WO2022121959A1 - siRNA分子及其在治疗冠状动脉疾病中的应用 - Google Patents
siRNA分子及其在治疗冠状动脉疾病中的应用 Download PDFInfo
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- sirna molecule
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
Definitions
- the invention relates to the field of biomedicine, in particular to a siRNA molecule and its application in the treatment of coronary artery disease.
- Coronary artery disease accounts for a large proportion of global morbidity and mortality, and it shows a clear upward trend with the acceleration of population aging.
- Blood enters the heart via the two main coronary arteries and feeds the heart through a network of blood vessels on the surface of the heart muscle. Cholesterol, fat deposits in the arteries, narrows the passages and causes atherosclerosis. Blood flowing in the artery can form a clot that blocks the artery.
- statin therapy is the first-line treatment option for primary prevention.
- PCSK9 inhibitors including monoclonal antibodies and siRNA drugs.
- ASO drugs targeting ApoB and LPA are the main drug treatment options: (1) According to the "2019 ACC/AHA Guidelines for Primary Prevention of Cardiovascular Diseases", for diabetic patients with elevated low-density lipoprotein cholesterol ( ⁇ 190 mg/dL) and aged 40-75 years , and in patients at sufficiently high risk of atherosclerotic cardiovascular disease (ASCVD) after a clinician-patient risk discussion, statin therapy is the first-line treatment option for primary prevention.
- PCSK9 inhibitors including monoclonal antibodies and siRNA drugs.
- ASO drugs targeting ApoB and LPA are the main drug treatment options.
- Statins are the drugs of choice for lowering serum total cholesterol.
- high-dose long-term use will produce side effects.
- the European Union and the US drug regulatory authorities have proved through a large number of data that statin use is associated with new-onset diabetes, increased glycosylated hemoglobin and/or elevated fasting blood glucose levels.
- myopathy mainly manifested as myalgia or muscle weakness, can lead to rhabdomyolysis and acute renal failure if continued medication is not detected in time.
- PCSK9 inhibitors are hailed as a milestone in the post-statin era, but long-term use will lead to drug resistance, and the beneficiary population still needs to be further expanded.
- Other innovative drugs are still in the clinical or preclinical research and development stage and cannot meet the unmet clinical needs for the time being. Therefore, finding innovative targets and developing new innovative drugs is an urgent need for medical and health care.
- the present invention aims to solve at least one of the technical problems existing in the prior art, and provides an siRNA molecule and its application in the treatment of coronary artery disease.
- Lipoprotein(a) (LPA, NM_005577.4) is a secreted protein expressed primarily in liver cells, and expression is restricted to humans and non-human primates. Studies have shown that the level of serum lipoprotein a is closely related to the degree and clinical type of coronary artery disease. The level of lipoprotein a in patients with angina pectoris, myocardial infarction, cerebral hemorrhage, etc. is significantly increased, which can also promote the formation of atherosclerosis pathologically. Lipoprotein a level is also a useful indicator for predicting the risk of recurrent cardiovascular events in patients with coronary heart disease.
- siRNA small interfering RNA
- RISC RNA-induced silencing complex
- the inventors designed a specific small interfering nucleic acid (siRNA) sequence for the lipoprotein (a) (LPA) gene target, synthesized siRNA, and used a transfection reagent to introduce the siRNA into cells to form an RNA-induced silencing complex (RNA- induce siliencing complex (RISC), which specifically recognizes and targets mRNA sequences that bind to target genes, and cuts mRNA between 10-11 clips from the 5' end, resulting in post-transcriptional gene silencing and regulation of lipoprotein (a) secretion .
- RISC RNA-induced silencing complex
- the present invention provides the following technical solutions:
- a double-stranded siRNA molecule for inhibiting the expression of lipoprotein a is provided, the sense strand of the siRNA molecule can specifically bind to the following region of the mRNA molecule encoding LPA At least one of: 80-109, 222-242, 263-283, 558-578, 3514-3534, 3659-3679, 4178-4198, 4627-4651, 4957-4993, 5033-5056, 5224-5244, 5466-5486, 5767-5789, 6058-6078;
- the sequence structure of the mRNA molecule encoding LPA is shown in genebank number NM_005577.4.
- the length of any one strand of the double-stranded siRNA molecule is 18-25 nucleotides.
- any strand of a double-stranded siRNA molecule can be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
- the present invention provides a siRNA molecule
- the siRNA molecule is selected from at least one of the following: (a) any nucleotide in SEQ ID NO: 1 to SEQ ID NO: 438 Sequence, preferably, at least one of SEQ ID NOs: 5-7, 24, 46-48, 61, 81-84, 104-109, 112-114, 129, 147, 178-179, 203; (b ) compared with any one of the nucleotide sequences in SEQ ID NO: 1 to SEQ ID NO: 438, the number of base differences is less than 3 nucleotide sequences.
- the provided siRNA molecule is selected from at least one of the following: compared with any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 438, the number of base differences is 2 or less nucleotide sequences. According to an embodiment of the present invention, the provided siRNA molecule is selected from at least one of the following: compared with any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 438, the number of base differences is 1 nucleotide sequence.
- the siRNA molecule includes at least one sense strand and at least one antisense strand;
- the sense strand is selected from at least one of the following: (a-1) SEQ ID NO: 1 to SEQ ID NO : at least one of 219; (b-1) compared with any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 219, the number of base differences is less than 3 nucleotide sequences , preferably a nucleotide sequence with a difference of 2 or less bases, more preferably a nucleotide sequence with a difference of 1 base;
- the antisense strand is paired with the sense strand in reverse complementary pairs, and the The antisense strand is selected from at least one of the following: (a-2) at least one of SEQ ID NO: 220 to SEQ ID NO: 438; (b-2) and SEQ ID NO: 220 to SEQ ID NO: Compared with any one of the 438 nucleotide sequences, the number of
- the siRNA molecule comprises at least one modified nucleotide.
- the modified nucleotides are selected from at least one of the following:
- 5'-phosphorothioate nucleotides 5-methylated cytosine nucleotides, 2'-O-methyl modified nucleotides, 2'-O-2-methoxyethyl modified Nucleotides, 2'-fluoro-modified nucleotides, 3'-nitrogen-modified nucleotides, 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides , locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, morpholino nucleotides, polypeptide nucleotides, phosphoramidates, and nucleotides including unnatural bases .
- the lengths of both the sense strand and the antisense strand in the siRNA molecule are no more than 25 bp. According to a preferred embodiment of the present invention, the lengths of the sense strand and the antisense strand in the siRNA molecule are 18-25 bp. According to a preferred embodiment of the present invention, the lengths of the sense and antisense strands in the siRNA molecule are 19-22 bp; according to a preferred embodiment of the present invention, the lengths of the sense and antisense strands in the siRNA molecule are 21 bp.
- the siRNA molecule is linked to a targeting ligand.
- the siRNA molecule is covalently linked to the targeting ligand
- the targeting ligand comprises at least one N-acetyl-galactosamine
- the targeting ligand is linked to the sense strand of the siRNA molecule
- the targeting ligand is attached to the 5' end of the sense strand of the siRNA molecule.
- the present invention provides a DNA sequence encoding the siRNA molecule according to any embodiment of the first aspect or the second aspect of the present invention.
- the present invention provides an expression vector, the expression vector comprising the siRNA molecule described in any embodiment of the first aspect or the second aspect of the present invention.
- the present invention provides a recombinant cell expressing the siRNA molecule described in any embodiment of the first aspect or the second aspect of the present invention.
- siRNA molecules can be introduced into recombinant cells by different methods, so that the recombinant cells can express siRNA, which can inhibit the expression of LPA gene, used to treat coronary artery disease or inhibit the expression of lipoproteins in liver cells.
- the recombinant cells can be eukaryotic cells. Available methods include, but are not limited to, calcium phosphate co-precipitation methods, electroporation methods, cationic liposome reagent methods, and the like. For example, the commonly used cationic liposome Lipofectamine 2000 can be used.
- the formed recombinant cells can also contain any form of carrier: such as polymer carrier, polypeptide carrier, high molecular polymer carrier, metal nanocarrier, ligand Various types of functions, etc.
- carrier such as polymer carrier, polypeptide carrier, high molecular polymer carrier, metal nanocarrier, ligand Various types of functions, etc.
- transfection reagents can also be used directly, such as Lipofectamine RNAiMAX transfection reagent, which can provide easy, Efficient siRNA delivery effect.
- the present invention provides an RNA-induced silencing complex, comprising the siRNA molecule, endonuclease and Argonatue protein according to any embodiment of the first aspect or the second aspect of the present invention.
- the mentioned RNA-induced silencing complex also known as RISC.
- the endonuclease (Dicer) in RISC has an RNaseIII domain, which is responsible for catalyzing the production of siRNA in the initial stage of RNAi, and plays a role in stabilizing the structure and function of RISC intermediates during RISC assembly.
- Argonaute protein is the core protein in RISC and has two main domains, PAZ and PIWI.
- siRNA molecule is the guide for RISC to complete the specific cleavage effect.
- mature RISC only contains one strand of siRNA molecule, the double-stranded structure of siRNA molecule during the formation of RISC is the decisive factor to ensure the effect of RNAi.
- the provided RNA-induced silencing complex can specifically recognize and target mRNA sequences bound to target genes, and cleave mRNA between clips 10-11 from the 5' end, resulting in post-transcriptional gene silencing and lower lipoprotein expression level as a treatment for patients with coronary artery disease.
- the present invention provides a kit or a kit comprising the siRNA molecule described in any embodiment of the first aspect or the second aspect of the present invention.
- the present invention provides a method for inhibiting the expression of intracellular lipoproteins in vitro, comprising: introducing siRNA molecules into cells, so as to reduce the expression of intracellular lipoproteins, so that The siRNA molecule is the siRNA molecule described in the first aspect or the second aspect of the present invention.
- the cells are liver cells.
- the cells are mammalian cells.
- the mammalian cells include, but are not limited to, humans.
- the present invention provides the use of an siRNA molecule in the preparation of a medicine for treating coronary artery disease, and the siRNA molecule is any one of the implementation of the first aspect or the second aspect of the present invention Example siRNA molecules.
- the present invention provides a drug for treating coronary artery disease, comprising an effective amount of the siRNA molecule described in the first or second aspect of the present invention and a pharmaceutically acceptable excipient.
- Drugs for treating coronary artery disease can be prepared into different formulations by conventional methods, for example, injections can be prepared by conventional methods using physiological saline or an aqueous solvent containing glucose and other adjuvants.
- the prepared different drugs can be administered in any convenient form, such as local, intravenous, intramuscular, subcutaneous, intradermal and other different routes. The dosage of the drug can be adjusted according to the actual situation.
- the eleventh aspect of the present invention also provides a method for treating coronary artery disease, comprising administering to a subject in need thereof a therapeutically effective amount of the above-mentioned siRNA molecule or the above-mentioned drug.
- the present invention provides an siRNA molecule and its application in the treatment of coronary artery disease.
- many siRNA molecules were designed and synthesized by determining the target gene expressing lipoprotein. By applying in vitro transfection technology, these siRNA molecules were introduced into liver cells, and the inhibitory effect of these siRNA molecules was verified.
- the inventors found that the siRNA molecules in SEQ ID NO: 1 to SEQ ID NO: 438 show excellent effects of regulating gene silencing, and can be applied to the treatment related to coronary artery disease.
- the present invention provides a siRNA molecule selected from at least one of the following: (a) any nucleoside in SEQ ID NO: 1 to SEQ ID NO: 438 acid sequence; (b) compared with any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 438, the number of base differences is 3 nucleotide sequences, for example, the number of base differences is A nucleotide sequence of two, for example, a nucleotide sequence in which the number of base differences is one.
- the siRNA molecule includes at least one sense strand and at least one antisense strand; the sense strand is selected from at least one of the following: (a-1) SEQ ID NO: 1 ⁇ At least one of SEQ ID NO: 219; (b-1) compared with any nucleotide sequence in SEQ ID NO: 1 to SEQ ID NO: 219, the number of base differences is less than 3 cores
- a nucleotide sequence preferably a nucleotide sequence with a difference of 2 or less bases, more preferably a nucleotide sequence with a difference of 1 base
- the antisense strand and the sense strand are paired with reverse complementarity
- the antisense strand is selected from at least one of the following: (a-2) at least one of SEQ ID NO:220 ⁇ SEQ ID NO:438; (b-2) and SEQ ID NO:220 ⁇ SEQ ID NO:438 Compared with any one of the nucleotide sequences in ID
- the sense strand of the provided double-stranded siRNA molecule can specifically bind to at least one of the following regions of the mRNA molecule encoding LPA: 80-109, 222-242, 80-109, 222-242, 263-283, 558-578, 3514-3534, 3659-3679, 4178-4198, 4627-4651, 4957-4993, 5033-5056, 5224-5244, 5466-5486, 5767-5789, 6058-6078.
- the structure of the mRNA molecule sequence encoding LPA is shown in genebank number NM_005577.4.
- the referenced region of the mRNA molecule encoding LPA is the nucleic acid sequence starting from the 5' end.
- the siRNA molecule inhibits the expression of the LPA gene.
- the provided siRNA molecule includes any one of SEQ ID NO: 1 to SEQ ID NO: 219 and its antisense strand.
- the sequence number of the antisense strand is any one of SEQ ID NO: 220 to SEQ ID NO: 438. Wherein, SEQ ID NO: 220 in the antisense strand corresponds to SEQ ID NO: 1, and SEQ ID NO: 221 in the antisense strand corresponds to SEQ ID NO: 2, and so on.
- the siRNA molecules provided are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 6, ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO :15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 , SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:28 ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
- the siRNA molecule may further include any one of the antisense strands corresponding to the sense strand.
- the antisense strand is reverse complementary to the sense strand, and the antisense strand is selected from at least one of SEQ ID NO: 220 to SEQ ID NO: 438.
- SEQ ID NO: 220 to SEQ ID NO: 438 are shown to inhibit the expression of LPA gene, and thus can be used to prevent or treat coronary artery disease.
- the antisense strands corresponding to these siRNA molecules will also be used as siRNA molecules for preventing or treating coronary artery disease, and are included within the scope of protection of the present invention.
- permission of the present invention will also be required.
- the provided siRNA molecule has in addition to any sequence in the above-mentioned SEQ ID NO: 1 ⁇ SEQ ID NO: 438, the nucleotide in the above-mentioned sequence can also be a modified nucleotide; this modified nucleotide It can be one, two, three or four, etc.
- Modified nucleotides include, but are not limited to: 5'-phosphorothioate nucleotides, 5'-methylated cytosine nucleotides, 2'-O-methyl modified nucleotides, 2'-methylated nucleotides -O-2-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 3'-nitrogen modified nucleotides, 2'-deoxy-2'-fluoro modified nucleosides Acids, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, morpholino nucleotides, polypeptide nucleotides, phosphoramidates , and nucleotides including unnatural bases, etc.
- the present invention also provides DNA sequences encoding the above-mentioned siRNA molecules.
- the present invention also provides an expression vector comprising the above-mentioned siRNA molecule.
- the expression vector may also contain promoters, terminators, marker genes and the like as required. Expression vectors are mainly used for the expression of siRNA molecules. Corresponding expression vectors can be prepared by using genetic engineering methods commonly used in the art.
- the present invention also provides recombinant cells expressing the above-mentioned siRNA molecules. In still other embodiments of the present invention, the present invention also provides the siRNA molecule, endonuclease and Argonature protein comprising the above-mentioned.
- Kits or kits can also be prepared using the provided siRNA molecules.
- the provided kit or kit can be used to detect, prevent or treat coronary artery disease.
- the coronary artery disease mentioned is generally understood in the art, and may include angina pectoris, myocardial infarction, cerebral hemorrhage and other diseases related to the elevated level of lipoprotein a in patients.
- a method for treating coronary artery disease comprising administering to a subject in need thereof a therapeutically effective amount of the above-mentioned siRNA molecule or the above-mentioned drug.
- the therapeutically effective amount required by the subject can be provided in conjunction with the condition of the subject.
- the subject mentioned can be a mammal or a human.
- the present invention provides a method of inhibiting the expression of a target gene in a cell performed in vitro, the method comprising the step of introducing the above-mentioned siRNA molecule.
- the target gene is a lipoprotein encoding gene.
- the term "treating" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease.
- Treatment encompasses diseases in mammals, including: (a) preventing the occurrence of a disease or disorder in individuals susceptible to but not yet diagnosed with the disease; (b) inhibiting the disease, eg, retarding the progression of the disease; or (c) Alleviate disease, such as reducing symptoms associated with disease.
- Treatment encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit the disease of the individual.
- siRNA small interfering RNA, small interfering RNA
- small interfering RNA refers to a short double-stranded RNA that specifically mediates effective gene expression inhibition (which may refer to gene silencing).
- antisense strand refers to a polynucleotide that is complementary to a portion or all of a target nucleic acid sequence.
- mRNA sequences, non-mRNA RNA sequences, or coding DNA sequences or non-coding DNA sequences can be complementary in their entirety or in part.
- the term "sense strand” as commonly understood in the art refers to a polynucleotide that is identical to a portion or all of a target nucleic acid sequence. For example, it can be identical to the whole or a portion of an mRNA sequence, a non-mRNA RNA sequence, or a coding DNA sequence or a non-coding DNA sequence.
- target nucleic acid can be mRNA, microRNA, piRNA, coding DNA sequence and non-coding DNA sequence, etc., of course, it is not limited thereto.
- nucleic acid molecules mentioned herein can be artificially synthesized by conventional methods. Of course, it doesn't stop there. Nucleic acid molecules can be synthesized chemically or enzymatically.
- the siRNA molecules can be combined with known liposomes, cationic polymers, antibodies, nanoparticles, etc., which can effectively deliver oligonucleotides into cells. Combined application of various vectors. The delivery can also be accomplished using a variety of known delivery methods.
- Example 1 Experiment 219 small interfering nucleic acids (siRNA) were designed for the lipoprotein gene (transcript information is NM_005577.4). The transcript information of the protein gene is recorded in the NCBI database.
- siRNA small interfering nucleic acids
- the designed siRNA sequences are shown in Table 1 below.
- the column of name and position in Table 1 shows the binding position of the siRNA molecule on the LPA target. The smaller the value of the corresponding position, the position near the upstream of the LPA gene.
- Example 2 In vitro cell model (Huh7 cells) to test the activity of small interfering nucleic acid (siRNA)
- Example 2 The activity of these siRNA sequences was tested using an in vitro cell model.
- the experimental process and experimental results are as follows:
- siRNA with a concentration of 50 ⁇ M in stock solution, dilute with DEPC water to obtain a 10 ⁇ M siRNA system, and dilute 50 ⁇ l Opti-MEM to obtain a 0.2 ⁇ M siRNA system, and mix by pipetting 3-5 times (final concentration 10 nM). Then dilute 0.5ul of 0.2 ⁇ M siRNA with 50 ⁇ l Opti-MEM to prepare a 0.002 ⁇ M siRNA system, and mix by pipetting 3-5 times (final concentration 0.1nM) to obtain a siRNA molecule dilution.
- RNA of the cells was extracted, and the expression of the LPA mRNA sequence in the cells was detected by real-time quantitative PCR (Quantitative Real-Time PCR), wherein the PCR primers used to amplify the internal reference genes PPIB and LPA are shown in Table 2:
- the inhibition rate of the small interfering nucleic acid mRNA expression level was calculated according to the following equation:
- Inhibition rate [1-(expression of LPA mRNA in experimental group/expression of PPIB mRNA in experimental group)/(expression of LPA mRNA in negative control group/expression of PPIB mRNA in negative control group)] ⁇ 100%.
- each experimental group is the cells treated with small interfering nucleic acid respectively;
- the negative control group (referred to as Blank) is the cells not treated with any small interfering nucleic acid.
- siRNA molecules can effectively inhibit the expression level of mRNA in LPA, and the specific data are shown in Table 3 below.
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Abstract
提供了siRNA分子及其在治疗冠状动脉疾病中的应用，该siRNA分子的正义链选自下列中的至少一种：SEQ ID NO:1～SEQ ID NO:219，该siRNA分子能够抑制脂蛋白的表达，尤其是肝脏细胞中脂蛋白的表达，从而可以应用于预防或者治疗冠状动脉疾病。
Description
相关申请的交叉引用
本发明要求于2020年12月09日提交的申请号为202011447329.4的中国专利申请的全部权益，并将它们的全部内容引入本文。
本发明涉及生物医药领域，具体涉及siRNA分子及其在治疗冠状动脉疾病中的应用。
冠状动脉疾病在全球发病率和死亡率中占比较大，并随着人口老龄化程度的加速，呈明显上升趋势。血液经由两条主要冠状动脉进入心脏，并经由心脏肌肉表面上的一个血管网络，使心脏得到养分。胆固醇，脂肪沉积在动脉中，使通道变窄，使之形成动脉粥样硬化。在动脉流动的血液会形成一种血栓，将动脉阻塞起来。目前，主要的药物治疗方案有：(1)根据《2019ACC/AHA心血管疾病一级预防指南》，对于低密度脂蛋白胆固醇升高(≥190mg/dL)、年龄在40-75岁的糖尿病患者，以及经临床医生与患者风险讨论后确定有足够高动脉粥样硬化性心血管疾病(ASCVD)风险的患者，他汀类药物治疗是一级预防的一线治疗方案。(2)PCSK9抑制剂，包括单抗及SiRNA药物。(3)靶向ApoB及LPA的ASO药物。
他汀类药物是目前常用的降低血清总胆固醇的首选药物。但随着该类药物的广泛使用，大剂量长期使用会产出副作用，欧盟和美国药品监督管理部门通过大量数据证明他汀使用与新发糖尿病、糖化血红蛋白和(或)空腹血糖水平升高存在较明确的相关性；另外，肌病，主要表现为肌痛或肌无力，若不及时发现仍旧继续用药，则可导致横纹肌溶解和急性肾功能衰竭。PCSK9抑制剂被誉为是后他汀时代的里程碑，不过长期使用会产生耐药性，受益人群仍需进一步扩展。其他创新药物仍处于临床或临床前研发阶段，暂不能满足临床未尽需求。因此，寻找创新靶点，开发新型创新药物是医疗健康的迫切需求。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一，提供了一种siRNA分子及其在治疗冠状动脉疾病中的应用。
脂蛋白(a)(LPA，NM_005577.4)是一种主要在肝脏细胞中表达的分泌蛋白，并且表达仅限于人类和非人灵长类动物。已有研究表明，血清脂蛋白a水平与冠状动脉病变程度及临床类型紧密相关，心绞痛、心肌梗死、脑溢血等患者脂蛋白a水平明显升高，病理上也会促进动脉粥样硬化的形成。脂蛋白a水平也是预测冠心病患者再发心血管事件风险的有用指标。因此，发明人创造性的想到：抑制LPA的活性可以有效预防或治疗冠状动脉疾病，设计合适的小干扰RNA(siRNA)序列可以特异性减少肝细胞合成LPA，同时避免脱靶效应。siRNA通过形成沉默复合体(RNA-induced silencing complex，RISC)，与靶标基因的mRNA的序列互补配对，使靶标基因的mRNA降解从而抑制靶标基因的表达。
发明人针对脂蛋白(a)(LPA)基因靶点，设计特异性的小干扰核酸(siRNA)序列，合成siRNA，利用转染试剂，将siRNA导入细胞内，形成RNA诱导沉默复合体(RNA-induce siliencing complex，RISC)，特异性识别并靶向结合靶基因的mRNA序列，并在距离5’端10-11位剪辑之间切割mRNA，从而导致转录后基因沉默，调控脂蛋白(a)分泌。通过siRNA分子靶向LPA基因，降低脂蛋白的表达量，有望成为治疗冠状动脉疾病患者，满足临床未尽的需求。
具体而言，本发明提供了如下技术方案：
在本发明的第一方面，提供了一种用于抑制脂蛋白a(LPA)表达的双链siRNA分子，所述的siRNA分子的正义链能够特异性的结合编码LPA的mRNA分子的如下区域中的至少一种：自5’端开始80-109、222-242、263-283、558-578、3514-3534、3659-3679、4178-4198、4627-4651、4957-4993、5033-5056、5224-5244、5466-5486、5767-5789、6058-6078；
所述编码LPA的mRNA分子的序列结构如genebank编号NM_005577.4所示。
根据本发明的实施例，所述双链siRNA分子的任意一条链的长度为18-25个核苷酸。例如双链siRNA分子的任意一条链的长度可以为18、19、20、21、22、23、24或者25个核苷酸。
在本发明的第二方面，本发明提供了一种siRNA分子，所述siRNA分子选自下列中的至少一种：(a)SEQ ID NO:1～SEQ ID NO:438中任意一条核苷酸序列，优选的，SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的至少一种；(b)与SEQ ID NO:1～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列。根据本发明的实施例，所提供的siRNA分子选自下列中的至少一种：与SEQ ID NO:1～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在2个以下的核苷酸序列。根据本发明的实施例，所提供的siRNA分子选自下列中的至少一种：与SEQ ID NO:1～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数为1个的核苷酸序列。
根据本发明的实施例，所述siRNA分子包括至少一条正义链和至少一条反义链；所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:1～SEQ ID NO:219中的至少一种；(b-1)与SEQ ID NO:1～SEQ ID NO:219中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列，优选碱基的差异数在2个以下的核苷酸序列，更优选碱基的差异数为1个的核苷酸序列；所述反义链与所述正义链反向互补配对，所述反义链选自下列中的至少一种：(a-2)SEQ ID NO:220～SEQ ID NO:438中的至少一种；(b-2)与SEQ ID NO:220～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列，优选碱基的差异数在2个以下的核苷酸序列，更优选碱基的差异数为1个的核苷酸序列。根据本发明的实施例，所述siRNA分子抑制LPA基因的表达。
根据本发明的实施例，所述siRNA分子包括至少一个被修饰的核苷酸。根据本发明的优选实施例，所述修饰的核苷酸选自下列至少之一：
5'-硫代磷酸酯基的核苷酸、5-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽核苷酸、氨基磷酸酯，以及包括非天然碱基的核苷酸。
根据本发明的实施例，所述siRNA分子中正义链和反义链的长度均不多于25bp。根据本发明的优选实施例，所述siRNA分子中正义链和反义链的长度为18～25bp。根据本发明的优选实施例，所述siRNA分子中正义链和反义链的长度为19～22bp；根据本发明的优选实施例，所述siRNA分子中正义链和反义链的长度为21bp。
根据本发明的实施例，所述siRNA分子与靶向配体连接。
根据本发明的实施例，所述siRNA分子与靶向配体通过共价键连接；
根据本发明的实施例，所述靶向配体包括至少一个N-乙酰基-半乳糖胺；
根据本发明的实施例，所述靶向配体与所述siRNA分子的正义链连接；
根据本发明的实施例，所述靶向配体与所述siRNA分子的正义链的5’末端连接。
在本发明的第三方面，本发明提供了一种DNA序列，所述DNA序列编码本发明第一方面或第二方面任一实施例所述的siRNA分子。
在本发明的第四方面，本发明提供了一种表达载体，所述表达载体包含本发明第一方面或第二方面任一实施例所述的siRNA分子。
在本发明的第五方面，本发明提供了一种重组细胞，所述重组细胞表达本发明第一方面或第二方面任一实施例所述的siRNA分子。可以通过不同的方法将siRNA分子导入到重组细胞中，从而使得重组细胞可以表达siRNA，从而可以抑制与LPA基因的表达，用来治疗冠状动脉疾病或者抑制肝脏细胞中脂蛋白的表达量。重组细胞可以是真核细胞。可用的方法包括但不限于：磷酸钙共沉淀的方法，电穿孔的方法，阳离子脂质体试剂的方法等等。例如可以采用常用的阳离子脂质体Lipofectamine 2000。所形成的重组细胞除了能够表达本发明第一方面所述的siRNA分子之外，还可以含有任何形式的载体：如多聚物载体、多肽载体、高分子聚合物载体、金属纳米载体、配体功能的各类载体等等。当然也可以直接利用转染试剂，例如Lipofectamine RNAiMAX转染试剂，该转染试剂能够在广泛的细胞系中(包括常见细胞类型、干细胞、原代细胞以及历来难转染的细胞类型)提供简便、高效的siRNA输送效果。
在本发明的第六方面，本发明提供了一种RNA诱导沉默复合体，包括本发明第一方面或第二方面任一实施例所述的siRNA分子、核酸内切酶和Argonatue蛋白。所提到的RNA诱导沉默复合体，也称为RISC。RISC中核酸内切酶(Dicer)具有RNaseIII结构域，在RNAi的起始阶段负责催化siRNA的产生，在RISC装配过程中起稳定RISC中间体结构和功能的作用。Argonaute蛋白是RISC中的核心蛋白，有PAZ和PIWI两个主要的结构域，前者为siRNA分子的传递提供结合位点，后者是RISC中的酶切割活性中心。siRNA分子是RISC完成特异性切割作用的向导，在成熟的RISC中虽然只包含siRNA分子的一条链，但siRNA分子在RISC形成过程中的双链结构是保证RNAi效应的决定因素。通过所提供的RNA诱导沉默复合体，可以特异性识别并靶向结合靶基因的mRNA序列，并在距离5’端10-11位剪辑之间切割mRNA，从而导致转录后基因沉默，降低脂蛋白的表达量，作为冠状动脉疾病患者的一种治疗手段。
在本发明的第七方面，本发明提供了一种试剂盒或药盒，所述试剂盒或者所述药盒包括本发明第一方面或第二方面任一实施例所述的siRNA分子。
在本发明的第八方面，本发明提供了一种在体外抑制细胞内脂蛋白表达量的方法，包括：将siRNA分子导入到细胞内，以便使得所述细胞内脂蛋白的表达量降低，所述siRNA分子为本发明第一方面或第二方面所述的siRNA分子。
根据本发明的实施例，所述细胞为肝脏细胞。根据本发明的实施例，所述细胞为哺乳动物细胞。所述哺乳动物细胞包括但不限于人。
在本发明的第九方面，本发明提供了一种siRNA分子在制备药物中的用途，所述药物用于治疗冠状动脉疾病，所述siRNA分子为本发明第一方面或第二方面任一实施例所述的siRNA分子。
在本发明的第十方面，本发明提供了一种治疗冠状动脉疾病的药物，包括有效量的本发明第一方面或第二方面所述的siRNA分子和药学上可接受的辅料。治疗冠状动脉疾病的药物可以采用常规方法制备成不同的制剂，例如可以采用生理盐水或者含有葡萄糖和其他辅剂的水溶剂通过常规方法制备成针剂。所制备成的不同的药物可以以任何方便的形式给药，例如可以通过局部、静脉、肌肉、皮下、皮内等不同的途径给药。药物的用量可以根据实际情况进行适应性调整。
本发明的第十一方面还提供了一种治疗冠状动脉疾病的方法，包括给予有需要的受试者治疗有效量 的上述所述的siRNA分子或者上述药物。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
下面详细描述本发明的实施例，需要说明的是，所描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明提供了一种siRNA分子及其在治疗冠状动脉疾病中的应用。实验通过确定表达脂蛋白的目的基因，设计合成了诸多siRNA分子。通过应用体外转染技术，将这些siRNA分子导入到肝脏细胞中，对于这些siRNA分子的抑制效果进行了验证。发明人发现：SEQ ID NO:1～SEQ ID NO:438中siRNA分子表现出优异的调控基因沉默的效果，可以将其应用于与冠状动脉疾病有关的治疗中。
在本发明的一些实施方式中，本发明提供了一种siRNA分子，所述siRNA分子选自下列中的至少一种：(a)SEQ ID NO:1～SEQ ID NO:438中任意一条核苷酸序列；(b)与SEQ ID NO:1～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数为3个的核苷酸序列，例如碱基的差异数为2个的核苷酸序列，再例如碱基的差异数为1个的核苷酸序列。
在本发明的至少一些实施方式中，所述siRNA分子包括至少一条正义链和至少一条反义链；所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:1～SEQ ID NO:219中的至少一种；(b-1)与SEQ ID NO:1～SEQ ID NO:219中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列，优选碱基的差异数在2个以下的核苷酸序列，更优选碱基的差异数为1个的核苷酸序列；所述反义链与所述正义链反向互补配对，所述反义链选自下列中的至少一种：(a-2)SEQ ID NO:220～SEQ ID NO:438中的至少一种；(b-2)与SEQ ID NO:220～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列，优选碱基的差异数在2个以下的核苷酸序列，更优选碱基的差异数为1个的核苷酸序列。
根据本发明的具体实施方式，所提供的双链siRNA分子的正义链能够特异性的结合编码LPA的mRNA分子的如下区域中的至少一种：自5’端开始80-109、222-242、263-283、558-578、3514-3534、3659-3679、4178-4198、4627-4651、4957-4993、5033-5056、5224-5244、5466-5486、5767-5789、6058-6078。所述编码LPA的mRNA分子序列的结构如genebank编号NM_005577.4所示。所提到的编码LPA的mRNA分子的区域为从5’端开始的核酸顺序。
根据本发明的优选实施例，所述siRNA分子抑制LPA基因的表达。根据本发明的优选实施例，所提供的siRNA分子包括SEQ ID NO:1～SEQ ID NO:219中的任意一条及其反义链。根据本发明的实施例，反义链的序列号为SEQ ID NO:220～SEQ ID NO:438中任一条。其中反义链中SEQ ID NO:220对应SEQ ID NO:1，反义链SEQ ID NO:221对应SEQ ID NO:2，依次类推。在本发明的实施例，所提供的siRNA分子为SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，SEQ ID NO:8，SEQ ID NO:9，SEQ ID NO:10，SEQ ID NO:11，SEQ ID NO:12，SEQ ID NO:13，SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16，SEQ ID NO:17，SEQ ID NO:18，SEQ ID NO:19，SEQ ID NO:20，SEQ ID NO:21，SEQ ID NO:22，SEQ ID NO:23，SEQ ID NO:24，SEQ ID NO:25，SEQ ID NO:26，SEQ ID NO:27，SEQ ID NO:28，SEQ ID NO:29，SEQ ID NO:30，SEQ ID NO:31，SEQ ID NO:32，SEQ ID NO:33，SEQ ID NO:34，SEQ ID NO:35，SEQ ID NO:36，SEQ ID NO:37，SEQ ID NO:38，SEQ ID NO:39，SEQ ID NO:40，SEQ ID NO:41，SEQ ID NO:42，SEQ ID NO:43，SEQ ID NO:44，SEQ ID NO:45，SEQ ID NO:46，SEQ ID NO:47，SEQ ID NO:48，SEQ ID NO:49，SEQ ID NO:50，SEQ ID NO:51，SEQ ID NO:52，SEQ ID NO:53，SEQ ID NO:54，SEQ ID NO:55，SEQ ID NO:56，SEQ ID NO:57，SEQ ID NO:58，SEQ ID NO:59，SEQ ID NO:60，SEQ ID NO:61，SEQ ID NO:62，SEQ ID NO:63，SEQ ID NO:64，SEQ ID NO:65，SEQ ID NO:66，SEQ ID NO:67，SEQ ID NO:68，SEQ ID NO:69，SEQ ID NO:70，SEQ ID NO:71，SEQ ID NO:72，SEQ ID NO:73，SEQ ID NO:74，SEQ ID NO:75，SEQ ID NO:76，SEQ ID NO:77，SEQ ID NO:78，SEQ ID NO:79，SEQ ID NO:80，SEQ ID NO:81，SEQ ID NO:82，SEQ ID NO:83，SEQ ID NO:84，SEQ ID NO:85，SEQ ID NO:86，SEQ ID NO:87，SEQ ID NO:88，SEQ ID NO:89，SEQ ID NO:90，SEQ ID NO:91，SEQ ID NO:92，SEQ ID NO:93，SEQ ID NO:94，SEQ ID NO:95，SEQ ID NO:96，SEQ ID NO:97，SEQ ID NO:98，SEQ ID NO:99，SEQ ID NO:100，SEQ ID NO:101，SEQ ID NO:102，SEQ ID NO:103，SEQ ID NO:104，SEQ ID NO:105，SEQ ID NO:106，SEQ ID NO:107，SEQ ID NO:108，SEQ ID NO:109，SEQ ID NO:110，SEQ ID NO:111，SEQ ID NO:112，SEQ ID NO:113，SEQ ID NO:114，SEQ ID NO:115，SEQ ID NO:116，SEQ ID NO:117，SEQ ID NO:118，SEQ ID NO:119，SEQ ID NO:120，SEQ ID NO:121，SEQ ID NO:122，SEQ ID NO:123，SEQ ID NO:124，SEQ ID NO:125，SEQ ID NO:126，SEQ ID NO:127，SEQ ID NO:128，SEQ ID NO:129，SEQ ID NO:130，SEQ ID NO:131，SEQ ID NO:132，SEQ ID NO:133，SEQ ID NO:134，SEQ ID NO:135，SEQ ID NO:136，SEQ ID NO:137，SEQ ID NO:138，SEQ ID NO:139，SEQ ID NO:140，SEQ ID NO:141，SEQ ID NO:142，SEQ ID NO:143，SEQ ID NO:144，SEQ ID NO:145，SEQ ID NO:146，SEQ ID NO:147，SEQ ID NO:148，SEQ ID NO:149，SEQ ID NO:150，SEQ ID NO:151，SEQ ID NO:152，SEQ ID NO:153，SEQ ID NO:154，SEQ ID NO:155，SEQ ID NO:156，SEQ ID NO:157，SEQ ID NO:158，SEQ ID NO:159，SEQ ID NO:160，SEQ ID NO:161，SEQ ID NO:162，SEQ ID NO:163，SEQ ID NO:164，SEQ ID NO:165，SEQ ID NO:166，SEQ ID NO:167，SEQ ID NO:168，SEQ ID NO:169，SEQ ID NO:170，SEQ ID NO:171，SEQ ID NO:172，SEQ ID NO:173，SEQ ID NO:174，SEQ ID NO:175，SEQ ID NO:176，SEQ ID NO:177，SEQ ID NO:178，SEQ ID NO:179，SEQ ID NO:180，SEQ ID NO:181，SEQ ID NO:182，SEQ ID NO:183，SEQ ID NO:184，SEQ ID NO:185，SEQ ID NO:186，SEQ ID NO:187，SEQ ID NO:188，SEQ ID NO:189，SEQ ID NO:190，SEQ ID NO:191，SEQ ID NO:192，SEQ ID NO:193，SEQ ID NO:194，SEQ ID NO:195，SEQ ID NO:196，SEQ ID NO:197，SEQ ID NO:198，SEQ ID NO:199，SEQ ID NO:200，SEQ ID NO:201，SEQ ID NO:202，SEQ ID NO:203，SEQ ID NO:204，SEQ ID NO:205，SEQ ID NO:206，SEQ ID NO:207，SEQ ID NO:208，SEQ ID NO:209，SEQ ID NO:210，SEQ ID NO:211，SEQ ID NO:212，SEQ ID NO:213，SEQ ID NO:214，SEQ ID NO:215，SEQ ID NO:216，SEQ ID NO:217，SEQ ID NO:218，SEQ ID NO:219。根据本发明的实施例，所述siRNA分子除了上述提到的任意一条正义链，还可以进一步包括所述正义链对应的任意一条反义链。根据本发明的实施例，所述反义链与所述正义链反向互补，所述反义链选自SEQ ID NO:220～SEQ ID NO:438中的至少一种。这些siRNA分子表现出抑制LPA基因的表达，从而可以用于预防或者治疗冠动脉疾病。需要说明的是，这些siRNA分子对应的反义链，也将用来作为预防或者治疗冠动脉疾病的siRNA分子，包含在本发明要求保护的范围之内。对于这些反义链分子序列SEQ ID NO:220～SEQ ID NO:438的应用，也将需要获得本发明的许可。
所提供的siRNA分子除了具有上述SEQ ID NO:1～SEQ ID NO:438中的任意一条序列之外，上述序列中的核苷酸还可以为修饰的核苷酸；这种修饰的核苷酸可以为一个，两个，三个或者四个等。修饰的核苷酸包括但不限于：5'-硫代磷酸酯基的核苷酸、5'-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽 核苷酸、氨基磷酸酯，以及包括非天然碱基的核苷酸等等。
在本发明的一些实施方式中，本发明还提供了编码上述siRNA分子的DNA序列。在本发明的另一些实施方式中，本发明还提供了包含上述siRNA分子的表达载体。根据本发明的实施例，表达载体中除了包含上述siRNA分子之外，还可以根据需要含有启动子、终止子、标记基因等等。表达载体主要用于siRNA分子的表达。可以采用本领域通用的基因工程手段制备相应的表达载体。
在本发明的又一些实施方式中，本发明还提供了表达上述siRNA分子的重组细胞。在本发明的又一些实施方式中，本发明还提供了包含上述siRNA分子、核酸内切酶和Argonature蛋白。
应用所提供的siRNA分子还可以制备试剂盒或者药盒。所提供的试剂盒或者药盒，可以用来检测、预防或者治疗冠状动脉疾病。本文中，所提到的冠状动脉疾病做本领域通常理解，可以包括心绞痛、心肌梗死、脑溢血等与患者脂蛋白a水平升高有关的疾病。
本发明的又一些实施方式中，还提供了一种治疗冠状动脉疾病的方法，包括给予有需要的受试者治疗有效量的上述siRNA分子或者上述药物。受试者所需要的治疗有效量可以结合受试者情况提供。所提到的受试者可以为哺乳动物或者人。
在本发明的一些实施方式中，本发明提供了一种在体外实施的抑制细胞内靶基因的表达的方法，所述方法包括导入上述siRNA分子的步骤。根据本发明的实施例，所述靶基因为脂蛋白编码基因。本发明所使用的术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药。
本文中，术语siRNA(small interfering RNA，小干扰RNA)分子作本领域通常意义理解，是指序列特异性地介导有效的基因表达抑制(可以指基因沉默)的短的双链的RNA。
本文中，术语“反义链”作本领域通常意义理解，是指与靶核酸序列一部分或者全部互补的多核苷酸。例如，可以mRNA序列、非mRNA的RNA序列、或者编码DNA序列或者非编码DNA序列的整体互补或者一部分序列互补。
本文中，术语“正义链”作本领域通常意义理解，是指与靶核酸序列一部分或者全部相同的多核苷酸。例如，可以与mRNA序列、非mRNA的RNA序列、或者编码DNA序列或者非编码DNA序列的整体相同或者一部分序列相同。
本文中，所提到的“靶核酸”可以为mRNA、microRNA、piRNA、编码DNA序列以及非编码DNA序列等等，当然并不限于此。
本文中所提到的核酸分子，可以采用通常的方法人工合成。当然，并不仅限于此。核酸分子可以通过化学合成或者酶法合成。
当然，为了能够使得所提供的siRNA分子能够有效实施体外或者体内传递，siRNA分子可以与已知的能够向细胞内有效传递寡核苷酸的脂质体、阳离子聚合物、抗体、纳米粒子等多种载体联合应用。同时也可以应用多种已知的传递方法实现传递。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
实施例1验针对脂蛋白基因(转录本信息为NM_005577.4)，设计了219条小干扰核酸(siRNA)。其中该蛋白基因的转录本信息记载在NCBI数据库中。
所设计的siRNA序列如下表1所示。其中表1中名称以及位置一栏示出了siRNA分子在LPA靶点上的结合位置，所对应的位置的数值越小，代表位于LPA基因的靠近上游的位置。
表1 siRNA序列
实施例2 体外细胞模型(Huh7细胞)测试小干扰核酸(siRNA)的活性
实施例2应用体外细胞模型测试了这些siRNA序列的活性。实验过程以及实验结果如下：
(1)悬浮转染试剂配制：
配制母液浓度为50μM的siRNA，用DEPC水稀释得到10μM siRNA体系，50μl Opti-MEM稀释得0.2μM siRNA体系，吹吸3-5次混匀(终浓度10nM)。然后用50μl Opti-MEM稀释0.5ul的0.2μM浓度的siRNA，配制0.002μM siRNA体系，吹吸3-5次混匀(终浓度0.1nM)，得到siRNA分子稀释液。
(2)用50μl Opti-MEM稀释2μl RNAiMAX转染试剂，吹吸3-5次混匀，得到稀释后的转染试剂。
(3)分别取稀释后的转染试剂和siRNA分子稀释液混合，吹吸3-5次混匀，室温下静置10min。
细胞处理：镜下观察Huh7细胞株汇合率>70％，进行细胞铺板，按2x10
5细胞/孔铺12孔板，每孔加入900μl含10％FBS DMEM培养基，将转染复合物加至12孔板中，置于37℃，5％CO2培养箱培养。
24h后，提取细胞的总RNA，通过实时定量PCR(Quantitative Real-Time PCR)检测细胞中LPA mRNA序列的表达情况，其中用于扩增内参基因PPIB、LPA的PCR引物如表2所示：
表2
小干扰核酸mRNA表达水平的抑制率按如下等式计算：
抑制率＝[1-(实验组LPA mRNA的表达量/实验组PPIB mRNA的表达量)/(阴性对照组LPA mRNA的表达量/阴性对照组PPIB mRNA的表达量)]×100％。
其中，各实验组为分别经小干扰核酸处理的细胞；阴性对照组(记为Blank)为未经任何小干扰核酸处理的细胞。
所提供的siRNA分子能有效抑制LPA中mRNA的表达水平，具体数据见下表3。
表3、部分siRNA分子对LPA的mRNA表达水平的抑制效果
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (28)
- 一种用于抑制脂蛋白a(LPA)表达的双链siRNA分子，其特征在于，所述的siRNA分子的正义链能够特异性的结合编码LPA的mRNA分子的如下区域中的至少一种：自5’端开始80-109、222-242、263-283、558-578、3514-3534、3659-3679、4178-4198、4627-4651、4957-4993、5033-5056、5224-5244、5466-5486、5767-5789、6058-6078；所述编码LPA的mRNA分子序列的结构如genebank编号NM_005577.4所示。
- 根据权利要求1所述的双链siRNA分子，其特征在于，所述的双链siRNA分子的任意一条链的长度为18-25个核苷酸。
- 一种siRNA分子，其特征在于，所述siRNA分子，所述siRNA分子包括至少一条正义链和至少一条反义链；所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:1～SEQ ID NO:219中的至少一种；(b-1)与SEQ ID NO:1～SEQ ID NO:219中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列；所述反义链与所述正义链反向互补配对，所述反义链选自下列中的至少一种：(a-2)SEQ ID NO:220～SEQ ID NO:438中的至少一种；(b-2)与SEQ ID NO:220～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列。
- 根据权利要求3所述的siRNA分子，其特征在于，所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:1～SEQ ID NO:219中的至少一种；(b-1)与SEQ ID NO:1～SEQ ID NO:219中的任意一条核苷酸序列相比，碱基的差异数在2个以下的核苷酸序列；所述反义链选自下列中的至少一种：(a-2)SEQ ID NO:220～SEQ ID NO:438中的至少一种；(b-2)与SEQ ID NO:220～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数在2个以下的核苷酸序列。
- 根据权利要求3所述的siRNA分子，其特征在于，所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:1～SEQ ID NO:219中的至少一种；(b-1)与SEQ ID NO:1～SEQ ID NO:219中的任意一条核苷酸序列相比，碱基的差异数在1个以下的核苷酸序列；所述反义链选自下列中的至少一种：(a-2)SEQ ID NO:220～SEQ ID NO:438中的至少一种；(b-2)与SEQ ID NO:220～SEQ ID NO:438中的任意一条核苷酸序列相比，碱基的差异数为1个的核苷酸序列。
- 根据权利要求3所述的siRNA分子，其特征在于，所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的至少一种；(b-1)与SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、 203中的任意一条核苷酸序列相比，碱基的差异数在3个以下的核苷酸序列。
- 根据权利要求3所述的siRNA分子，其特征在于，所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的至少一种；(b-1)与SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的任意一条核苷酸序列相比，碱基的差异数在2个以下的核苷酸序列。
- 根据权利要求3所述的siRNA分子，其特征在于，所述正义链选自下列中的至少一种：(a-1)SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的至少一种；(b-1)与SEQ ID NO:5-7、24、46-48、61、81-84、104-109、112-114、129、147、178-179、203中的任意一条核苷酸序列相比，碱基的差异数为1个的核苷酸序列。
- 根据权利要求3～8中任一项所述的siRNA分子，其特征在于，所述siRNA分子抑制LPA基因的表达。
- 根据权利要求1～9中任一项所述的siRNA分子，其特征在于，所述siRNA分子包括至少一个被修饰的核苷酸。
- 根据权利要求10所述的siRNA分子，其特征在于，所述修饰的核苷酸选自下列至少之一：5'-硫代磷酸酯基的核苷酸、5'-甲基化胞嘧啶核苷酸、2'-O-甲基修饰的核苷酸、2'-O-2-甲氧乙基修饰的核苷酸、2'-氟代修饰的核苷酸、3'-氮取代修饰的核苷酸、2'-脱氧-2'-氟修饰的核苷酸、2'-脱氧修饰的核苷酸、锁定的核苷酸、脱碱基核苷酸、2'-氨基修饰的核苷酸、吗啉代核苷酸、多肽核苷酸、氨基磷酸酯，以及包括非天然碱基的核苷酸。
- 根据权利要求1～11中任一项所述的siRNA分子，其特征在于，所述siRNA分子中正义链和反义链的长度均不多于25bp。
- 根据权利要求12所述的siRNA分子，其特征在于，所述siRNA分子中正义链和反义链的长度为18～25bp。
- 根据权利要求12所述的siRNA分子，其特征在于，所述siRNA分子中正义链和反义链的长度为19～22bp。
- 根据权利要求12所述的siRNA分子，其特征在于，所述siRNA分子中正义链和反义链的长度为21bp。
- 根据权利要求1～15中任一项所述的siRNA分子，其特征在于，所述siRNA分子与靶向配体连接。
- 根据权利要求16所述的siRNA分子，其特征在于，所述siRNA分子与靶向配体通过共价键连接。
- 根据权利要求16所述的siRNA分子，其特征在于，所述靶向配体包括至少一个N-乙酰基-半乳糖胺。
- 根据权利要求16所述的siRNA分子，其特征在于，所述靶向配体与所述siRNA分子的正义链连接。
- 根据权利要求16所述的siRNA分子，其特征在于，所述靶向配体与所述siRNA分子的正义链的5’末端连接。
- 一种RNA诱导沉默复合体，其特征在于，包括权利要求1～20中任一项所述的siRNA分 子、核酸内切酶和Argonatue蛋白。
- 一种试剂盒或药盒，其特征在于，所述试剂盒或者所述药盒包括权利要求1～20中任一项所述的siRNA分子。
- 一种在体外抑制细胞内脂蛋白表达量的方法，其特征在于，包括：将siRNA分子导入到细胞内，以便使得所述细胞内脂蛋白的表达量降低，所述siRNA分子为权利要求1～20中任一项所述的siRNA分子。
- 根据权利要求23所述的方法，其特征在于，所述细胞为肝脏细胞。
- 根据权利要求23所述的方法，其特征在于，所述细胞为哺乳动物细胞。
- siRNA分子在制备药物中的用途，所述药物用于治疗冠状动脉疾病，所述siRNA分子为权利要求1～20中任一项所述的siRNA分子。
- 一种治疗冠状动脉疾病的药物，其特征在于，包括有效量的权利要求1～20中任一项所述的siRNA分子和药学上可接受的辅料。
- 一种治疗冠状动脉疾病的方法，其特征在于，包括：给予有需要的受试者治疗有效量的权利要求1～10中任一项所述的siRNA分子或者权利要求27所述的药物。
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