WO2022068923A9 - 血管生成素样3(ANGPTL3)的siRNA及其用途 - Google Patents

血管生成素样3(ANGPTL3)的siRNA及其用途 Download PDF

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WO2022068923A9
WO2022068923A9 PCT/CN2021/122118 CN2021122118W WO2022068923A9 WO 2022068923 A9 WO2022068923 A9 WO 2022068923A9 CN 2021122118 W CN2021122118 W CN 2021122118W WO 2022068923 A9 WO2022068923 A9 WO 2022068923A9
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sirna
seq
nucleotides
sense strand
antisense strand
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PCT/CN2021/122118
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English (en)
French (fr)
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WO2022068923A1 (zh
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陈平
刘兆贵
张捷婷
王芮
徐娟
付中国
张海霖
陈璞
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纳肽得(青岛)生物医药有限公司
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Priority claimed from CN202110008013.3A external-priority patent/CN114716489A/zh
Application filed by 纳肽得(青岛)生物医药有限公司 filed Critical 纳肽得(青岛)生物医药有限公司
Priority to EP23173354.4A priority Critical patent/EP4331608A1/en
Priority to CN202180022902.3A priority patent/CN115516092A/zh
Priority to EP21874577.6A priority patent/EP4223875A1/en
Priority to CN202211333934.8A priority patent/CN116333013A/zh
Priority to JP2022578882A priority patent/JP2023536685A/ja
Publication of WO2022068923A1 publication Critical patent/WO2022068923A1/zh
Publication of WO2022068923A9 publication Critical patent/WO2022068923A9/zh
Priority to US18/092,202 priority patent/US20230257750A1/en
Priority to JP2023054421A priority patent/JP2023121159A/ja
Priority to US18/130,418 priority patent/US20240067971A1/en

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Definitions

  • the present disclosure relates to the technical field of genetic engineering, in particular, the present disclosure relates to angiopoietin-like 3 (ANGPTL3) siRNA and uses thereof.
  • ANGPTL3 angiopoietin-like 3
  • Hyperlipidemia also known as dyslipidemia, is a systemic disease in which fat metabolism or operation is abnormal, making plasma lipids higher than normal.
  • the clinical manifestations of dyslipidemia mainly include two aspects: (1) xanthoma caused by lipid deposition in the dermis; (2) atherosclerosis caused by lipid deposition in the vascular endothelium, resulting in coronary heart disease and peripheral vascular disease Wait.
  • hyperlipidemia is not uncommon in my country. According to the survey, about 10% to 20% of adults have elevated blood total cholesterol (TC) or triglyceride (TG), and even nearly 10% of children have elevated blood lipids.
  • TC total cholesterol
  • TG triglyceride
  • the increase of serum cholesterol levels in the population will lead to an increase of about 9.2 million cardiovascular disease events in my country from 2010 to 2030, which is closely related to the obvious improvement of people's living standards and changes in eating habits.
  • the existing drugs for the treatment of dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probucol, fibrates, niacin and its derivatives.
  • statins are the first choice drugs for lowering serum total cholesterol. It is also used to treat patients with simple elevated serum total cholesterol levels. The total cholesterol level was mainly increased, and the serum triglyceride level was slightly increased.
  • Such drugs mainly include lovastatin (Melon), simvastatin (Zocon), pravastatin (Plagu), fluvastatin (Lexco), atorvastatin (Lipitor) And cerivastatin (Basting) and so on.
  • Such as long-term use may have abdominal distension, diarrhea, constipation, headache, insomnia, rash, thrombotic thrombocytopenic purpura (see diffuse ecchymosis on the face, chest, extremities, with decreased platelet count).
  • paresthesias mostly in the face, scalp, tongue and limbs, manifested as numbness, burning sensation, skin irritation or pain.
  • serum transaminases The most serious adverse reaction is rhabdomyolysis, which is manifested as muscle weakness, myalgia, anuria, and increased serum creatine kinase level, with an incidence of about 1 ⁇ . If the drug is not detected and stopped in time, it will cause severe myopathy and even kidney failure.
  • an object of the present disclosure is to provide an siRNA for inhibiting the expression of ANGPTL3.
  • the inventors of the present disclosure target ANGPTL3 by designing suitable specific small interfering RNA sequences and siRNA conjugates, and degrade ANGPTL3 in cells by degrading ANGPTL3. Therefore, the siRNA provided by the present disclosure can be used to prevent and/or treat dyslipidemia diseases.
  • the siRNA includes a sense strand and an antisense strand
  • the antisense strand includes a region of complementarity paired with the sense strand
  • the sense strand is selected from the group consisting of SEQ ID NO:
  • the nucleotide sequence of each strand in 1 to SEQ ID NO: 154 is different from the nucleotide sequence of no more than 5 nucleotides
  • the antisense strand is selected from SEQ ID NO: 155 to SEQ ID NO: 308
  • the nucleotide sequence of each strand differs by no more than 5 nucleotides in nucleotide sequence.
  • Angiopoietin-like protein 3 (ANGPTL3, NM_014495.4) is a secreted protein mainly expressed in liver cells.
  • ANGPTL3 angiopoietin-like protein 3
  • TG triglyceride
  • siRNA forms a silencing complex (RNA-induced silencing complex, RISC), which is complementary to the mRNA sequence of the target gene (ANGPTL3 gene), and degrades the mRNA of the target gene to inhibit the expression of the target gene, thereby reducing LDL-C and VLDL.
  • RISC RNA-induced silencing complex
  • the present disclosure also provides an siRNA selected from any pair of siRNAs in any of the following groups:
  • [Corrected 15.12.2021 in accordance with Rule 91] (4) can specifically target the 304-388 nucleotides of the angiopoietin-like protein 3 sequence, preferably, can specifically target the 304-359 nucleotides of the angiopoietin-like protein 3 sequence acid; more preferably, the sense strand of the siRNA is selected from SEQ ID NO:27, and the antisense strand is selected from SEQ ID NO:181,
  • the sense strand of the siRNA is selected from SEQ ID NO:29, and the antisense strand is selected from SEQ ID NO:183,
  • the sense strand of the siRNA is selected from SEQ ID NO:31, and the antisense strand is selected from SEQ ID NO:185,
  • the sense strand of the siRNA is selected from SEQ ID NO:32, and the antisense strand is selected from SEQ ID NO:186,
  • the sense strand of the siRNA is selected from SEQ ID NO:35, and the antisense strand is selected from SEQ ID NO:189,
  • the sense strand of the siRNA is selected from SEQ ID NO:36, and the antisense strand is selected from SEQ ID NO:190;
  • the sense strand of the siRNA is selected from SEQ ID NO:44, and the antisense strand is selected from SEQ ID NO:198;
  • [Corrected 15.12.2021 in accordance with Rule 91] (6) can specifically target the 1360-1430 nucleotides of the angiopoietin-like protein 3 sequence, preferably, can specifically target the 1397-1430 nucleotides of the angiopoietin-like protein 3 sequence acid; more preferably, the sense strand of the siRNA is selected from SEQ ID NO: 145, and the antisense strand is selected from SEQ ID NO: 299,
  • the sense strand of the siRNA is selected from SEQ ID NO:150, and the antisense strand is selected from SEQ ID NO:304,
  • the sense strand of the siRNA is selected from SEQ ID NO:151, and the antisense strand is selected from SEQ ID NO:305,
  • the sense strand of the siRNA is selected from SEQ ID NO:152, and the antisense strand is selected from SEQ ID NO:306,
  • the sense strand of the siRNA is selected from SEQ ID NO:154, and the antisense strand is selected from SEQ ID NO:308.
  • the siRNA includes at least one modified nucleotide
  • 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 length of the complementary region is at least 17 bp
  • the length of the complementary region is 18-21 bp;
  • the complementary region is 19 bp in length.
  • the length of the sense strand and the antisense strand in the siRNA is no more than 25 bp;
  • the length of the sense strand and the antisense strand in the siRNA is 18-25 bp;
  • the length of the sense and antisense strands in the siRNA is 21 bp.
  • the bases in the sense strand and the antisense strand in the siRNA may be one-to-one complementary pairing, or may be offset by several bases, but have a complementary region of at least 17 bp.
  • siRNA conjugate comprising the aforementioned siRNA and a targeting ligand, wherein the siRNA is covalently linked to the targeting ligand;
  • the targeting ligand is connected to the sense strand in the siRNA
  • the targeting ligand is linked to the 5' end of the sense strand in the siRNA through a phosphorothioate bond.
  • the targeting ligand includes at least one N-acetyl-galactosamine.
  • the targeting ligand is a GalNAC target compound.
  • the GalNAC target compounds are 1043, 1046, and 1048, and their structures are shown in the following formulas 1-3,
  • the targeting ligand is attached to the sense strand in the siRNA.
  • the pharmaceutical composition includes the aforementioned siRNA and/or the aforementioned siRNA conjugate, and optionally, the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant.
  • compositions according to embodiments of the present disclosure can be used to inhibit cellular synthesis of ANGPTL3, thereby reducing the levels of LDL-C, VLDL-C, HDL-C, and triglycerides (TG), for therapeutic prevention and/or treatment Hyperlipidemia and hypertriglyceridemia.
  • kits includes the siRNA and/or the siRNA conjugate.
  • kits according to embodiments of the present disclosure can be used to inhibit ANGPTL3 gene expression in cells, thereby reducing the levels of LDL-C, VLDL-C, HDL-C, and triglycerides (TG), for therapeutic prevention and/or Treatment of hyperlipidemia and hypertriglyceridemia.
  • ANGPTL3 triglycerides
  • Yet another aspect of the present disclosure provides a method of inhibiting ANGPTL3 gene expression in a subject, the method comprising: administering the aforementioned siRNA and/or the aforementioned siRNA conjugate to the subject to inhibit the expression of the ANGPTL3 gene.
  • Yet another aspect of the present disclosure provides a method for inhibiting ANGPTL3 gene expression in a cell.
  • the method comprises: transfecting the cell with the siRNA and/or the siRNA conjugate to inhibit the expression of the ANGPTL3 gene in the cell.
  • siRNA is used to form a silencing complex, which is complementary to the sequence of the mRNA of the target gene ANGPTL3 gene, and the mRNA of the target gene is degraded to inhibit the expression of the target gene, thereby reducing LDL.
  • silencing complex which is complementary to the sequence of the mRNA of the target gene ANGPTL3 gene, and the mRNA of the target gene is degraded to inhibit the expression of the target gene, thereby reducing LDL.
  • TG triglycerides
  • the cell is derived from a mammal
  • the cells are derived from a human;
  • the cells are liver cells.
  • a silencing complex is formed in human liver cells, which is complementary to the mRNA sequence of the ANGPTL3 gene, and the mRNA of the ANGPTL3 gene is degraded to inhibit its expression, thereby reducing LDL-C, VLDL-C, and HDL. -C and triglyceride (TG) levels.
  • Another aspect of the present disclosure provides the use of the siRNA and/or the siRNA conjugate in the preparation of a medicament or a kit.
  • the medicine or kit is used to inhibit ANGPTL3 gene expression.
  • the siRNA provided by the present disclosure is used to prepare a drug or a kit, and the drug or kit can reduce the expression level of the ANGPTL3 gene in cells through the siRNA therein, thereby preventing and/or treating dyslipidemia.
  • the medicine or kit is used to prevent and/or treat dyslipidemia
  • the dyslipidemia disease includes hyperlipidemia and hypertriglyceridemia
  • the medicament or kit is used to inhibit ANGPTL3 gene expression in cells.
  • Yet another aspect of the present disclosure provides a method for preventing and/or treating dyslipidemia.
  • the method comprises: administering the siRNA and/or the siRNA conjugate to the subject.
  • the dyslipidemia disease includes hyperlipidemia and hypertriglyceridemia.
  • Figure 1 shows the expression results of ANGPTL3 gene (abbreviated as ANL3 in the figure) in the cells detected by real-time quantitative PCR after some siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 0.1 nM;
  • Figure 2 shows the expression results of the ANGPTL3 gene (abbreviated as ANL3 in the figure) in the cells detected by real-time quantitative PCR after some siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 10 nM;
  • FIG. 3 shows the GalNAc-siRNA conjugate synthesized in Example 3
  • Figure 4 shows the activity test results (EC 50 values) of each conjugate in Example 4.
  • “Pharmaceutically acceptable carriers” are art-recognized and include pharmaceutically acceptable materials, compositions or carriers suitable for administering the compounds of the present disclosure to mammals. Such carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that participate in carrying or transferring the host substance from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient.
  • materials that can be used as pharmaceutically acceptable carriers include: carbohydrates such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, Ethyl cellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, Olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycols; esters, such as ethyl oleate and ethyl laurate; agar; buffers , such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; Ringer's solution; ethanol; phosphate buffer; and other
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring, releasing, coating, sweetening, flavoring and perfuming agents can also be present in the compositions , preservatives and antioxidants.
  • compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to prepare a unit dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, in one percent units, this amount will be from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.
  • 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, particularly humans, including: (a) preventing the development 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) alleviating the disease, eg reducing symptoms associated with the disease.
  • Treatment encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in the individual, including but not limited to administration of a drug containing a compound described herein to an individual in need thereof.
  • the siRNA includes a sense strand and an antisense strand
  • the antisense strand includes a region of complementarity paired with the sense strand
  • the sense strand is selected from the group consisting of SEQ ID NO:
  • the nucleotide sequence of each strand in 1 to SEQ ID NO: 154 is different from the nucleotide sequence of no more than 5 nucleotides
  • the antisense strand is selected from SEQ ID NO: 155 to SEQ ID NO: 308
  • the nucleotide sequence of each strand differs by no more than 5 nucleotides in nucleotide sequence.
  • the sense strands include, in addition to SEQ ID NO: 1 to SEQ ID NO: 154 shown in Table 2, 1, 2, 3 and 3 of the sense strands shown in Table 2 Contiguous nucleotide sequences that differ by 1, 4, and 5 nucleotides.
  • the antisense strands include, in addition to SEQ ID NO: 155 to SEQ ID NO: 308 shown in Table 2, 1 or 2 antisense strands as shown in Table 2 , 3, 4, and 5 nucleotides distinct contiguous nucleotide sequences.
  • the siRNA includes 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 length of the complementary region is 18-21 bp, for example, 19 bp.
  • the length of the sense strand and the antisense strand in the siRNA is 18-25 bp, for example, 21 bp.
  • the length of the sense strand and the antisense strand in the siRNA is 21 bp, and the bases in the sense strand and the antisense strand are complementary one by one, or the sense strand and the antisense strand in the siRNA are complementary There are 19 consecutive bases complementary to each other, that is, the length of the complementary region is 19 bp.
  • liver cells are transfected with the siRNA to inhibit the expression of the ANGPTL3 gene in the cells.
  • RNA- induce siliencing complex RISC
  • the siRNA is covalently linked to the targeting ligand.
  • the targeting ligand includes at least one N-acetyl-galactosamine.
  • the targeting ligand is attached to the sense strand in the siRNA.
  • Embodiments of the present disclosure are described in detail below.
  • the embodiments described below are exemplary only for explaining the present disclosure and should not be construed as limiting the present disclosure. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used.
  • the reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
  • Example 1 In vitro cell model (Hep 3B cells) to test the activity of small interfering nucleic acid (siRNA)
  • the concentration of siRNA stock solution is 50 ⁇ M, diluted with DEPC water to obtain 10 ⁇ M siRNA system, 50 ⁇ l Opti-MEM diluted to 0.2 ⁇ M siRNA system, and mixed by pipetting 3-5 times (final concentration 10 nM).
  • RNA of the cells was extracted, and the expression of the ANGPTL3 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 ANGPTL3 are shown in Table 1. :
  • inhibition rate [1-(expression of ANGPTL3 mRNA in experimental group/expression of PPIB mRNA in experimental group)/(expression of ANGPTL3 mRNA in negative control group amount/expression amount 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.
  • Figures 1 and 2 respectively show the results of the expression of ANGPTL3 gene in Hep 3B cells detected by real-time quantitative PCR after some siRNAs in Table 2 were transfected at a concentration of 0.1 nM or 10 nM. It is shown that the siRNA shown in the attached figure can significantly reduce the expression of ANGPTL3 gene whether Hep 3B cells are transfected with a concentration of 0.1 nM or 10 nM.
  • N-tert-butoxycarbonyl-1,4-butanediamine (4.87 g, 25.89 mmol) was then added, the temperature was raised to 25° C. and the reaction was stirred for 16 hours. TLC showed that the starting material had basically disappeared.
  • NC-4 (2.6 g, 4.7 mmol) was dissolved in DCM (200 mL), cooled to 0-5 °C in an ice-water bath, HATU (5.6 g, 14.83 mmol) and DIEA (4.85 °C) were added. g, 37.6 mmol) and stirred for 20 minutes,
  • NC-6-02 (69.5 g, 0.29 mol) and tert-butyl bromoacetate (187 g, 0.96 mol) were added to tetrahydrofuran (700 mL) and purified water (350 mL), stirred, and cooled to 5°C in an ice-water bath Next, potassium carbonate (322 g, 2.34 mol) was added. The reaction was stirred at 25°C for 14 hours, and the conversion of the starting materials was detected by TLC.
  • NC-6-03 (23 g, 39.6 mmol) was dissolved in 1,4-dioxane (200 mL), concentrated hydrochloric acid (40 mL) was added, the temperature was raised to 60° C. and reacted for 2 hours, TLC detected that the raw materials were basically consumed complete.
  • NC-6 1.5 g, 3.6 mmol
  • HBTU 4.5 g, 12.0 mmol
  • DIEA 4.75 g, 36 mmol
  • LCMS detected that the starting material was basically consumed.
  • GN-18-01 (45.52 g, 70 mmol) was added to HCl/EtOAc solution (2N, 500 mL) in batches, stirred at 25° C. for 2 hours, LCMS detection showed that the raw material disappeared.
  • oligonucleotide sequence portions of the antisense and sense strands of the following RNAi agent duplexes, as well as the ligation of targeting ligands and RNAs, are in accordance with J.Org.Chem.2012,77,4566-4577; Curr.
  • the phosphoramidite coupling technique reported in Protoc. Nucleic Acid Chem., 81, e107 is synthesized on solid phase for oligonucleotide synthesis.
  • the targeting ligands 1046, 1048, and 1043 were all linked to the 5' end of the siRNA sense strand through a phosphorothioate bond.
  • the synthesized GalNAc-siRNA conjugates are described in the table in Figure 3.
  • the conjugate structure in the second column of the table includes three parts.
  • the structure of G1043-S2A2-A265 is: 1043 target head through phosphorothioate
  • the bond is connected to the 5' end of the siRNA sense strand numbered A265.
  • S2A2 is the modification type of the siRNA of A265.
  • the specific modification groups and modification methods are:
  • Ao represents adenosine
  • Uo represents uridine
  • Go represents guanosine
  • Co represents cytosine nucleoside.
  • a G C T (A for 2'-deoxyadenosine, T for 2'-deoxythymidine, G for 2'-deoxyguanosine, C for 2'-deoxycytidine );
  • 2'-F aF gF cF uF (aF means 2'-fluoroadenosine, uF means 2'-fluorouridine, gF means 2'-fluoroguanosine, cF means 2' - fluorocytosine);
  • 2'-OMe aM gM cM uM (aM represents 2'-O-methyladenosine, uM represents 2'-O-methyluridine, gM represents 2'-O-methylguanine nucleus glycoside, cM represents 2'-O-methylcytosine);
  • the y and z in the sequence represent the position of the target head.
  • Example 4 In vitro cell model (Hep 3B cells) to test the activity of the conjugate
  • the final concentration of siRNA is 10nM/1nM/0.5nM/0.25nM/0.1nM/0.05nM/0.01nM.
  • the transfection group used siNC as a negative control, and its sequence was:
  • Antisense 5'-ACGUGACACGUUCGGAGAATT-3'.
  • RNA of the cells was extracted, and the expression of the ANGPTL3 mRNA sequence in the cells was detected by real-time quantitative PCR (Quantitative Real-Time PCR).
  • the PCR primers used to amplify the internal reference genes PPIB and ANGPTL3 are shown in Table 1:
  • EC50 values were calculated using nonlinear regression of graphpad prism and represent the amount of conjugate that inhibits half of the target mRNA (ANGPTL3) expression.
  • mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and were SPF animals. Before administration, the above mice were weighed and the state was observed, and animals with uniform body weight and no abnormal state were selected for follow-up experiments.
  • mice Each mouse was injected with 2.5*10 ⁇ 11 titer virus solution, 100ul through the tail vein. After 7 days, the experimental animals were randomly divided into groups, and each test substance was subcutaneously administered at a dose of 5 mg/kg. Seventy-two hours after administration, the animals were sacrificed by cervical dislocation, and liver tissue was collected for RNA extraction and quantification.

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Abstract

提供一种血管生成素样3(ANGPTL3)的siRNA及其用途。通过设计合适的特异性小干扰RNA序列以及siRNA缀合物，靶向ANGPTL3，通过降解细胞中ANGPTL3基因的转录物，从而降低ANGPTL3蛋白表达量。所述的siRNA可用于预防和/或治疗血脂异常疾病。

Description

血管生成素样3(ANGPTL3)的siRNA及其用途
优先权信息
本申请请求2020年09月30日向中国国家知识产权局提交的、专利申请号为202011061038.1的专利申请，2021年01月05日向中国国家知识产权局提交的、专利申请号为202110008013.3的专利申请，以及2021年04月13日向中国国家知识产权局提交的、专利申请号为202110397429.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开涉及基因工程技术领域，具体地，本公开涉及血管生成素样3(ANGPTL3)的siRNA及其用途。
背景技术
高脂血症又称血脂异常，是脂肪代谢或运转异常，使血浆脂质高于正常值的一种全身性疾病。血脂异常的临床表现主要包括两大方面：(1)脂质在真皮内沉积所引起的黄色瘤；(2)脂质在血管内皮沉积所引起的动脉粥样硬化，产生冠心病和周围血管病等。高血脂症在我国已不少见，据调查，成人中血总胆固醇(TC)或甘油三酯(TG)升高者约占10％至20％，甚至儿童中也有近10％者血脂升高，人群血清胆固醇水平的升高将导致2010年～2030年期间我国心血管病事件约增加920万，这与我国人民的生活水平明显提高、饮食习惯发生改变等原因有密切关系。现有的治疗血脂异常的药物主要有他汀类、胆固醇吸收抑制剂、树脂类、普罗步考、贝特类和烟酸及其衍生物。
目前治疗药物使用后或多或少都会有一些禁忌症和副反应，例如，他汀类药是目前常用的降低血清总胆固醇的首选药物，治疗单纯性血清总胆固醇水平增高者，也用于以血清总胆固醇水平增高为主，且伴有血清三酰甘油水平轻度增高者。这类药物主要包括洛伐他汀(美降之)、辛伐他汀(舒降之)、普伐他汀(普拉固)、氟伐他汀(来适可)、阿托伐他汀(立普妥)和西立伐他汀(拜斯亭)等。如长期服用可有腹胀、腹泻、便秘、头痛、失眠、皮疹、血栓性血小板减少性紫癜(见于面部、胸部、肢端有弥漫性瘀斑、伴血小板计数减少)。此外，还有精神抑郁、感觉异常、多发生于面部、头皮、舌头和四肢，表现为麻木感、烧灼感、皮肤过敏或疼痛。并可引起脱皮、血清转氨酶升高。最严重的不良反应为横纹肌溶解，表现为肌无力、肌痛、无尿、血清肌酸激酶水平升高等，发生率约为1‰。若未及时被发现停药，会产生严重的肌病，甚至会造成肾功能衰竭。
因此，亟需开发一种可长期服用且副作用小的治疗血脂异常的药物。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本公开的一个目的在于提供一种用于抑制ANGPTL3表达的siRNA，本公开的发明人通过设计合适的特异性小干扰RNA序列以及siRNA缀合物，靶向ANGPTL3，通过降解细胞中ANGPTL3基因的转录物，从而降低ANGPTL3蛋白表达量，因此本公开提供的siRNA可用于预防和/或治疗血脂异常疾病。
为此，本公开一方面提供一种siRNA。根据本公开的实施例，所述siRNA包括一条正义链和一条反义链，所述反义链包括与所述正义链互补配对的互补性区域，其中所述正义链选自与SEQ ID NO:1～SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列，所述反义链选自与SEQ ID NO:155～SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
血管生成素样蛋白3(ANGPTL3，NM_014495.4)是一种主要在肝脏细胞中表达的分泌蛋白。已有研究表明，血管生成素样蛋白3(ANGPTL3)是LDL-C、HDL-C和甘油三酯代谢的关键调节因子，具有多种潜在的作用节点，ANGPTL3的功能缺失突变可导致LDL-C、VLDL-C、HDL-C和甘油三酯(TG)降低，从而使基于GWAS的心血管疾病风险降低，且没有已知的遗传缺陷的不良表型。因此，抑制ANGPTL3的活性可以有效预防或治疗血脂异常，本公开的发明人通过设计合适的小干扰RNA(siRNA)序列特异性减少肝细胞合成ANGPTL3，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(ANGPTL3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，继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
根据本公开实施例的siRNA，还可以具有以下附加技术特征的至少之一：
本公开还提供一种siRNA，所述siRNA选自如下任一组中的任一对siRNA：
[根据细则91更正 15.12.2021]　
(1)能够特异性的靶向血管生成素样蛋白3序列的第60-80位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:10，反义链选自SEQ ID NO:165；
[根据细则91更正 15.12.2021]　
(2)能够特异性的靶向血管生成素样蛋白3序列的第107-133位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:17，反义链选自SEQ ID NO:171，或者所述siRNA的正义链选自SEQ ID NO:18，反义链选自SEQ ID NO:172；
[根据细则91更正 15.12.2021]　
(3)能够特异性的靶向血管生成素样蛋白3序列的第163-187位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:19，反义链选自SEQ ID NO:173；
[根据细则91更正 15.12.2021]　
(4)能够特异性的靶向血管生成素样蛋白3序列的第304-388位核苷酸，优选的，能够特异性的靶向血管生成素样蛋白3序列的第304-359位核苷酸；更优选的，所述的siRNA的正义链选自SEQ ID NO:27，反义链选自SEQ ID NO:181，
或者，所述的siRNA的正义链选自SEQ ID NO:29，反义链选自SEQ ID NO:183，
或者，所述的siRNA的正义链选自SEQ ID NO:31，反义链选自SEQ ID NO:185，
或者，所述的siRNA的正义链选自SEQ ID NO:32，反义链选自SEQ ID NO:186，
或者，所述的siRNA的正义链选自SEQ ID NO:35，反义链选自SEQ ID NO:189，
或者，所述的siRNA的正义链选自SEQ ID NO:36，反义链选自SEQ ID NO:190；
[根据细则91更正 15.12.2021]　
(5)能够特异性的靶向血管生成素样蛋白3序列的第430-459位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:43，反义链选自SEQ ID NO:197，
或者，所述的siRNA的正义链选自SEQ ID NO:44，反义链选自SEQ ID NO:198；
[根据细则91更正 15.12.2021]　
(6)能够特异性的靶向血管生成素样蛋白3序列的第1360-1430位核苷酸，优选的，能够特异性的靶向血管生成素样蛋白3序列的第1397-1430位核苷酸；更优选的，所述的siRNA的正义链选自SEQ ID NO:145，反义链选自SEQ ID NO:299，
或者，所述的siRNA的正义链选自SEQ ID NO:150，反义链选自SEQ ID NO:304，
或者，所述的siRNA的正义链选自SEQ ID NO:151，反义链选自SEQ ID NO:305，
或者，所述的siRNA的正义链选自SEQ ID NO:152，反义链选自SEQ ID NO:306，
或者，所述的siRNA的正义链选自SEQ ID NO:154，反义链选自SEQ ID NO:308。
根据本公开的实施例，所述siRNA包括至少一个被修饰的核苷酸；
任选地，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。
根据本公开的实施例，所述互补性区域的长度至少为17bp；
任选地，所述互补性区域的长度为18～21bp；
任选地，所述互补性区域的长度为19bp。
根据本公开的实施例，所述siRNA中正义链和反义链的长度不多于25bp；
任选地，所述siRNA中正义链和反义链的长度为18～25bp；
任选地，所述siRNA中正义链和反义链的长度为21bp。
根据本公开的实施例，所述siRNA中正义链和反义链中的碱基可以是一一互补配对，也可以是错位几个碱基，但具有至少17bp的互补性区域。
本公开另一方面提供一种siRNA缀合物，所述siRNA缀合物包括前面所述的siRNA和靶向配体，其中，所述siRNA与所述靶向配体共价连接；
优选的，所述靶向配体连接于所述siRNA中正义链；
更优选的，所述靶向配体通过硫代磷酸酯键与所述siRNA中正义链的5’端连接。
根据本公开的实施例，所述靶向配体包括至少一个N-乙酰基-半乳糖胺。
根据本公开的实施例，所述靶向配体为GalNAC靶头化合物。
根据本公开的实施例，所述GalNAC靶头化合物为1043、1046、1048，其结构如下式1-3所示，
Figure PCTCN2021122118-appb-000001
Figure PCTCN2021122118-appb-000002
根据本公开的实施例，所述靶向配体连接于所述siRNA中正义链。
本公开另一方面提供一种药物组合物。根据本公开的实施例，所述药物组合物包括前述的siRNA和/或前述的siRNA缀合物，任选地，所述药物组合物还包括药学上可接受的辅料。
由此，根据本公开实施例的药物组合物可以用于抑制细胞合成ANGPTL3，从而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平，以治疗预防和/或治疗高血脂症和高甘油三酯血症。
本公开又一方面提供一种试剂盒。根据本公开的实施例，所述试剂盒包括所述的siRNA和/或siRNA缀合物。
由此，根据本公开实施例的试剂盒可以用于抑制细胞中ANGPTL3基因表达，从而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平，以治疗预防和/或治疗高血脂症和高甘油三酯血症。
本公开又一方面提供一种抑制受试者ANGPTL3基因表达的方法，所述方法包括：向受试者施用前述的siRNA和/或前述的siRNA缀合物，以抑制ANGPTL3基因的表达。
本公开又一方面提供一种抑制细胞中ANGPTL3基因表达的方法。根据本公开的实施例，所述方法包括：用所述的siRNA和/或所述的siRNA缀合物转染所述细胞，以抑制所述细胞中ANGPTL3基因的表达。
根据本公开实施例的抑制细胞中ANGPTL3基因表达的方法，利用siRNA形成沉默复合体，与靶标基因ANGPTL3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，继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
根据本公开的实施例，所述细胞源自哺乳动物；
任选地，所述细胞源自人；
任选地，所述细胞为肝脏细胞。
利用本公开提供的siRNA，在人的肝脏细胞中形成沉默复合体，与ANGPTL3基因的mRNA的序列互补配对，使ANGPTL3基因的mRNA降解从而抑制其表达，继而降低LDL-C、VLDL-C、HDL-C和甘油三酯(TG)的水平。
本公开又一方面提供所述的siRNA和/或所述的siRNA缀合物在制备药物或试剂盒中的用途。根据本公开的实施例，所述药物或试剂盒用于抑制ANGPTL3基因表达。
利用本公开提供的siRNA制备药物或试剂盒，所述药物或试剂盒通过其中的siRNA以降低细胞中ANGPTL3基因的表达水平，从而预防和/或治疗血脂异常疾病。
根据本公开的实施例，所述药物或试剂盒用于预防和/或治疗血脂异常疾病；
任选地，所述血脂异常疾病包括高血脂症和高甘油三酯血症；
任选地，所述药物或试剂盒用于抑制细胞中ANGPTL3基因表达。
本公开又一方面提供一种预防和/或治疗血脂异常疾病的方法。根据本公开的实施例，所述方法包括： 向受试者施用所述的siRNA和/或所述的siRNA缀合物。
根据本公开的实施例，所述血脂异常疾病包括高血脂症和高甘油三酯血症。
本公开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，或通过本公开的实践了解到。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了表2中部分siRNA以0.1nM浓度转染Hep 3B细胞后，利用实时定量PCR检测的细胞中ANGPTL3基因(图中简写为ANL3)的表达量结果；
图2显示了表2中部分siRNA以10nM浓度转染Hep 3B细胞后，利用实时定量PCR检测的细胞中ANGPTL3基因(图中简写为ANL3)的表达量结果；
图3显示了实施例3中合成的GalNAc-siRNA缀合物；
图4显示了实施例4中各个缀合物的活性测试结果(EC 50值)。
发明详细描述
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
“药学可接受的载体”在本领域中是公认的，包括适于将本公开的化合物施用于哺乳动物的药学可接受的材料、组合物或载体。所述载体包括参与携带主体物质或将其从一个器官或机体的一部分转移到另一个器官或机体的另一部分的液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料。各载体在与制剂中的其它成分相容和对患者无害的意义上必须是“可接受的”。可用作药学可接受的载体的材料的一些实例包括：糖类，如乳糖、葡萄糖和蔗糖；淀粉类，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和醋酸纤维素、粉状西黄蓍胶、麦芽、明胶、滑石粉，赋形剂如可可脂和栓剂蜡类；油类，如花生油、棉子油、红花油、芝麻油、橄榄油、玉米油和豆油；二醇类，如丙二醇；多元醇类，如甘油、山梨醇、甘露醇和聚乙二醇；酯类，如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；海藻酸；无热原的水；林格氏溶液；乙醇；磷酸盐缓冲液；和药物制剂中所用的其它无毒的可相容的物质。
在组合物中也可以存在润湿剂、乳化剂和润滑剂如十二烷基硫酸钠和硬脂酸镁，以及着色剂、释放剂、包衣剂、甜味剂、矫味剂和芳香剂、防腐剂和抗氧化剂。
本公开的药物组合物包括适于口服、鼻、局部、口含、舌下、直肠和/或胃肠外施用的那些。制剂可以方便地以单位剂型形式存在并且可以通过药学领域公知的任何方法来制备。可以与载体物质组合来制备单剂量形式的活性成分的量一般是产生治疗作用的化合物的量。一般而言，以百分之一为单位，该量为约1％至约99％活性成分，优选约5％至约70％，最优选约10至约30％。
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、特别是人的疾病，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，包括但不限于将含本文所述化合物的药物给予有需要的个体。
本公开提供一种用于抑制ANGPTL3表达的siRNA。根据本公开的实施例，所述siRNA包括一条正义链和一条反义链，所述反义链包括与所述正义链互补配对的互补性区域，其中所述正义链选自与SEQ ID NO:1～SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列，所述反义链选自与SEQ ID NO:155～SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
根据本公开的实施例，所述正义链除了包括表2中所示的SEQ ID NO:1～SEQ ID NO:154，还包括与表2中所示的正义链有1个、2个、3个、4个、5个核苷酸区别的连续核苷酸序列。
根据本公开的实施例，所述反义链除了包括表2中所示的SEQ ID NO:155～SEQ ID NO:308，还包括与表2中所示的反义链有1个、2个、3个、4个、5个核苷酸区别的连续核苷酸序列。
根据本公开的实施例，所述siRNA包括至少一个被修饰的核苷酸；
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。
根据本公开的实施例，所述互补性区域的长度为18～21bp，例如为19bp。
根据本公开的实施例，所述siRNA中正义链和反义链的长度为18～25bp，例如为21bp。
根据本公开具体的实施例，所述siRNA中正义链和反义链的长度为21bp，所述正义链和反义链中的碱基一一互补，或者所述siRNA中正义链和反义链中具有19个连续的碱基互补，即所述互补性区域的长度为19bp。
根据本公开的实施例，用所述的siRNA转染肝脏细胞，以便抑制细胞中ANGPTL3基因的表达。
针对血管生成素样3(ANGPTL3)基因靶点，本公开的发明人设计合适的小干扰核酸(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，调控血管生成素样3分泌蛋白表达。
根据本公开的实施例，所述siRNA与靶向配体通过共价键连接。
根据本公开的实施例，所述靶向配体包括至少一个N-乙酰基-半乳糖胺。
根据本公开的实施例，所述靶向配体连接于所述siRNA中正义链。
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。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。
本实施例的部分合成路线可参考CN202110397429.9、CN202110008013.3；本申请的实施例以源引的方式加入上述两件专利申请。
实施例1体外细胞模型(Hep 3B细胞)测试小干扰核酸(siRNA)的活性
1)悬浮转染试剂配制：siRNA母液浓度为50μM，DEPC水稀释得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)。50μl Opti-MEM稀释0.5ul 0.2μMSiRNA得0.002μM siRNA体系，吹吸3-5次混匀(终浓度0.1nM)；50μl Opti-MEM稀释2μl RNAiMAX，吹吸3-5次混匀。分别混合转染试剂和小干扰核酸稀释液，吹吸3-5次混匀，室温下静置10min。
2)处理细胞：镜下观察Hep 3B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％CO 2培养箱培养。
3)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ANGPTL3 mRNA序列的表达情况，其中用于扩增内参基因PPIB、ANGPTL3的PCR引物如表1所示：
表1：用于扩增内参基因PPIB、ANGPTL3的PCR引物序列
Figure PCTCN2021122118-appb-000003
4)小干扰核酸对ANGPTL3表达水平的抑制率按如下等式计算：抑制率＝[1-(实验组ANGPTL3 mRNA的表达量/实验组PPIB mRNA的表达量)/(阴性对照组ANGPTL3 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。
利用上述方法获得表2中154对siRNA分别以0.1nM和10nM的浓度转染Hep 3B细胞后，对ANGPTL3基因(NM_014495.4)表达的抑制率结果。
表2：154对靶向ANGPTL3的siRNA序列
Figure PCTCN2021122118-appb-000004
Figure PCTCN2021122118-appb-000005
Figure PCTCN2021122118-appb-000006
Figure PCTCN2021122118-appb-000007
Figure PCTCN2021122118-appb-000008
附图1和2分别显示了表2中部分siRNA以0.1nM或10nM浓度转染Hep 3B细胞后，利用实时定量PCR检测的细胞中ANGPTL3基因的表达量结果。表明附图中所示的siRNA不论是以0.1nM还是10nM浓度转染Hep 3B细胞，均能够明显降低ANGPTL3基因的表达。
实施例2、GalNAc连接靶头的合成
一、GalNAc靶头1043的合成
按照以下方法，合成了TO-23(1043靶头)及TP-23(1043靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-17-01的合成
Figure PCTCN2021122118-appb-000009
(1)在N 2氛围下，将GC-1(12g，25.89mmol)溶于DCM(200mL)中，冰水浴降温至0～5℃，加入HBTU(11.78g，31mmol)和DIEA(10g，77.67mmol)，搅拌10分钟，
(2)随后加入N-叔丁氧羰基-1,4-丁二胺(4.87g，25.89mmol)，升温至25℃搅拌反应16小时，TLC显示原料基本消失。
(3)加入饱和的氯化铵溶液(100mL)淬灭，分液，DCM(100mL×2)萃取，
(4)合并有机相并用饱和食盐水(100mL)洗涤，无水Na 2SO 4干燥，过滤并浓缩。经柱层析纯化(DCM/MeOH＝20/1)得白色固体化合物GN-17-01(15g，收率91％)。
2、中间体GN-17的合成
Figure PCTCN2021122118-appb-000010
(1)将GN-17-01(15g，23.67mmol)溶于DCM(150mL)中，加入TFA(50mL)，25℃搅拌1小时，TLC显示原料基本消失，浓缩，
(2)用乙腈(100mL×3)与TFA共沸除去多余TFA，得泡沫状固体GN-17(TFA盐，12.6g)。
3、中间体TO-23-01的合成
Figure PCTCN2021122118-appb-000011
(1)在N 2氛围下，将NC-4(2.6g，4.7mmol)溶于DCM(200mL)中，冰水浴降温至0～5℃，加入HATU(5.6g，14.83mmol)和DIEA(4.85g，37.6mmol)搅拌20分钟，
(2)随后加入GN-17(8.45g，15.5mmol)，升温至25℃搅拌反应4小时。TLC检测，原料基本消失，
(3)加入饱和氯化铵溶液(50mL)淬灭，分液，DCM(100mL×2)萃取，
(4)合并有机相并用饱和食盐水(100mL)洗涤，无水Na 2SO 4干燥，
(5)过滤并浓缩得粗制品。经柱层析纯化(DCM/MeOH＝10/1)得白色固体TO-23-01(6.3g，收率63.1％)。
4、化合物TO-23(1043靶头)的合成
Figure PCTCN2021122118-appb-000012
(1)向TO-23-01(6.3g，3.0mmol)的MeOH(100mL)溶液中加入10％Pd/C(600mg)和Pd(OH) 2/C(600mg)，H 2置换3次，25℃搅拌反应3小时，TLC(DCM/MeOH＝8/1)检测原料基本消失，
(2)过滤，浓缩得粗制品。经柱层析纯化(DCM/MeOH/TEA＝10/1/0.1)得白色固体TO-23(4.5g，收率75％)。
1H NMR(400MHz,DMSO-d 6)δ7.88-7.81(m,9H),7.14(s,1H),5.21(d,J＝3.4Hz,3H),4.95(dd,J＝11.2,3.4Hz,3H),4.53(d,J＝8.5Hz,3H),4.07-3.97(m,9H),3.88(dt,J＝11.0,9.0Hz,3H),3.77-3.71(m,3H),3.63-3.50(m,24H),3.49-3.41(m,8H),3.38-3.35(m,2H),3.08-2.98(m,12H),2.35–2.25(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(s,12H).
MS(ESI):m/z[1/2M+H] +理论值1000.5，实测值1000.3。
5、化合物TP-23(1043靶头连接siRNA的前体)的合成
Figure PCTCN2021122118-appb-000013
(1)N 2氛围下，将TO-23(2.3g，1.15mmol)溶于干燥DCM(40mL)中，加入DIEA(0.86mL，5.2mmol)，利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.46mL，2.1mmol)的干燥DCM(2mL)溶液。25℃反应1小时。TLC检测，原料基本消失，
(2)加入饱和NaHCO 3(20mL)淬灭，分液，有机相用饱和NaHCO 3(20mL)溶液，饱和食盐水(20mL)洗涤，无水MgSO4干燥，过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5％TEA/DCM碱化，DCM/MeOH/TEA＝15/1/0.1)得白色固体TP-23(1.8g，收率71.1％)。
1H NMR(400MHz,DMSO-d 6)δ7.91-7.79(m,9H),7.15(s,1H),5.21(d,J＝3.4Hz,3H),4.95(dd,J＝11.2,3.4Hz,3H),4.53(d,J＝8.5Hz,3H),4.06-3.97(m,9H),3.88(dt,J＝11.1,8.9Hz,3H),3.78-3.66(m,6H),3.63-3.41(m,36H),3.07-2.98(m,12H),2.76(t,J＝5.9Hz,2H),2.35-2.24(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(m,12H),1.13(dd,J＝6.7,4.1Hz,12H)；
31P NMR(162MHz,DMSO-d 6)δ147.81；
MS(ESI):m/z[1/2M+Na] +理论值1122.5，实测值1122.4。
二、GalNAc靶头1046的合成
按照以下方法，合成了TO25(1046靶头)及TP-25(1046靶头连接siRNA的前体)的一种非对映异构体。
1、中间体NC-6-01的合成
Figure PCTCN2021122118-appb-000014
(1)N 2氛围下，向1000mL三口瓶中加入干燥的THF(300mL)，冰浴降温至0-5℃搅拌，分批加入60％NaH(14g，354.8mmol)，随后缓慢滴加2-氯乙氧基乙醇(40g，322.5mmol)的THF溶液(200mL)，保温反应30分钟，再向反应瓶中滴加溴化苄(60.3g，354.8mmol)，升至25℃搅拌16小时，TLC监测原料基本消耗完毕。
(2)缓慢滴加饱和氯化铵溶液(150mL)淬灭，分液，水相用EtOAc(100mL×2)萃取，合并有机相并用饱和食盐水(300mL)洗涤、无水Na 2SO 4干燥，过滤浓缩获得粗制产品。粗产品用硅胶柱层析纯化(石油醚/EtOAc＝5/1)得淡黄色油状物化合物NC-6-01(53g，收率78％)。
MS(ESI):m/z[M+H] +理论值215.1，实测值215.1。
2、中间体NC-6-02的合成
Figure PCTCN2021122118-appb-000015
(1)将乙二胺(196g，3.26mol)置于2000mL三口瓶中，加入乙腈(1000mL)、碳酸钾(90g，0.65mol)和碘化钠(60.6g，0.33mol)搅拌。随后将NC-6-01(70g，0.33mol)的乙腈(100mL)溶液缓慢滴加至反应瓶中，升温至60℃搅拌16小时，TLC检测原料基本消耗完毕。
(2)停止反应，浓缩，加入纯化水(300mL)，浓盐酸调节pH至4-5，EtOAc(200mL×3)萃取三次，水相加入氢氧化钠固体调节pH至13-14，经DCM(200mL×3)萃取三次，合并有机相并用饱和食盐水(300mL)洗涤、无水Na 2SO 4干燥，过滤浓缩得淡黄色油状物NC-6-02(69.5g，87％)。
MS(ESI):m/z[M+H] +理论值239.2，实测值239.1。
3、中间体NC-6-03的合成
Figure PCTCN2021122118-appb-000016
(1)将NC-6-02(69.5g，0.29mol)和溴乙酸叔丁酯(187g，0.96mol)加入到四氢呋喃(700mL)和纯化水(350mL)中，搅拌，冰水浴降温至5℃以下，加入碳酸钾(322g，2.34mol)。25℃搅拌反应14小时，TLC检测原料转化完全。
(2)反应液加入纯化水(300mL)，静置分层，分出有机相，水相经EtOAc(200mL×2)萃取两次，合并有机相，加入饱和食盐水(500mL)洗涤、无水Na 2SO 4干燥，过滤浓缩得淡黄色油状物NC-6-03(201g)。
MS(ESI):m/z[M+H] +理论值581.4，实测值581.3。
4、中间体NC-6的合成
Figure PCTCN2021122118-appb-000017
(1)将NC-6-03(23g，39.6mmol)溶于1,4-二氧六环(200mL)中，加入浓盐酸(40mL)，升温至60℃反应2小时，TLC检测原料基本消耗完毕。
(2)浓缩，再次加入1,4-二氧六环(200mL)浓缩，得白色固体粗品，将粗品加入到乙酸乙酯(200mL)中打浆2小时，抽滤，收集滤饼，50℃真空烘干得得白色固体化合物NC-6(22.6g，96.9％)。
(3)MS(ESI):m/z[M+H] +理论值413.2，实测值413.1。
5、中间体TO-25-01的合成
Figure PCTCN2021122118-appb-000018
(1)N 2氛围下，将NC-6(1.5g，3.6mmol)、HBTU(4.5g，12.0mmol)和DIEA(4.75g，36mmol)加入到DCM(50mL)中搅拌30分钟，随后滴加GN-17(6.4g，12.0mmol)与DIEA(4.75g，36mmol)的DCM(50mL)溶液，25℃搅拌16小时，LCMS检测，原料基本消耗完毕。
(2)加入DCM(100mL)稀释，向反应液中加入1N盐酸溶液(80mL×2)洗涤，合并有机相，经饱和碳酸氢钠(100mL)洗涤、饱和食盐水(100mL)洗涤、无水Na 2SO 4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH＝7/1)得白色固体化合物TO-25-01(4.3g，收率60％)。
(3)MS(ESI):m/z[M/2+H] +理论值980.0，实测值979.9。
6、中间体TO-25(1046靶头)的合成
Figure PCTCN2021122118-appb-000019
(1)将TO-25-01(4.3g，2.2mmol)溶于甲醇(80mL)中，加入10％钯碳(1.0g)，H 2置换三次，25℃搅拌2小时，LCMS检测，原料基本消失。
(2)过滤，浓缩，加入DCM(20mL)溶解，缓慢滴加至MTBE(300mL)中，搅拌析晶30分钟，抽滤，得到白色固体化合物TO-25(3.7g，收率90％)。
1H NMR(400MHz,DMSO-d 6)δ8.48(d,J＝5.6Hz,1H),8.06(t,J＝5.7Hz,2H),7.85(dd,J＝11.7,6.8Hz,6H),5.21(d,J＝3.3Hz,3H),4.95(dd,J＝11.2,3.3Hz,3H),4.53(d,J＝8.5Hz,3H),4.08-3.83(m,14H),3.75(p,J＝4.8Hz,5H),3.68-3.26(m,28H),3.21-2.95(m,14H),2.30(q,J＝7.9,6.7Hz,6H),1.94-1.78(m,,36H),1.41-1.38(m,12H)；
MS(ESI):m/z[1/2M+H] +理论值934.9，实测值934.8。
7、TP-25的(1046靶头连接siRNA的前体)合成
Figure PCTCN2021122118-appb-000020
(1)N 2氛围下，将TO-25(700mg，0.37mmol)溶于干燥DCM(10mL)中，加入DIEA(0.31mL，1.9mmol)，利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.19mL，0.74mmol)的干燥DCM(1mL)溶液，25℃反应30分钟，TLC检测，原料基本消失。
(2)加入饱和NaHCO 3(10mL)淬灭，DCM(10mL)稀释，分液，有机相用饱和NaHCO 3(10mL)溶液，饱和食盐水(10mL)洗涤，无水NaSO 4干燥，过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5％TEA/DCM碱化，DCM/MeOH/TEA＝15/1/0.1)得白色固体TP-25(405mg，收率53％)。
1H NMR(400MHz,DMSO-d 6)δ8.12(t,J＝6.0Hz,2H),7.98-7.75(m,7H),5.21(d,J＝3.4Hz,3H),4.96(dd,J＝11.2,3.4Hz,2H),4.54(d,J＝8.4Hz,2H),4.02(q,J＝5.3,4.5Hz,9H),3.95-3.83(m,3H),3.82-3.50(m,23H),3.40-3.26(m,4H),3.12-2.94(m,27H),2.76-2.59(m,7H),2.29(t,J＝6.7Hz,5H),2.11-1.78(m,38H),1.38(s,12H),1.16(d,J＝7.5Hz,12H)；
31P NMR(162MHz,DMSO-d6)δ147.97；
MS(ESI):m/z[1/2M+Na] +理论值1057.0，实测值1057.4。
三、GalNAc靶头1048的合成
按照以下方法，合成了TO26(1048靶头)及TP-26(1048靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-18-01的合成
Figure PCTCN2021122118-appb-000021
(1)N 2氛围下，将GC-2(20.1g，39.7mmol)溶于DCM(200mL)中，分批加入CDI(7.09g，73.7mmol)，25℃搅拌3小时，随后将N-Boc-乙二胺(7.0g，43.7mmol)和三乙胺(12.05g，119.1mmol)加入反应液中，反应16小时，LCMS检测显示原料消失。
(2)加入饱和碳酸氢钠溶液(200mL)淬灭，分液，水相经DCM(100mL×3)萃取，合并有机相并使用饱和氯化铵溶液(200mL)、饱和氯化钠溶液(200mL)洗涤，无水Na 2SO 4干燥，过滤浓缩获得粗制产品。粗产品经甲基叔丁基醚(100mL)洗涤，油状产物浓缩得白色固体化合物GN-18-01(24.43g，收率95.1％)。
MS(ESI):m/z[M+H] +理论值650.3，实测值650.5。
2、中间体GN-18的合成
Figure PCTCN2021122118-appb-000022
(1)将GN-18-01(45.52g，70mmol)分批加入到HCl/EtOAc溶液(2N，500mL)中，25℃搅拌2小时，LCMS检测显示原料消失。
(2)倾倒出溶剂，固体浓缩得粗制产品。粗产品经甲基叔丁基醚(200mL)打浆纯化，过滤，滤饼40℃真空干燥得白色固体GN-18(49.6g)。
MS(ESI):m/z[M+H] +理论值550.3，实测值550.5。
3、中间体TO-26-01的合成
Figure PCTCN2021122118-appb-000023
(1)N 2氛围下，将NC-6(1.5g，3.6mmol)、PyBOP(6.2g，12.0mmol)和DIEA(4.75g，36mmol)加入到DCM(50mL)中搅拌30分钟，随后滴加GN-18(6.6g，12.0mmol)与DIEA(4.75g，36mmol)的DCM(50mL)溶液，25℃搅拌16小时，LCMS检测，原料基本消耗完毕。
(2)加入DCM(100mL)稀释，向反应液中加入1N盐酸溶液(80mL×2)洗涤，合并有机相，经饱和碳酸氢钠(100mL)、饱和食盐水(100mL)洗涤、无水Na 2SO 4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH＝7/1)得白色固体化合物TO-26-01(4.7g，收率65％)。
MS(ESI):m/z[M/2+H] +理论值1004.0，实测值1004.2。
4、中间体TO-26(1048靶头)的合成
Figure PCTCN2021122118-appb-000024
(1)将TO-26-01(4.0g，2.0mmol)溶于甲醇(80mL)中，加入10％钯碳(1.0g)，H 2置换三次，25℃搅拌2小时，LCMS检测，原料基本消失。
(2)过滤，浓缩，加入DCM(20mL)溶解，缓慢滴加至MTBE(200mL)中，搅拌析晶30分钟，抽滤，得到白色固体化合物TO-26(3.5g，收率91％)。
1H NMR(400MHz,DMSO-d 6)δ8.54(s,1H),8.14(s,2H),7.95-7.92(m,3H),7.84(d,J＝7.8Hz,3H),5.21(d,J＝3.4Hz,3H),4.97(dd,J＝11.2,3.4Hz,3H),4.54(d,J＝8.5Hz,3H),4.13-3.66(m,21H),3.60-3.44(m,37H),3.14(d,J＝13.8Hz,15H),2.31(t,J＝6.4Hz,6H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H).
MS(ESI):m/z[1/2M+H] +理论值958.9，实测值959.1。
5、TP-26(1048靶头连接siRNA的前体)的合成
Figure PCTCN2021122118-appb-000025
(1)N 2氛围下，将TO-26(900mg，0.47mmol)溶于干燥DCM(12mL)中，加入DIEA(0.39mL，0.44mmol)，利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(277mg，1.17mmol)的干燥DCM(1mL)溶液，25℃反应30分钟，TLC检测，原料基本消失。
(2)加入饱和NaHCO 3(10mL)淬灭，DCM(10mL)稀释，分液，有机相用饱和NaHCO 3(10mL)溶液，饱和食盐水(10mL)洗涤，无水NaSO 4干燥，过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5％TEA/DCM碱化，DCM/MeOH/TEA＝15/1/0.1)得白色固体TP-26(600mg，收率60％)。
1H NMR(400MHz,DMSO-d6) 1H NMR(400MHz,DMSO-d 6)δ8.15(s,2H),7.94-7.81(m,7H),5.22(d,J＝3.4Hz,3H),4.97(dd,J＝11.2,3.4Hz,3H),4.55(d,J＝8.5Hz,3H),4.03(s,8H),3.88(dt,J＝11.2,8.9Hz,3H),3.81-3.67(m,7H),3.64-3.46(m,30H),3.11(d,J＝13.1Hz,19H),2.76(t,J＝5.9Hz,3H),2.65-2.54(m,7H),2.31(t,J＝6.6Hz,7H),2.11(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H),1.13(d,J＝6.8,12H). 31P NMR(162MHz,DMSO-d6)δ147.89；
MS(ESI):m/z 1/2[M-i-Pr 2N]理论值1007.9，实测值1008.2。
实施例3、体外构建偶联GalNAc靶头偶联的(修饰的)siRNA缀合物
以下RNAi剂双链体的反义链和正义链的寡核苷酸序列部分，以及靶向性配体与RNA的连接，均按照J.Org.Chem.2012,77,4566-4577；Curr.Protoc.Nucleic Acid Chem.,81,e107报道的亚磷酰胺偶联技术在用于寡核苷酸合成的固相上合成。靶向性配体1046、1048、1043均通过硫代磷酸酯键与siRNA正义链5’末端连接。
合成的GalNAc-siRNA缀合物如图3中表格所述，表中第二列的的缀合物结构包括三部分，例如，G1043-S2A2-A265的结构为：1043靶头通过硫代磷酸酯键与编号为A265的siRNA正义链5’末端连接，S2A2为对A265的siRNA的修饰类型，具体的修饰基团和修饰方式为：
核酸序列中，Ao表示腺嘌呤核苷，Uo表示尿嘧啶核苷，Go表示鸟嘌呤核苷，Co表示胞嘧啶核苷，直接相邻的核苷酸之间没有符号，表示以正常的磷酸酯键连接。
DNA：A G C T(A表示2’-脱氧腺嘌呤核苷，T表示2’-脱氧胸腺嘧啶核苷，G表示2’-脱氧鸟嘌呤核苷，C表示2’-脱氧胞嘧啶核苷)；
2'-F：aF gF cF uF(aF表示2’-氟代腺嘌呤核苷，uF表示2’-氟代尿嘧啶核苷，gF表示2’-氟代鸟嘌呤核苷，cF表示2’-氟代胞嘧啶核苷)；
2'-OMe：aM gM cM uM(aM表示2’-O-甲基腺嘌呤核苷，uM表示2’-O-甲基尿嘧啶核苷，gM表示2’-O-甲基鸟嘌呤核苷，cM表示2’-O-甲基胞嘧啶核苷)；
*：表示以硫代磷酸酯键连接；
序列中的y、z代表靶头的位置。
实施例4：体外细胞模型(Hep 3B细胞)测试缀合物的活性
人肝癌Hep3B细胞(中国科学院上海细胞库)，培养于添加10％胎牛血清(FBS)(Gibco，US)的DMEM(Gibco，US)中，于37℃,5％CO2条件下培养(il60,Thermo Fisher)。转染实验进行当天，使用0.25％Trysin(Gibco，US)消化细胞，计数并以450μL/孔、5万/孔的密度接种于24孔板。随后，以lipofectmine2000(Thermo Fisher)转染方式加入受试样品。按RNAiMAX试剂说明说书标准流程转染，siRNA终浓度为10nM/1nM/0.5nM/0.25nM/0.1nM/0.05nM/0.01nM。转染组以siNC为阴性对照，其序列为：
正义链(sense)：5’-UUCUCCGAACGUGUCACGUTT-3’
反义链(antisense)：5’-ACGUGACACGUUCGGAGAATT-3’。
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ANGPTL3 mRNA序列的表达情况，其中用于扩增内参基因PPIB、ANGPTL3的PCR引物如表1所示：
各个缀合物的活性测试结果(EC 50值)见图4。
EC 50值利用graphpad prism的非线性回归计算，表示抑制一半目标mRNA(ANGPTL3)表达量时的缀合物的用量。
结果可见，选取的缀合物在体外活性测试的实验中，表现出良好的降低ANGPTL3相对表达水平的结果。
实施例5：AAV-hANGPTL3小鼠模型的构建及给药测试
实验动物基本信息：
实验动物购自济南朋悦实验动物繁育有限公司，为SPF级动物。给药前对上述小鼠称重并观察状态，选取体重均一、状态无异常的动物进行后续实验。
种属 性别 年龄 体重 来源
C57小鼠 雄性 4周 20±2g 济南朋悦
饲养条件：非SPF级饲养条件。正常饲养条件下动物自由进食饮水。动物购进后，进行3-7天适应性培养后开始实验。
造模及给药：每只小鼠通过尾静脉注射2.5*10^11滴度的病毒溶液，100ul。7天后，对实验动物进行随机分组，每个受试物按5mg/kg的剂量进行皮下给药。给药后72小时，颈椎脱臼牺牲动物，取肝组织，进行RNA的提取与定量。
各个缀合物的结果见表3。
表3、各个缀合物的小鼠模型给药测试结果
Figure PCTCN2021122118-appb-000026
Figure PCTCN2021122118-appb-000027
结果可见，选取的缀合物在体内活性测试的实验中，也表现出良好的降低ANGPTL3相对表达水平的结果。
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公开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须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公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (12)

  1. 一种siRNA，其特征在于，所述siRNA包括一条正义链和一条反义链，所述反义链包括与所述正义链互补配对的互补性区域，其中所述正义链选自与SEQ ID NO:1～SEQ ID NO:154中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列，所述反义链选自与SEQ ID NO:155～SEQ ID NO:308中每一条链的核苷酸序列区别不多于5个核苷酸的核苷酸序列。
  2. [根据细则91更正 15.12.2021]
    根据权利要求1所述的siRNA，其特征在于，所述siRNA选自如下任一组中的任一对siRNA：
    (1)能够特异性的靶向血管生成素样蛋白3序列的第60-80位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:10，反义链选自SEQ ID NO:165；
    (2)能够特异性的靶向血管生成素样蛋白3序列的第107-133位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:17，反义链选自SEQ ID NO:171，或者所述siRNA的正义链选自SEQ ID NO:18，反义链选自SEQ ID NO:172；
    (3)能够特异性的靶向血管生成素样蛋白3序列的第163-187位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:19，反义链选自SEQ ID NO:173；
    (4)能够特异性的靶向血管生成素样蛋白3序列的第304-388位核苷酸，优选的，能够特异性的靶向血管生成素样蛋白3序列的第304-359位核苷酸；更优选的，所述的siRNA的正义链选自SEQ ID NO:27，反义链选自SEQ ID NO:181，
    或者，所述的siRNA的正义链选自SEQ ID NO:29，反义链选自SEQ ID NO:183，
    或者，所述的siRNA的正义链选自SEQ ID NO:31，反义链选自SEQ ID NO:185，
    或者，所述的siRNA的正义链选自SEQ ID NO:32，反义链选自SEQ ID NO:186，
    或者，所述的siRNA的正义链选自SEQ ID NO:35，反义链选自SEQ ID NO:189，
    或者，所述的siRNA的正义链选自SEQ ID NO:36，反义链选自SEQ ID NO:190；
    (5)能够特异性的靶向血管生成素样蛋白3序列的第430-459位核苷酸；优选的，所述的siRNA的正义链选自SEQ ID NO:43，反义链选自SEQ ID NO:197，
    或者，所述的siRNA的正义链选自SEQ ID NO:44，反义链选自SEQ ID NO:198；
    (6)能够特异性的靶向血管生成素样蛋白3序列的第1360-1430位核苷酸，优选的，能够特异性的靶向血管生成素样蛋白3序列的第1397-1430位核苷酸；更优选的，所述的siRNA的正义链选自SEQ ID NO:145，反义链选自SEQ ID NO:299，
    或者，所述的siRNA的正义链选自SEQ ID NO:150，反义链选自SEQ ID NO:304，
    或者，所述的siRNA的正义链选自SEQ ID NO:151，反义链选自SEQ ID NO:305，
    或者，所述的siRNA的正义链选自SEQ ID NO:152，反义链选自SEQ ID NO:306，
    或者，所述的siRNA的正义链选自SEQ ID NO:154，反义链选自SEQ ID NO:308。
  3. 根据权利要求1或2所述的siRNA，其特征在于，所述siRNA包括至少一个被修饰的核苷酸；
    任选地，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。
  4. 根据权利要求1或2所述的siRNA，其特征在于，所述互补性区域的长度至少为17bp；
    任选地，所述互补性区域的长度为18～21bp；
    任选地，所述互补性区域的长度为19bp。
  5. 一种siRNA缀合物，其特征在于，所述siRNA缀合物包括权利要求1～4中任一项所述的siRNA和靶向配体，其中，所述siRNA与所述靶向配体共价连接；
    优选的，所述靶向配体连接于所述siRNA中正义链；
    更优选的，所述靶向配体通过硫代磷酸酯键与所述siRNA中正义链的5’端连接。
  6. 根据权利要求5所述的siRNA缀合物，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；
    优选的，所述靶向配体为GalNAC靶头化合物；
    更优选的，所述GalNAC靶头化合物为1043、1046、1048，
    Figure PCTCN2021122118-appb-100001
  7. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1～4中任一项所述的siRNA和/或权利要求5或6所述的siRNA缀合物，任选地，所述药物组合物还包括药学上可接受的辅料。
  8. 一种抑制受试者ANGPTL3基因表达的方法，其特征在于，所述方法包括：向受试者施用权利要求1～4中任一项所述的siRNA和/或权利要求5或6所述的siRNA缀合物，以抑制ANGPTL3基因的表达。
  9. 一种抑制细胞中ANGPTL3基因表达的方法，其特征在于，所述方法包括：用权利要求1～4中任一项所述的siRNA和/或权利要求5或6所述的siRNA缀合物转染所述细胞，以抑制所述细胞中ANGPTL3基因的表达。
  10. 权利要求1～4中任一项所述的siRNA和/或权利要求5或6所述的siRNA缀合物在制备药物或试剂盒中的用途，其特征在于，所述药物或试剂盒用于抑制ANGPTL3基因表达。
  11. 根据权利要求10所述的用途，其特征在于，所述药物或试剂盒用于预防和/或治疗血脂异常疾病；
    任选地，所述血脂异常疾病包括高血脂症和高甘油三酯血症；
    任选地，所述药物或试剂盒用于抑制细胞中ANGPTL3基因表达。
  12. 一种预防和/或治疗血脂异常疾病的方法，其特征在于，所述方法包括向受试者施用权利要求1～4中任一项所述的siRNA和/或权利要求5或6所述的siRNA缀合物；
    任选地，所述血脂异常疾病包括高血脂症和高甘油三酯血症。
PCT/CN2021/122118 2020-09-30 2021-09-30 血管生成素样3(ANGPTL3)的siRNA及其用途 WO2022068923A1 (zh)

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