siRNA for angiopoietin-like 3 (ANGPTL3) and its uses

Abstract

The present invention relates to the field of genetic engineering, specifically to angiopoietin-like 3 (ANGPTL3) siRNA and its use. The inventors of the present invention have designed an appropriate specific small interfering RNA sequence and siRNA conjugate to target ANGPTL3 and degrade the transcript of the ANGPTL3 gene in cells, thereby reducing the expression level of ANGPTL3 protein, and thus the siRNA according to the present invention is useful for the prevention and / or treatment of dyslipidemia.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and benefit of a patent application filed at the China Patent Office on September 30, 2020, bearing application number 202011061038.1, a patent application filed at the China Patent Office on January 5, 2021, bearing application number 202110008013.3, and a patent application filed at the China Patent Office on April 13, 2021, bearing application number 202110397429.9, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of genetic engineering technology; specifically, the present invention relates to siRNA for angiopoietin-like 3 (ANGPTL3) and its use. [Background technology]

[0003] Hyperlipidemia, also known as dyslipidemia, is a systemic disease in which plasma lipids are higher than normal due to abnormal fat metabolism or driving. Clinical symptoms of dyslipidemia can be broadly divided into (1) xanthomas caused by intradermal deposition of lipids, and (2) atherosclerosis, coronary heart disease, and peripheral vascular disease caused by endothelial deposition of lipids. Hyperlipidemia is not uncommon in Japan, and according to a survey, the increase in total blood cholesterol (TC) or triglyceride (TG) in adults is about 10% to 20%, and hyperlipidemia is seen in about 10% of children. It is said that the increase in serum cholesterol levels in the general population is closely related to the increase in cardiovascular disease cases in Japan to about 9.2 million between 2010 and 2030, the remarkable increase in the standard of living of the people in Japan, and changes in eating habits. Conventional drugs for treating dyslipidemia were mainly statins, cholesterol absorption inhibitors, resins, probenazoles, fibrates, and nicotinic acid and its derivatives.

[0004] After using traditional therapeutic drugs, there are more or less contraindications and side effects. For example, statin drugs are currently the main drugs commonly used to lower serum total cholesterol, and drugs that treat simple elevation of serum total cholesterol level are also used to treat diseases that mainly involve elevation of serum total cholesterol level and slight elevation of serum triacylglycerol level. Such drugs mainly include lovastatin (Metaplateau), simvastatin (Losplateau), pravastatin (Plateau), fluvastatin (LaiShiKe), atorvastatin (Rivastatin), and simvastatin (Baystachyon) etc. For example, over a long period of administration, there are abdominal distension, diarrhea, constipation, headache, insomnia, rash, thrombotic thrombocytopenic purpura (diffuse congestion in face, chest, extremities, accompanied by a decrease in platelet count). It also causes mental depression, paresthesia, and numbness, burning sensation, skin allergy, or pain in the face, scalp, tongue, and limbs. It also causes shedding and an increase in serum transaminases. The worst side effect is rhabdomyolysis, which manifests as muscle weakness, muscle pain, urination, and an increase in serum creatine kinase levels, with an incidence of about 1‰. If the drug is not discontinued in a timely manner, severe myopathy may occur and even lead to renal insufficiency.

[0005] Therefore, there is an urgent need for the development of a drug for treating dyslipidemia that can be taken for a long period of time and has few side effects. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to solve at least to some extent one of the technical problems in related art.To this end, one of the objectives of the present invention is to provide siRNA for inhibiting the expression of ANGPTL3, and the inventors of the present invention design suitable specific small interfering RNA sequence and siRNA conjugate to target ANGPTL3, and degrade the transcript of ANGPTL3 gene in cells, thereby reducing the expression level of ANGPTL3 protein, so that the siRNA according to the present invention is useful for preventing and / or treating dyslipidemia. [Means for solving the problem]

[0007] To this end, one aspect of the present invention provides an siRNA, according to an embodiment of the present invention, the siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region that complementarily pairs with the sense strand, the sense strand being selected from a nucleotide sequence that differs by 5 nucleotides or less from the nucleotide sequence of each strand of SEQ ID NO:1 to SEQ ID NO:154, and the antisense strand being selected from a nucleotide sequence that differs by 5 nucleotides or less from the nucleotide sequence of each strand of SEQ ID NO:155 to SEQ ID NO:308.

[0008] Angiopoietin-like protein 3 (ANGPTL3, NM_014495.4) is a secreted protein that is mainly expressed in liver cells. Studies have shown that angiopoietin-like protein 3 (ANGPTL3) is an important regulator of LDL-C, HDL-C and triglyceride metabolism, with various potential action nodes, and loss-of-function mutations in ANGPTL3 result in lowering of LDL-C, VLDL-C, HDL-C and triglycerides (TG), lowering the risk of cardiovascular disease based on GWAS, and without any known adverse phenotypes of genetic defects. Therefore, inhibition of ANGPTL3 activity is effective in preventing or treating dyslipidemia. The inventors of the present invention specifically reduce the synthesis of ANGPTL3 in liver cells by designing an appropriate small interfering RNA (siRNA) sequence, while avoiding the detargeting effect. siRNA forms a silencing complex (RNA-induced silencing complex: RISC), complementarily pairs with the mRNA sequence of the target gene (ANGPTL3 gene), degrades the mRNA of the target gene, inhibits the expression of the target gene, and further reduces the levels of LDL-C, VLDL-C, HDL-C, and triglyceride (TG).

[0009] The siRNA according to the embodiments of the present invention may further have at least one of the following additional technical features:

[0010] The present invention further provides an siRNA, the siRNA being selected from any pair of siRNAs in any of the following groups: (1) capable of specifically targeting nucleotides 60-80 of the angiopoietin-like protein 3 sequence, preferably, the sense strand of the siRNA is selected from SEQ ID NO: 10 and the antisense strand is selected from SEQ ID NO: 165; (2) A siRNA capable of specifically targeting nucleotides 107-133 of the angiopoietin-like protein 3 sequence, wherein preferably the sense strand of the siRNA is selected from SEQ ID NO: 17 and the antisense strand is selected from SEQ ID NO: 171, or the sense strand of the siRNA is selected from SEQ ID NO: 18 and the antisense strand is selected from SEQ ID NO: 172; (3) capable of specifically targeting nucleotides 163-187 of the angiopoietin-like protein 3 sequence, preferably, the sense strand of the siRNA is selected from SEQ ID NO: 19 and the antisense strand is selected from SEQ ID NO: 173; (4) A siRNA capable of specifically targeting nucleotides 304-388 of the angiopoietin-like protein 3 sequence, preferably nucleotides 304-359 of the angiopoietin-like protein 3 sequence, more preferably wherein the sense strand of the siRNA is selected from SEQ ID NO: 27 and the antisense strand is selected from SEQ ID NO: 181; Or, the sense strand of the siRNA is selected from SEQ ID NO: 29 and the antisense strand is selected from SEQ ID NO: 183; Or, the sense strand of the siRNA is selected from SEQ ID NO: 31 and the antisense strand is selected from SEQ ID NO: 185; Or, the sense strand of the siRNA is selected from SEQ ID NO: 32 and the antisense strand is selected from SEQ ID NO: 186; Or, the sense strand of the siRNA is selected from SEQ ID NO: 35 and the antisense strand is selected from SEQ ID NO: 189; or the sense strand of the siRNA is selected from SEQ ID NO: 36 and the antisense strand is selected from SEQ ID NO: 190; (5) A siRNA capable of specifically targeting nucleotides 430-459 of the angiopoietin-like protein 3 sequence, wherein the sense strand of the siRNA is selected from SEQ ID NO: 43 and the antisense strand is selected from SEQ ID NO: 197; or the sense strand of the siRNA is selected from SEQ ID NO: 44 and the antisense strand is selected from SEQ ID NO: 198; (6) A siRNA capable of specifically targeting nucleotides 1360-1430 of the angiopoietin-like protein 3 sequence, preferably nucleotides 1397-1430 of the angiopoietin-like protein 3 sequence, more preferably wherein the sense strand of the siRNA is selected from SEQ ID NO: 145 and the antisense strand is selected from SEQ ID NO: 299; Or, the sense strand of the siRNA is selected from SEQ ID NO: 150 and the antisense strand is selected from SEQ ID NO: 304; Or, the sense strand of the siRNA is selected from SEQ ID NO: 151 and the antisense strand is selected from SEQ ID NO: 305; Or, the sense strand of the siRNA is selected from SEQ ID NO: 152 and the antisense strand is selected from SEQ ID NO: 306; Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:154 and the antisense strand is selected from SEQ ID NO:308.

[0011] According to an embodiment of the invention, the siRNA comprises at least one modified nucleotide, Optionally, the modified nucleotide is: The modified nucleotide is selected from at least one of a 5'-phosphorothioated nucleotide, a 5-methylated cytosine nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-2-methoxyethyl modified nucleotide, a 2'-fluoro modified nucleotide, a 3'-nitrogen substituted modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a morpholino nucleotide, a polypeptide nucleotide, a phosphoramidate, and a nucleotide comprising a non-natural base.

[0012] According to an embodiment of the invention, the length of the complementary region is at least 17 bp, Optionally, the length of the complementary region is 18-21 bp; Optionally, the length of the complementary region is 19 bp.

[0013] According to an embodiment of the present invention, the length of the sense strand and the antisense strand in the siRNA is 25 bp or less; Optionally, the length of the sense strand and the antisense strand in the siRNA is 18 to 25 bp; Optionally, the length of the sense strand and the antisense strand in the siRNA is 21 bp.

[0014] According to an embodiment of the present invention, the bases of the sense strand and the antisense strand in the siRNA may be complementary paired one-to-one, or may be shifted by several bases but have a complementary region of at least 17 bp.

[0015] Another aspect of the invention provides a siRNA conjugate, comprising the siRNA described above and a targeting ligand, wherein the siRNA is covalently linked to the targeting ligand; Preferably, the targeting ligand is linked to the sense strand of the siRNA; More preferably, the targeting ligand is linked to the 5' end of the sense strand in the siRNA via a phosphorothioate bond.

[0016] According to an embodiment of the present invention, the targeting ligand comprises at least one N-acetyl-galactosamine.

[0017] According to an embodiment of the invention, the targeting ligand is a GalNAC targeting head compound.

[0018] According to an embodiment of the present invention, the GalNAC targeting head compounds are 1043, 1046, and 1048, the structures of which are shown in Formula 1-3 below.

[0019] [ka] GalNAC target head 1043

[0020] [ka] GalNAC target head 1046

[0021] [ka] GalNAC targeting head 1048

[0022] According to an embodiment of the present invention, the targeting ligand is linked to a sense strand of the siRNA.

[0023] Another aspect of the present invention provides a pharmaceutical composition, according to an embodiment of the present invention, comprising the siRNA and / or the siRNA conjugate, and optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0024] As a result, the pharmaceutical compositions according to embodiments of the present invention can be used to prevent and / or treat hyperlipidemia and hypertriglyceridemia by inhibiting the synthesis of ANGPTL3 in cells, thereby reducing LDL-C, VLDL-C, HDL-C and triglyceride (TG) levels.

[0025] Another aspect of the present invention provides a kit, according to an embodiment of the present invention, comprising the siRNA and / or siRNA conjugate.

[0026] As a result, the kit according to an embodiment of the present invention can be used to prevent and / or treat hyperlipidemia and hypertriglyceridemia by inhibiting expression of the ANGPTL3 gene in cells, thereby reducing LDL-C, VLDL-C, HDL-C and triglyceride (TG) levels.

[0027] Another aspect of the present invention provides a method for inhibiting expression of the ANGPTL3 gene in a subject, the method comprising administering to the subject the siRNA and / or the siRNA conjugate to inhibit expression of the ANGPTL3 gene.

[0028] Another aspect of the present invention provides a method for inhibiting expression of the ANGPTL3 gene in a cell. According to an embodiment of the present invention, the method comprises transfecting the cell with the siRNA and / or the siRNA conjugate to inhibit expression of the ANGPTL3 gene in the cell.

[0029] A method for inhibiting expression of the ANGPTL3 gene in a cell according to an embodiment of the present invention uses siRNA to form a silencing complex, which complementarily pairs with 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, and further reduces the levels of LDL-C, VLDL-C, HDL-C and triglyceride (TG).

[0030] According to an embodiment of the invention, the cell is derived from a mammal, Optionally, the cell is derived from a human, Optionally, the cell is a liver cell.

[0031] The siRNA of the present invention forms a silencing complex in human liver cells, complementarily pairs with the sequence of the mRNA of the ANGPTL3 gene, degrades the mRNA of the ANGPTL3 gene, inhibits its expression, and further reduces the levels of LDL-C, VLDL-C, HDL-C, and triglyceride (TG).

[0032] Another aspect of the present invention provides a use of the siRNA and / or the siRNA conjugate for the manufacture of a drug or kit. According to an embodiment of the present invention, the drug or kit is used for inhibiting the expression of the ANGPTL3 gene.

[0033] The siRNA according to the present invention is used to produce a drug or kit, which prevents and / or treats dyslipidemia by reducing the expression level of the ANGPTL3 gene in cells using the siRNA.

[0034] According to an embodiment of the present invention, the medicament or kit is used for the prevention and / or treatment of dyslipidemia, Optionally, the dyslipidemia comprises hyperlipidemia and hypertriglyceridemia; Optionally, the agent or kit is used to inhibit expression of the ANGPTL3 gene in a cell.

[0035] Another aspect of the present invention provides a method for preventing and / or treating dyslipidemia, according to an embodiment of the present invention, the method comprising administering the siRNA and / or the siRNA conjugate to a subject.

[0036] According to an embodiment of the present invention, the dyslipidemia includes hyperlipidemia and hypertriglyceridemia.

[0037] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief description of the drawings]

[0038] The above and / or additional aspects and advantages of the present invention will become apparent and will be readily understood from the following detailed description of the embodiments, which are given with reference to the following drawings, in which: [Figure 1] The results show that some of the siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 0.1 nM, and then the expression level of the ANGPTL3 gene (abbreviated as ANL3 in the drawings) in the cells was detected using real-time quantitative PCR. [Diagram 2] The results show that some of the siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 10 nM, and then the expression level of the ANGPTL3 gene (abbreviated as ANL3 in the drawings) in the cells was detected using real-time quantitative PCR. [Diagram 3] 1 shows the GalNAc-siRNA conjugate synthesized in Example 3. [Figure 4] 1 shows the activity test results (EC50 value) of each conjugate in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The following examples of the present invention are described in detail. The following examples are illustrative and are used only to explain the present invention, but should not be construed as limiting the present invention.

[0040] A "pharmaceutically acceptable carrier" is known in the art and includes any pharma- ceutically acceptable material, composition, or carrier suitable for administration of a compound of the invention to a mammal. The carrier includes liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that are involved in carrying the host material or transferring the host material 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 other ingredients in the formulation and not harmful to the patient. Some examples of materials useful as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as tragacanth powder, malt, gelatin, talc, cocoa butter, and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyhydric alcohols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar-agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, Ringer's solution, ethanol, phosphate buffers, and other non-toxic compatible substances used in pharmaceutical formulations.

[0041] In the compositions, wetting agents, emulsifying agents, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants may also be present.

[0042] The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with carrier materials to prepare a single dosage form is generally the amount of compound that performs a therapeutic function. Generally, in hundredths, this amount is about 1% to about 99%, preferably about 5% to about 70%, and most preferably about 10% to about 30% of active ingredient.

[0043] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of adverse effects resulting from a partial or complete cure of a disease. As used herein, "treatment" covers mammalian, particularly human, diseases, including (a) prevention of the onset of a disease or condition in an individual susceptible to the disease but in whom the disease has not yet been definitively diagnosed, (b) inhibition of the disease, e.g., delaying the progression of the disease, or (c) alleviation of the disease, e.g., alleviation of symptoms associated with the disease. As used herein, "treatment" covers any use of administering a drug or compound to an individual to treat, cure, ameliorate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administering a drug, including a compound as described herein, to an individual in need thereof.

[0044] The present invention provides an siRNA for inhibiting the expression of ANGPTL3. According to an embodiment of the present invention, the siRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region that complementarily pairs with the sense strand, wherein the sense strand is selected from a nucleotide sequence that differs from the nucleotide sequence of each strand of SEQ ID NO:1 to SEQ ID NO:154 by 5 nucleotides or less, and the antisense strand is selected from a nucleotide sequence that differs from the nucleotide sequence of each strand of SEQ ID NO:155 to SEQ ID NO:308 by 5 nucleotides or less.

[0045] According to an embodiment of the present invention, the sense strand, in addition to including SEQ ID NO:1 to SEQ ID NO:154 shown in Table 2, further includes a contiguous nucleotide sequence that differs from the sense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0046] According to an embodiment of the present invention, the antisense strand, in addition to including SEQ ID NO:155 to SEQ ID NO:308 shown in Table 2, further includes a consecutive nucleotide sequence that differs from the antisense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0047] According to an embodiment of the invention, the siRNA comprises at least one modified nucleotide, The modified nucleotide is The modified nucleotide is selected from at least one of a 5'-phosphorothioated nucleotide, a 5-methylated cytosine nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-2-methoxyethyl modified nucleotide, a 2'-fluoro modified nucleotide, a 3'-nitrogen substituted modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a morpholino nucleotide, a polypeptide nucleotide, a phosphoramidate, and a nucleotide comprising a non-natural base.

[0048] According to an embodiment of the present invention, the length of the complementary region is 18-21 bp, for example, 19 bp.

[0049] According to an embodiment of the present invention, the length of the sense strand and the antisense strand in the siRNA is 18 to 25 bp, for example, 21 bp.

[0050] According to a specific embodiment of the present invention, 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 paired one-to-one, or the sense strand and the antisense strand in the siRNA have 19 consecutive complementary bases, i.e., the length of the complementary region is 19 bp.

[0051] According to an embodiment of the present invention, the siRNA is used to transfect liver cells to inhibit the expression of the ANGPTL3 gene in the cells.

[0052] For the angiopoietin-like 3 (ANGPTL3) gene target, the inventors of the present invention designed an appropriate specific small interfering nucleic acid (siRNA) sequence, synthesized the siRNA, and introduced the siRNA into cells using a transfection reagent to form a silencing complex (RNA-induced silencing complex: RISC), which specifically recognizes and binds to the mRNA sequence of the target gene, and cleaves the mRNA between the clips at positions 10-11 from the 5' end, thereby causing post-transcriptional gene silencing and regulating the expression of angiopoietin-like 3 secreted protein.

[0053] According to an embodiment of the invention, the siRNA is covalently linked to a targeting ligand.

[0054] According to an embodiment of the present invention, the targeting ligand comprises at least one N-acetyl-galactosamine.

[0055] According to an embodiment of the present invention, the targeting ligand is linked to a sense strand of the siRNA.

[0056] The following examples of the present invention are described in detail. The examples described below are illustrative and are only for the purpose of interpreting the present invention, and should not be understood as limiting the present invention. In the examples, specific techniques and conditions are not specified, and are carried out according to techniques and conditions described in the literature in the relevant technical field, or according to product specifications. Reagents or equipment used that are not specified by manufacturer are all commercially available and ordinary.

[0057] Some synthetic routes of this embodiment may refer to CN202110397429.9, CN202110008013.3. The embodiments of this application are incorporated by source into the above two patent applications.

[0058] Example 1 Testing the activity of small interfering nucleic acids (siRNAs) in an in vitro cell model (Hep 3B cells)

[0059] 1) Preparation of suspended transfection reagent: The concentration of siRNA mother solution was 50μM, which was diluted with DEPC water to obtain 10μM siRNA system, and diluted with 50μl Opti-MEM to obtain 0.2μM siRNA system, and mixed uniformly by blowing 3-5 times (final concentration 10nM). 0.5ul 0.2μM SiRNA was diluted with 50μl Opti-MEM to obtain 0.002μM siRNA system, and mixed uniformly by blowing 3-5 times (final concentration 0.1nM). 2μl RNAiMAX was diluted with 50μl Opti-MEM, and mixed uniformly by blowing 3-5 times. Transfection reagent and small interfering nucleic acid dilution solution were mixed respectively, and mixed uniformly by blowing 3-5 times, and left at room temperature for 10min.

[0060] 2) Cell treatment: Hep3B cell line confluence rate >70% was observed by microscope, cells were plated, 2 × 10 5The cells were plated in a 12-well plate at 10% FBS-containing DMEM medium (900 μl) was added to each well, and the transfection complex was added to the 12-well plate and cultured at 37° C. in a 5% CO 2 incubator.

[0061] 3) After 24 hours, the total RNA of the cells was extracted and the expression status of the ANGPTL3 mRNA sequence in the cells was detected by quantitative real-time PCR. The PCR primers used to amplify the internal control genes PPIB and ANGPTL3 are shown in Table 1.

[0062] Table 1: PCR primer sequences used to amplify the internal control genes PPIB and ANGPTL3. JPEG0007607957000004.jpg112144

[0063] 4) The inhibition rate of small interfering nucleic acid against ANGPTL3 expression level is calculated according to the following formula: Inhibition rate = [1-(Experimental group ANGPTL3 mRNA expression amount / Experimental group PPIB mRNA expression amount) / (Negative control group ANGPTL3 mRNA expression amount / Negative control group PPIB mRNA expression amount)] x 100%. Here, each experimental group is a cell treated with a small interfering nucleic acid, and the negative control group (referred to as Blank) is a cell not treated with any small interfering nucleic acid.

[0064] Using the above method, the results of inhibition rate on the expression of ANGPTL3 gene (NM_014495.4) in Table 2 are obtained after transfecting Hep 3B cells with 154 pairs of siRNAs at concentrations of 0.1 nM and 10 nM, respectively.

[0065] Table 2: 154 pairs of siRNA sequences targeting ANGPTL3 JPEG0007607957000005.jpg182143JPEG0007607957000006.jpg177143JPEG0007607957000007.jpg177143JPEG00076079570 00008.jpg177143JPEG0007607957000009.jpg177143JPEG0007607957000010.jpg177143JPEG0007607957000011.jpg138143

[0066] Figures 1 and 2 respectively show the results of the expression level of ANGPTL3 gene in Hep 3B cells detected by real-time quantitative PCR after transfecting the cells with some siRNAs in Table 2 at a concentration of 0.1 nM or 10 nM. It is clear that the siRNAs shown in the figures can significantly reduce the expression of ANGPTL3 gene when transfecting Hep 3B cells at either a concentration of 0.1 nM or 10 nM.

[0067] Example 2. Synthesis of GalNAc-linked targeting head

[0068] 1. Synthesis of GalNAc targeting head 1043 Diastereoisomers of TO-23 and TP-23 (precursors of 1043-targeting head-binding siRNA) were synthesized according to the following method.

[0069] 1. Synthesis of intermediate GN-17-01

[0070] JPEG0007607957000012.jpg15135

[0071] (1) In a N2 atmosphere, GC-1 (12 g, 25.89 mmol) was dissolved in DCM (200 mL), the temperature was lowered to 0-5 °C in an ice-water bath, HBTU (11.78 g, 31 mmol) and DIEA (10 g, 77.67 mmol) were added, and the mixture was stirred for 10 minutes.

[0072] (2) Next, N-tert-butoxycarbonyl-1,4-butanediamine (4.87 g, 25.89 mmol) was added, the temperature was raised to 25° C., and the reaction was carried out with stirring for 16 hours. It was confirmed by TLC that the raw materials had substantially disappeared.

[0073] (3) The reaction was quenched by adding saturated ammonium chloride solution (100 mL), separated, and extracted with DCM (100 mL x 2).

[0074] (4) The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated. Purification by column chromatography (DCM / MeOH = 20 / 1) gave the white solid compound GN-17-01 (15 g, yield 91%).

[0075] 2. Synthesis of intermediate GN-17

[0076] JPEG0007607957000013.jpg15135

[0077] (1) GN-17-01 (15 g, 23.67 mmol) was dissolved in DCM (150 mL), TFA (50 mL) was added, and the mixture was stirred at 25° C. for 1 hour. After confirming that the raw materials had essentially disappeared by TLC, the mixture was concentrated.

[0078] (2) Excess TFA was removed by azeotropic distillation with acetonitrile (100 mL × 3) and TFA to obtain foam-like solid GN-17 (TFA salt, 12.6 g).

[0079] 3. Synthesis of intermediate TO-23-01

[0080] JPEG0007607957000014.jpg42129

[0081] (1) In a N2 atmosphere, NC-4 (2.6 g, 4.7 mmol) was dissolved in DCM (200 mL), the temperature was lowered to 0-5 °C in an ice-water bath, HATU (5.6 g, 14.83 mmol) and DIEA (4.85 g, 37.6 mmol) were added, and the mixture was stirred for 20 min.

[0082] (2) Next, GN-17 (8.45 g, 15.5 mmol) was added, the temperature was raised to 25° C., and the reaction was carried out with stirring for 4 hours. TLC detection confirmed that the raw materials had substantially disappeared.

[0083] (3) The reaction was quenched by adding saturated ammonium chloride solution (50 mL), separated, and extracted with DCM (100 mL x 2).

[0084] (4) The organic phase was combined, washed with saturated saline (100 mL), and dried over anhydrous Na2SO4.

[0085] (5) Filtration and concentration gave the crude product, which was purified by column chromatography (DCM / MeOH = 10 / 1) to give TO-23-01 (6.3 g, 63.1% yield) as a white solid.

[0086] 4. Synthesis of compound TO-23 (1043 target head)

[0087] JPEG0007607957000015.jpg31130

[0088] (1) 10% Pd / C (600 mg) and Pd(OH)2 / C (600 mg) were added to a solution of TO-23-01 (6.3 g, 3.0 mmol) in MeOH (100 mL), and the mixture was purged with H2 three times. The mixture was stirred at 25°C for 3 hours to react, and TLC (DCM / MeOH = 8 / 1) detection confirmed that the raw materials had essentially disappeared.

[0089] (2) Filtration and concentration gave the crude product, which was purified by column chromatography (DCM / MeOH / TEA = 10 / 1 / 0.1) to give a white solid TO-23 (4.5 g, 75% yield).

[0090] 1 H NMR (400 MHz, DMSO-d6) δ 7.88-7.81 (m, 9H), 7.14 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 4.95 (dd, J = 11.2, 3.4 Hz, 3H), 4.53 (d, J = 8.5 Hz, 3H), 4.07-3.97 (m, 9H), 3.88 (dt, J = 11.0, 9.0 Hz, 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).

[0091] MS (ESI): m / z [1 / 2M + H] + The theoretical value is 1000.5 and the measured value is 1000.3.

[0092] 5. Synthesis of compound TP-23 (precursor of 1043-targeting head-binding siRNA)

[0093] JPEG0007607957000016.jpg27138

[0094] (1) In a N2 atmosphere, TO-23 (2.3 g, 1.15 mmol) was dissolved in dry DCM (40 mL), DIEA (0.86 mL, 5.2 mmol) was added, and a solution of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.46 mL, 2.1 mmol) in dry DCM (2 mL) was slowly added dropwise using a syringe. The reaction was carried out at 25°C for 1 hour. TLC detection confirmed that the raw material had essentially disappeared.

[0095] (2) The reaction was quenched with saturated NaHCO3 (20 mL), separated, and the organic phase was washed with saturated NaHCO3 (20 mL) solution, saturated brine (20 mL), dried over anhydrous MgSO4, filtered, and concentrated to give the crude product. Purification by column chromatography (silica column prebasified with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) gave a white solid TP-23 (1.8 g, 71.1% yield).

[0096] 1 H NMR (400 MHz, DMSO-d6) δ 7.91-7.79 (m, 9H), 7.15 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 4.95 (dd, J = 11.2, 3.4 Hz, 3H), 4.53 (d, J = 8.5 Hz, 3H), 4.06-3.97 (m, 9H), 3.88 (dt, J = 11.1, 8.9 Hz, 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, 4.1 Hz, 12H),

[0097] 31 P NMR (162 MHz, DMSO-d6) δ 147.81,

[0098] MS (ESI): m / z [1 / 2M + Na] + The theoretical value is 1122.5 and the measured value is 1122.4.

[0099] II. Synthesis of GalNAc-targeting head 1046

[0100] Diastereoisomers of TO25 and TP-25 (precursor of 1046-targeting head-binding siRNA) were synthesized according to the following method.

[0101] 1. Synthesis of intermediate NC-6-01

[0102] JPEG0007607957000017.jpg12128

[0103] (1) In a N2 atmosphere, dry THF (300 mL) was added to a 1000 mL three-neck flask, the temperature was lowered to 0-5°C in an ice bath, and the mixture was stirred. 60% NaH (14 g, 354.8 mmol) was gradually added, and then a THF solution (200 mL) of 2-chloroethyloxyethanol (40 g, 322.5 mmol) was gradually added dropwise. The mixture was allowed to react for 30 minutes while keeping the temperature at 25°C. Furthermore, benzyl bromide (60.3 g, 354.8 mmol) was added dropwise to the reaction flask, the temperature was raised to 25°C, and the mixture was stirred for 16 hours. TLC detection confirmed that the raw materials had been substantially consumed.

[0104] (2) The reaction was quenched by slowly adding saturated ammonium chloride solution (150 mL) dropwise, separated, the aqueous phase was extracted with EtOAc (100 mL x 2), the organic phase was combined, washed with saturated brine (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified by silica column chromatography (petroleum ether / EtOAc = 5 / 1) to obtain pale yellow oily compound NC-6-01 (53 g, 78% yield).

[0105] MS (ESI): m / z [M + H] + The theoretical value is 215.1 and the measured value is 215.1.

[0106] 2. Synthesis of intermediate NC-6-02

[0107] JPEG0007607957000018.jpg14133

[0108] (1) Ethylenediamine (196g, 3.26mol) was placed in a 2000mL three-neck flask, acetonitrile (1000mL), potassium carbonate (90g, 0.65mol) and sodium iodide (60.6g, 0.33mol) were added and stirred. Then, a solution of NC-6-01 (70g, 0.33mol) in acetonitrile (100mL) was slowly added dropwise to the reaction flask, the temperature was raised to 60°C, and the mixture was stirred for 16 hours. TLC detection confirmed that the raw material was substantially consumed.

[0109] (2) The reaction was stopped, concentrated, purified water (300 mL) was added, the pH was adjusted to 4-5 with concentrated hydrochloric acid, extracted three times with EtOAc (200 mL × 3), the aqueous phase was added solid sodium hydroxide to adjust the pH to 13-14, extracted three times with DCM (200 mL × 3), the organic phase was combined, washed with saturated brine (300 mL), dried over anhydrous Na2SO4, filtered and concentrated to give pale yellow oil NC-6-02 (69.5 g, 87%).

[0110] MS (ESI): m / z [M + H] + The theoretical value is 239.2 and the kph value is 239.1.

[0111] 3. Synthesis of intermediate NC-6-03

[0112] JPEG0007607957000019.jpg30128

[0113] (1) 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, cooled to below 5°C in an ice-water bath, and potassium carbonate (322 g, 2.34 mol) was added. The reaction was stirred at 25°C for 14 hours, and TLC detection confirmed that the raw materials had been completely converted.

[0114] (2) Purified water (300 mL) was added to the reaction solution, and the solution was allowed to stand to separate the organic phase. The aqueous phase was extracted twice with EtOAc (200 mL x 2). The organic phases were combined, washed with saturated saline (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a pale yellow oily substance NC-6-03 (201 g).

[0115] MS (ESI): m / z [M + H] + The theoretical value is 581.4 and the measured value is 581.3.

[0116] 4. Synthesis of intermediate NC-6

[0117] JPEG0007607957000020.jpg34128

[0118] (1) NC-6-03 (23 g, 39.6 mmol) was dissolved in 1,4-dioxane (200 mL), concentrated hydrochloric acid (40 mL) was added, and the mixture was heated to 60° C. and reacted for 2 hours. TLC detection confirmed that the raw material was substantially consumed.

[0119] (2) Concentration, re-addition of 1,4-dioxane (200 mL) and concentration gave a white solid crude product, which was added to ethyl acetate (200 mL), pulped for 2 hours, filtered with suction, and the cake was collected and dried in vacuum at 50° C. to give white solid compound NC-6 (22.6 g, 96.9%).

[0120] (3)MS (ESI): m / z [M+H] + The theoretical value is 413.2 and the measured value is 413.1.

[0121] 5. Synthesis of intermediate TO-25-01

[0122] JPEG0007607957000021.jpg28147

[0123] (1) Under a N2 atmosphere, NC-6 (1.5 g, 3.6 mmol), HBTU (4.5 g, 12.0 mmol) and DIEA (4.75 g, 36 mmol) were added to DCM (50 mL) and stirred for 30 minutes. Then, a solution of GN-17 (6.4 g, 12.0 mmol) and DIEA (4.75 g, 36 mmol) in DCM (50 mL) was added dropwise and stirred at 25°C for 16 hours. LCMS detection confirmed that the raw materials were substantially consumed.

[0124] (2) DCM (100 mL) was added to dilute the reaction mixture, and 1 N hydrochloric acid solution (80 mL x 2) was added to wash the reaction mixture. The organic phases were combined, washed with saturated sodium bicarbonate (100 mL), washed with saturated saline (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified by silica column chromatography (DCM / MeOH = 7 / 1) to obtain white solid compound TO-25-01 (4.3 g, yield 60%).

[0125] (3)MS (ESI): m / z [M / 2 + H] + The theoretical value is 980.0 and the measured value is 979.9.

[0126] 6. Synthesis of intermediate TO-25

[0127] JPEG0007607957000022.jpg27141

[0128] (1) TO-25-01 (4.3 g, 2.2 mmol) was dissolved in methanol (80 mL), 10% palladium carbon (1.0 g) was added, the mixture was purged with H2 three times, and the mixture was stirred at 25°C for 2 hours. LCMS detection confirmed that the raw materials had essentially disappeared.

[0129] (2) The mixture was filtered and concentrated, dissolved in DCM (20 mL), slowly added dropwise to MTBE (300 mL), stirred for 30 minutes to crystallize, and filtered with suction to obtain white solid compound TO-25 (3.7 g, yield 90%).

[0130] 1 H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J = 5.6 Hz, 1H), 8.06 (t, J = 5.7 Hz, 2H), 7.85 (dd, J = 11.7, 6.8 Hz, 6H), 5.21 (d, J = 3.3 Hz, 3H), 4.95 (dd, J = 11.2, 3.3 Hz, 3H), 4.53 (d, J = 8.5 Hz, 3H), 4.08-3.83 (m, 14H), 3.75 (p, J = 4.8 Hz, 5H), 3.68-3.26 (m, 28H), 3.21-2.95 (m, 14H), 2.30 (q, J = 7.9, 6.7 Hz, 6H), 1.94-1.78 (m,, 36H), 1.41-1.38 (m, 12H),

[0131] MS (ESI): m / z [1 / 2M + H] + The theoretical value is 934.9 and the measured value is 934.8.

[0132] 7. Synthesis of TP-25 (precursor of 1046-targeting head-binding siRNA)

[0133] JPEG0007607957000023.jpg28148

[0134] (1) In a N2 atmosphere, TO-25 (700 mg, 0.37 mmol) was dissolved in dry DCM (10 mL), DIEA (0.31 mL, 1.9 mmol) was added, and a solution of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.19 mL, 0.74 mmol) in dry DCM (1 mL) was slowly added dropwise using a syringe, and the reaction was carried out at 25°C for 30 minutes. TLC detection confirmed that the raw materials had essentially disappeared.

[0135] (2) The reaction was quenched by adding saturated NaHCO3 (10 mL), diluted with DCM (10 mL), separated, and the organic phase was washed with saturated NaHCO3 (10 mL) solution, saturated brine (10 mL), dried over anhydrous NaSO4, filtered, and concentrated to give the crude product. Purification by column chromatography (silica column prebasified with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) gave TP-25 (405 mg, 53% yield) as a white solid.

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 8.12 (t, J = 6.0 Hz, 2H), 7.98-7.75 (m, 7H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 2H), 4.54 (d, J = 8.4 Hz, 2H), 4.02 (q, J = 5.3, 4.5 Hz, 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 = 6.7 Hz, 5H), 2.11-1.78 (m, 38H), 1.38 (s, 12H), 1.16 (d, J = 7.5 Hz, 12H),

[0137] 31 P NMR (162 MHz, DMSO-d6) δ 147.97,

[0138] MS (ESI): m / z [1 / 2M + Na] + The theoretical value is 1057.0 and the measured value is 1057.4.

[0139] 3. Synthesis of GalNAc-targeting head 1048

[0140] Diastereoisomers of TO26 and TP-26 (precursor of 1048-targeting head-binding siRNA) were synthesized according to the following method.

[0141] 1. Synthesis of intermediate GN-18-01

[0142] JPEG0007607957000024.jpg17137

[0143] (1) In a N2 atmosphere, GC-2 (20.1 g, 39.7 mmol) was dissolved in DCM (200 mL), CDI (7.09 g, 73.7 mmol) was added little by little, and the mixture was stirred at 25°C for 3 hours. Subsequently, N-Boc-ethylenediamine (7.0 g, 43.7 mmol) and triethylamine (12.05 g, 119.1 mmol) were added to the reaction solution, and the mixture was reacted for 16 hours. LCMS detection confirmed that the raw materials had substantially disappeared.

[0144] (2) The reaction was quenched by adding saturated sodium bicarbonate solution (200 mL), separated, the aqueous phase was extracted with DCM (100 mL x 3), the organic phase was combined, washed with saturated ammonium chloride solution (200 mL), saturated sodium chloride solution (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was washed with methyl tert-butyl ether (100 mL), and the oily product was concentrated to obtain a white solid compound GN-18-01 (24.43 g, yield 95.1%).

[0145] MS (ESI): m / z [M + H] + The theoretical value is 650.3 and the measured value is 650.5.

[0146] 2. Synthesis of intermediate GN-18

[0147] JPEG0007607957000025.jpg14133

[0148] (1) GN-18-01 (45.52 g, 70 mmol) was added portionwise to a HCl / EtOAc solution (2N, 500 mL) and stirred at 25° C. for 2 hours. LCMS detection confirmed that the raw material had essentially disappeared.

[0149] (2) The solvent was poured off and the solid was concentrated to give a crude product, which was purified by pulping with methyl tert-butyl ether (200 mL), filtered, and the filter cake was dried under vacuum at 40° C. to give a white solid GN-18 (49.6 g).

[0150] MS (ESI): m / z [M + H] + The theoretical value is 550.3 and the measured value is 550.5.

[0151] 3. Synthesis of intermediate TO-26-01

[0152] JPEG0007607957000026.jpg30146

[0153] (1) In a N2 atmosphere, NC-6 (1.5 g, 3.6 mmol), PyBOP (6.2 g, 12.0 mmol) and DIEA (4.75 g, 36 mmol) were added to DCM (50 mL) and stirred for 30 minutes, and then a solution of GN-18 (6.6 g, 12.0 mmol) and DIEA (4.75 g, 36 mmol) in DCM (50 mL) was added dropwise and stirred at 25°C for 16 hours. LCMS detection confirmed that the raw materials were substantially consumed.

[0154] (2) DCM (100 mL) was added to dilute the reaction solution, and 1N hydrochloric acid solution (80 mL x 2) was added to wash the reaction solution. The organic phases were combined, washed with saturated sodium bicarbonate (100 mL) and saturated saline (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica column chromatography (DCM / MeOH = 7 / 1) to obtain a white solid compound TO-26-01 (4.7 g, yield 65%).

[0155] MS (ESI): m / z [M / 2 + H] + The theoretical value is 1004.0 and the measured value is 1004.2.

[0156] 4. Synthesis of intermediate TO-26 (1048 target head)

[0157] JPEG0007607957000027.jpg33140

[0158] (1) TO-26-01 (4.0 g, 2.0 mmol) was dissolved in methanol (80 mL), 10% palladium carbon (1.0 g) was added, the mixture was purged with H2 three times, and the mixture was stirred at 25°C for 2 hours. LCMS detection confirmed that the raw materials had substantially disappeared.

[0159] (2) The mixture was filtered and concentrated, dissolved in DCM (20 mL), slowly added dropwise to MTBE (200 mL), stirred for 30 minutes to crystallize, and filtered with suction to obtain white solid compound TO-26 (3.5 g, yield 91%).

[0160] 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.14 (s, 2H), 7.95-7.92 (m, 3H), 7.84 (d, J = 7.8 Hz, 3H), 5.21 (d, J = 3.4 Hz, 3H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 4.54 (d, J = 8.5 Hz, 3H), 4.13-3.66 (m, 21H), 3.60-3.44 (m, 37H), 3.14 (d, J = 13.8 Hz, 15H), 2.31 (t, J = 6.4 Hz, 6H), 2.10 (s, 9H), 2.00 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H).

[0161] MS (ESI): m / z [1 / 2M + H] + The theoretical value is 958.9 and the measured value is 959.1.

[0162] 5. Synthesis of TP-26 (precursor of 1048-targeting head-binding siRNA)

[0163] JPEG0007607957000028.jpg34146

[0164] (1) In a N2 atmosphere, TO-26 (900 mg, 0.47 mmol) was dissolved in dry DCM (12 mL), DIEA (0.39 mL, 0.44 mmol) was added, and a solution of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (277 mg, 1.17 mmol) in dry DCM (1 mL) was slowly added dropwise using a syringe, and the reaction was carried out at 25°C for 30 minutes. TLC detection confirmed that the raw materials had essentially disappeared.

[0165] (2) The reaction was quenched by adding saturated NaHCO3 (10 mL), diluted with DCM (10 mL), separated, and the organic phase was washed with saturated NaHCO3 (10 mL) solution, saturated brine (10 mL), dried over anhydrous NaSO4, filtered, and concentrated to give the crude product. Purification by column chromatography (silica column was prebasified with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) gave a white solid TP-26 (600 mg, 60% yield).

[0166] 1 H NMR (400 MHz, DMSO-d6) 1H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 2H), 7.94-7.81 (m, 7H), 5.22 (d, J = 3.4 Hz, 3H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (d, J = 8.5 Hz, 3H), 4.03 (s, 8H), 3.88 (dt, J = 11.2, 8.9 Hz, 3H), 3.81- 3.67 (m, 7H), 3.64 - 3.46 (m, 30H), 3.11 (d, J = 13.1 Hz, 19H), 2.76 (t, J = 5.9 Hz, 3H), 2.65 - 2.54 (m, 7H), 2.31 (t, J = 6.6 Hz, 7H), 2.11 (s, 9H), 2.00 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.13 (d, J = 6.8, 12H).

[0167] 31 P NMR (162 MHz, DMSO-d6) δ 147.89,

[0168] MS (ESI): m / z 1 / 2[Mi-Pr2N] theoretical value is 1007.9, found value is 1008.2.

[0169] Example 3. Construction of GalNAc-targeted head-linked (modified) siRNA conjugates in vitro

[0170] The oligonucleotide sequence portions of the antisense and sense strands of the following RNAi agent duplexes, and the linkage between the targeting ligand and the RNA are all synthesised on a solid phase for oligonucleotide synthesis by phosphoramidite coupling techniques as described in J. Org. Chem. 2012, 77, 4566-4577, Curr. Protoc. Nucleic Acid Chem., 81, e107. The targeting ligands 1046, 1048, and 1043 are all linked to the 5' end of the siRNA sense strand via a phosphorothioate bond.

[0171] JPEG0007607957000029.jpg48141

[0172] JPEG0007607957000030.jpg49134

[0173] JPEG0007607957000031.jpg45132

[0174] The GalNAc-siRNA conjugates synthesized are as shown in the table in Figure 3. The conjugate structure in the second row in the table includes three parts. For example, the structure of G1043-S2A2-A265 is that the 1043 target head is linked to the 5' end of the siRNA sense strand numbered A265 through a thiophosphate ester bond, and S2A2 is the modification type of siRNA for A265, and the specific modification groups and modification methods are as follows:

[0175] In nucleic acid sequences, Ao represents an adenine nucleotide, Uo represents a uridine nucleotide, Go represents a guanosine nucleotide, and Co represents a cytosine nucleotide, with no sign between directly adjacent nucleotides indicating that they are linked by a normal phosphate bond.

[0176] DNA:AGCT (A represents a 2'-deoxyadenine nucleotide, T represents a 2'-deoxycythymidine nucleotide, G represents a 2'-deoxyguanosine nucleotide, and C represents a 2'-deoxycytosine nucleotide).

[0177] 2'-F: aF gF cF uF (aF represents a 2'-fluoro adenine nucleotide, uF represents a 2'-fluoro uridine nucleotide, gF represents a 2'-fluoro guanosine nucleotide, and cF represents a 2'-fluoro guanosine nucleotide).

[0178] 2'-OMe: aM gM cM uM, where aM represents a 2'-O-methyl adenine nucleotide, uM represents a 2'-O-methyl uridine nucleotide, gM represents a 2'-O-methyl guanosine nucleotide, and cM represents a 2'-O-methyl cytosine nucleotide.

[0179] *: indicates that the linkage is via a thiophosphate bond.

[0180] The y,z in the array represent the position of the target head.

[0181] Example 4: Testing the activity of the conjugates in an in vitro cell model (Hep 3B cells)

[0182] Human hepatoma Hep3B cells (Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM (Gibco, US) supplemented with 10% fetal bovine serum (FBS) (Gibco, US) at 37°C and 5% CO2 (il60, Thermo Fisher). On the day of the transfection experiment, the cells were digested with 0.25% Trysin (Gibco, US), counted, and seeded into 24-well plates at 450 μL / well and a density of 50,000 / well. Then, the test samples were added using the lipofectmine2000 (Thermo Fisher) transfection method. Transfection was performed according to the standard flow in the RNAiMAX reagent manual, and the final concentrations of siRNA were 10 nM / 1 nM / 0.5 nM / 0.25 nM / 0.1 nM / 0.05 nM / 0.01 nM. For the transfected group, siNC was used as a negative control, and its sequence was as follows:

[0183] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0184] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0185] After 24 hours, total RNA was extracted from the cells, and the expression status of ANGPTL3 mRNA sequence in the cells was detected by quantitative real-time PCR, where the internal control gene PPIB and the PCR primers used to amplify ANGPTL3 are as shown in Table 1.

[0186] Activity test results for each conjugate (EC 50 The values ​​are as shown in Figure 4.

[0187] EC 50 Values ​​were calculated by nonlinear regression in GraphPad Prism and represent the amount of conjugate used to inhibit half the expression level of the target mRNA (ANGPTL3).

[0188] As can be seen from the results, the selected conjugates were shown to successfully reduce the relative expression level of ANGPTL3 in in vitro activity test experiments.

[0189] Example 5: Construction of AAV-hANGPTL3 mouse model and administration test

[0190] Basic information on the experimental animals is shown in Table 3.

[0191] The experimental animals were purchased from Jinan Peng Yue Experimental Animal Breeding Co., Ltd. and were SPF animals. Before administration, the mice were weighed and observed. Animals with uniform weight and normal condition were selected for further experiments.

[0192] Table 3: Basic information of experimental animals JPEG0007607957000032.jpg19136

[0193] The rearing conditions were non-SPF grade rearing conditions. Under normal rearing conditions, the animals had free access to food and water. After purchasing the animals, they were cultured for 3-7 days for adaptation before the test was started.

[0194] Model construction and administration: Each mouse was injected with 2.5*10^11 titer virus solution via tail vein, 100ul. After 7 days, the experimental animals were randomly grouped and subcutaneously administered with a dose of 5mg / kg to each subject. 72 hours after administration, the animals were sacrificed by cervical dislocation, and liver tissue was collected for RNA extraction and quantification.

[0195] The results for each conjugate are shown in Table 4.

[0196] Table 4: Test results of administration of each conjugate to a mouse model JPEG0007607957000033.jpg248165

[0197] As can be seen from the results, the selected conjugates also successfully reduced the relative expression level of ANGPTL3 in the in vivo activity test experiment.

[0198] In the description of this specification, the description of terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "several examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, general descriptions of the above terms are not necessarily directed to the same embodiment or example. And, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. It should be noted that a person skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification, as long as they are not mutually inconsistent.

[0199] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An siRNA comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region that complementarily pairs with the sense strand, the sense strand having a nucleotide sequence of SEQ ID NO: 1, and the antisense strand having a nucleotide sequence of SEQ ID NO: 155; The siRNA is characterized in that the siRNA targets angiopoietin-like 3 (ANGPTL3).

2. 11. An siRNA conjugate comprising the siRNA of claim 1 and a targeting ligand, wherein the siRNA is covalently linked to the targeting ligand.

3. The siRNA conjugate of claim 2 , wherein the targeting ligand is linked to a sense strand of the siRNA.

4. The siRNA conjugate of claim 3 , wherein the targeting ligand is linked to the 5′ end of the sense strand of the siRNA via a phosphorothioate bond.

5. The siRNA conjugate of claim 2, wherein the targeting ligand comprises at least one N-acetyl-galactosamine.

6. The siRNA conjugate of claim 5 , wherein the targeting ligand is a GalNAC targeting head compound.

7. The target ligand is the following: １０４３、 １０４６、 １０４８ The siRNA conjugate of claim 6 ,

8. A pharmaceutical composition comprising the siRNA of claim 1 and / or the siRNA conjugate of claim 2, characterized in that the pharmaceutical composition further comprises a pharma- ceutical acceptable additive.

9. Use of the siRNA of claim 1 and / or the siRNA conjugate of claim 2 in the manufacture of a drug or kit, characterized in that the drug or kit is used for inhibiting expression of the ANGPTL3 gene.

10. The drug or kit is used for preventing and / or treating dyslipidemia, The dyslipidemia includes hyperlipidemia and hypertriglyceridemia, The drug or kit is characterized in that it is used for inhibiting expression of the ANGPTL3 gene in a cell.

10. The use according to claim 9.

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