heart tissue of patients with dilated cardiomyopathy. lncRNA DCRT is mainly localized in nucleus of myocardial cells and significantly reduced in myocardial cells of patients with dilated cardiomyopathy. Subsequently, it's confirmed by animal experiments that upregulating lncRNA DCRT has an effect of improving central function impairment and pathological increase in myocardial cell area in a cardiac dilation model induced by thoracic aortic constriction surgery.
In order to achieve the therapeutic purpose of dilated cardiomyopathy, the present invention uses lncRNA DCRT in animal experiments. It has been confirmed in animal experiments that a synthetic of lncRNA DCRT can significantly improve cardiac function impairment and myocardial cell contraction dysfunction induced by thoracic aortic constriction, to achieve a goal of assisting in treating dilated cardiomyopathy. Therefore, based on above findings and results, the present invention provides a drug for clinically treating dilated cardiomyopathy with lncRNA DCRT as a therapeutic target.
Based on a base sequence of lncRNA DCRT, the present invention designs a sequence for expressing lncRNA DCRT, and synthesizes it. A use of lncRNA DCRT synthetic can significantly improve cardiac function impairment induced by thoracic aortic constriction, myocardial cell contractile dysfunction, and calcium ion instability. This indicates that lncRNA DCRT has a potential to improve dilated cardiomyopathy.
The present invention found through experiments that the expression of lncRNA DCRT in heart tissue of patients with dilated cardiomyopathy was reduced (
Expression detection methods of cTNT, Col1a1, and CD31, as well as DAPI staining operation, can refer to the methods and steps recorded in the article “LncRNA ZNF593-AS Alleviates Contract Dysfunction in Dilated Cardiopathy”.
Operation of overexpressing GFP, construction method of Sham mice, and construction method of TAC disease model mice can refer to the methods and steps recorded in the article “LncRNA ZNF593-AS Alleviates Contract Dysfunction in Dilated Cardiopathy”.
The vertical axis of the left figure in
The technical solution of the present invention will be described clearly and completely in the following content by combining with specific examples and experimental examples. Obviously, the described experimental examples and examples are only a part of the present invention's experimental examples and examples, not all of them. Based on the experimental examples and examples of the present invention, all other examples obtained by a person skilled in the art without creative work fall within protection scope of the present invention.
The reverse transcription kit was purchased from Thermofish Company;
Peripheral blood of patients with dilated cardiomyopathy comes from hospitalized patients at Tongji Hospital in Wuhan from 2012 to 2018, all of whom have signed informed consent forms;
Examples group 1. use of lncRNA DCRT as drug of the present invention
This group of examples provides use of lncRNA DCRT with sequence shown as SEQ ID NO.1 as drug.
In some embodiments, a reverse complementary sequence of lncRNA DCRT is shown as SEQ ID NO.2.
In specific embodiments, the drug refers to a drug for treating cardiovascular disease.
In more specific embodiments, the cardiovascular disease is dilated cardiomyopathy.
The present invention for the first time reveals the pharmaceutical use of lncRNA DCRT shown as SEQ ID NO.1. Based on disclosure of the present invention, those skilled in the art can receive guidance and inspiration to use lncRNA DCRT shown as SEQ ID NO.1 in pharmacy. Any act of synthesizing, amplifying, preparing, producing, using, expressing, secreting, enriching, propagating, selling, promising for sale, importing, or exporting lncRNA DCRT shown as SEQ ID NO.1 for medicinal or commercial purposes falls within protection scope of the present invention.
The drug is not limited to treating cardiovascular diseases. Based on the disclosed content of the present invention, a person skilled in the art can receive guidance and inspiration to use lncRNA DCRT shown as SEQ ID NO.1 as drug and treating diseases other than cardiovascular diseases. Any use of lncRNA DCRT shown as SEQ ID NO.1 as a drug target for treating any disease falls within protection scope of the present invention.
Examples group 2. drug for treating cardiovascular diseases of the present invention.
This group of examples provides a drug for treating cardiovascular disease. All embodiments of this group possesses the following common features: a active pharmaceutical ingredient of the drug is taking lncRNA DCRT with a sequence shown as SEQ ID NO.1 as target.
In specific embodiments, the drug for treating cardiovascular disease, comprising: a active pharmaceutical ingredient, characterized in that, the active pharmaceutical ingredient comprises: a substance regulating expression of lncRNA DCRT.
In some examples, said regulating refers to up-regulating; preferably, the substance regulating expression of lncRNA DCRT is selected from a group consisting of primers upregulating or overexpressing lncRNA DCRT, a recombinant expression vector upregulating or overexpressing lncRNA DCRT, and a transformant upregulating or overexpressing lncRNA DCRT;
In specific embodiments, the expression vector is selected from: pAAV vector;
A more specific embodiment of this group provides a drug for treating dilated cardiomyopathy, in which all embodiments have the following features: the active pharmaceutical ingredient of the drug for treating dilated cardiomyopathy is taking lncRNA DCRT as the drug target, and its therapeutic effect for treating dilated cardiomyopathy is achieved by upregulating expression of lncRNA DCRT. The present invention found through experiments that sequence of lncRNA DCRT is highly conserved, and content of lncRNA DCRT in patients with dilated cardiomyopathy is significantly lower than that in control group. The lncRNA DCRT in main myocardial cells is significantly reduced. Subsequently, it's confirmed by animal experiments that upregulating lncRNA DCRT has an effect of improving central function impairment and myocardial cell contractility reduction induced by thoracic aortic constriction surgery, to achieve a goal of assisting in treating dilated cardiomyopathy.
In further embodiments, the active pharmaceutical ingredient of the drug include substances that upregulate expression of lncRNA DCRT; alternatively, the active pharmaceutical ingredient of the drug can degrade or inhibit a substance that downregulates expression of lncRNA DCRT. As a segment of RNA obtained externally in animals, lncRNA DCRT can be upregulated through molecular biotechnology methods to achieve therapeutic effects. Other methods, such as certain chemical molecules, can also be used to inhibit its degradation and achieve the same goal of upregulating its level.
In a preferred embodiment, the active pharmaceutical ingredient of the drug comprises a full-length fragment of sequence of lncRNA DCRT, thereby increasing its expression level and further treating it.
In other embodiments, the drug further comprises pharmaceutically acceptable excipients and/or reagents for buffering, synthesizing, and/or purifying the lncRNA DCRT sequence fragments. A person skilled in the art can add various pharmaceutically acceptable adjuvants/excipients to the anti dilated cardiomyopathy drug of the present invention according to objective needs, and make various dosage forms for easy sales or promotion.
In specific embodiments, the pharmaceutically acceptable excipients or pharmaceutical excipients are selected from: solvents, projectiles, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavor correction agents, preservatives, suspension aids, coating materials, aromatics, anti adhesives, integrators, penetration enhancers, pH regulators, buffering agents Plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, moisturizers, absorbers, diluents, flocculants, anti flocculants, filter aids, release blockers.
A person skilled in the art can make routine selections or adjustments to pharmaceutical excipients based on actual production needs, combined with conventional technical means or basic knowledge of pharmaceutical production processes (such as the «Encyclopedia of Pharmaceutical Formulation Technology», «Pharmaceutical Formulation Technology», etc.), in order to produce drugs with different dosage forms, storage conditions, and shelf life. This is not a technical barrier for the person skilled in the art, and can be easily achieved.
Those skilled in the art can select and allocate above excipients, and make the lncRNA DCRT of this invention and/or the substances regulating expression of lncRNA DCRT into different dosage forms, such as powder, tablet, oral liquid, capsule, granule, spray, gel, paste, decoction, pill, spray, inhaler, aerosol, injection, etc.
This group of embodiments provides a method of preparing a drug for treating cardiovascular diseases, characterized in that, comprising the following step: a substance upregulating or overexpressing lncRNA DCRT is used as the active pharmaceutical ingredient of the drug; sequence of lncRNA DCRT is shown as SEQ ID NO.1.
In specific embodiments, the method of preparing a drug for treating cardiovascular diseases also comprising the following step: a substance upregulating or overexpressing lncRNA DCRT is mixed with a medicinal excipient to produce the drug;
In specific embodiments, the expression vector is selected from: pAAV vector;
The present invention provides use of a substance capable of upregulating the expression of lncRNA DCRT in preparing anti-dilated cardiomyopathy drug. Any act of packaging above substance in commercial packaging labeled with anti-dilated cardiomyopathy use for any scale falls within protection scope of the present invention.
The present invention provides a method for preparing an anti dilated cardiomyopathy drug, comprising: using a substance that can upregulate expression of lncRNA DCRT as the active pharmaceutical ingredient of the anti dilated cardiomyopathy drug.
The specific experimental operation steps of this group of examples are shown in experiment example 2 below.
Examples group 4. Screening Method for Drug of the present invention.
This group of examples provides A screening method of a drug for treating cardiovascular diseases. All examples of this group possesses the following common features, said method comprising the following step: screening candidate substance with lncRNA DCRT as target; sequence of lncRNA DCRT is shown as SEQ ID NO.1.
In further embodiments, a screening method for a drug for treating cardiovascular disease further comprises the following step: further screening substance regulating lncRNA DCRT expression from the candidate substance as active pharmaceutical ingredient of the drug;
In preferred embodiments, primers upregulating or overexpressing lncRNA DCRT include:
In specific embodiments, the expression vector is selected from: pAAV vector;
The most specific example of this group provides a screening method for anti dilated cardiomyopathy drug, comprising the following steps: detecting whether candidate substance can upregulate expression of lncRNA DCRT, and screening for substances that can promote expression of lncRNA DCRT.
The specific experimental operation steps of this example can be found in experiment example 2.
Collecting heart tissues from 14 patients with dilated cardiomyopathy and 7 healthy heart transplant donors, and storing them in a −80° C. freezer. Adding 1 ml TRIZOL (Invitrogen company) to every 50 mg of heart, grinding, and extracting RNA. Using Nanodrop® ND-1000 to detect RNA quality.
It's shown by results that the expression of lncRNA DCRT in the heart tissue of patients with dilated cardiomyopathy decreased (
1. Packaging and purification of overexpressing lncRNA DCRT transformants
This experimental example requires packaging two viruses: rAAV-GFP (negative control) carrying green fluorescent protein (GFP) and rAAV-DCRT (upregulated DCRT) carrying lncRNA DCRT; a reverse complementary sequence of lncRNA DCRT is shown as SEQ ID NO.2, which was delivered to Shanghai Sangon Biotechnology Service Co., Ltd to synthesize by adding Hind III and Not I adhesive ends to oligonucleotide single strand respectively. Annealing forms double chains, with the following system:
After mixing and centrifuging the system, carrying out annealing reaction on PCR instrument with reaction procedure of: 95° C. for 2 minutes, decrease by 1° C. every 90 seconds, decrease to 25° C., and reserve at 4° C.
Putting a digestion product to 1% agarose gel for electrophoresis, cutting the gel block containing target fragment under ultraviolet light, and using a gel recovery kit to purify nucleic acid fragment with the following steps:
Mixing and centrifuging the system (10 ul), and connecting it overnight at 16° C.
Taking T1 competent bacteria out of a −80° C. refrigerator and placing it on ice for 30 minutes to thaw. Adding above connecting products to the competent bacteria and placing on ice for 30 minutes. Heating them in a 42° C. water bath for 45 seconds, followed by an ice bath for 5 minutes. Adding 200 ul of antibiotic free LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.4) and shaking on a shaking table at 37° C. for 1 hour at 100 rpm. Spreading bacterial solution onto a solid LB (pancreatic peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar powder 15 g/L) culture plate containing 100 ug/ml ampicillin, and incubating overnight in a 37° C. incubator. Selecting the grown monoclonal colonies and amplifying them in liquid LB medium containing ampicillin. Sending the bacterial solution to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing, and amplifying and preserving the correctly sequenced strains.
Amplifying constructed pAAV-DCRT bacterial solution, pAAV-GFP bacterial solution, pXX9 bacterial solution, and phelper bacterial solution in TB medium containing 100 ug/ml ampicillin (tryptone 12 g/L, yeast extract 24 g/L, K2HPO4·3H2O 16.43 g/L, KH2PO4 2.31 g/L, 0.4% glycerol) for 14-16 hours. Extracting plasmids by using an endotoxin free plasmid extraction kit from Tiangen Biochemical Technology Co., Ltd with the following specific steps:
This experiment used a three plasmid calcium phosphorus co transfection method to package virus in HEK 293T cells. Passaging HEK 293T in a cell culture dish with a diameter of 15 cm. After cells grew to about 85%, replacing complete culture medium. Adding each reagent to prepare plasmid transfection solution was prepared in the following order (for each culture dish):
Droping above plasmid transfection solution into HEK 293T cell dishes, culturing for 12 hours, then replacing with high sugar DMEM medium containing 5% fetal bovine serum, continuing to culture for 48-60 hours, collecting cells, and storing them in a −80° C. freezer.
Repeatedly freezing and thawing cells between −80° C. and 37° C., centrifuging at 8000 g for 15 minutes, and transferring supernatant to a clean round bottom centrifuge tube. The remaining steps are as follows:
After mixing above systems, centrifuging them instantaneously, incubating at 70° C. for 10 minutes, then icing them for 2 minutes, and then adding the following reagents:
2. Protective effect of lncRNA DCRT on cardiac function in mice with thoracic aortic constriction surgery induced cardiac dilation:
After high expression of lncRNA DCRT in mouse hearts, detecting cardiac function by using ultrasound and cardiac catheterization. It's shown by results that full length of lncRNA DCRT had an effect on improving central function impairment in a cardiac dilation model induced by thoracic aortic constriction surgery (
3. Protective effect of lncRNA DCRT on pathological increase of myocardial cells induced by thoracic aortic constriction surgery in mice with cardiac dilation:
After high expression of lncRNA DCRT in mouse hearts, measuring area of myocardial cells in mice. It's shown by results that, full length of lncRNA DCRT had an effect on improving the pathological increase in myocardial cell area induced by thoracic aortic constriction surgery. (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310099726.4 | Feb 2023 | CN | national |
