SiRNA of angiopoietin-like 3 and use thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to siRNA of angiogenin-like 3 (ANGPTL3) and application of the siRNA. According to the present invention, by designing the suitable specific small interfering RNA sequence and the siRNA conjugate, the ANGPTL3 is targeted, and by degrading the transcript of the ANGPTL3 gene in the cell, the ANGPTL3 protein expression quantity is reduced, such that the siRNA provided by the present invention can be used for preventing and / or treating the dyslipidemia disease.

Description

[0001] This invention is a divisional application of the invention patent with the application date of September 30, 2021, application number 202180022902.3, and invention title "siRNA of angiopoietin-like 3 (ANGPTL3) and its uses".

[0002] This application claims the priority and rights of the patent application filed with the China National Intellectual Property Administration on September 30, 2020, with the patent application number 202011061038.1, the patent application filed with the China National Intellectual Property Administration on January 5, 2021, with the patent application number 202110008013.3, and the patent application filed with the China National Intellectual Property Administration on April 13, 2021, with the patent application number 202110397429.9, and incorporates the full text thereof herein by reference. Technical Field

[0003] This disclosure relates to the field of genetic engineering technology. Specifically, this disclosure relates to siRNA of angiopoietin-like 3 (ANGPTL3) and its uses. Background Art

[0004] Hyperlipidemia, also known as dyslipidemia, is a systemic disease in which fat metabolism or transport is abnormal, causing plasma lipids to be higher than normal values. 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 diseases, etc. According to investigations, about 10% to 20% of adults have elevated total blood cholesterol (TC) or triglyceride (TG), and even nearly 10% of children have elevated blood lipids. Existing drugs for treating dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probucol, fibrates, and niacin and its derivatives.

[0005] Currently, more or less, there are some contraindications and side effects after the use of current therapeutic drugs. For example, statins are currently the first-choice drugs for reducing serum total cholesterol. They are used to treat patients with simple elevated serum total cholesterol levels, and also for those with mainly elevated serum total cholesterol levels accompanied by slightly elevated serum triglyceride levels. Such drugs mainly include lovastatin (Mevacor), simvastatin (Zocor), pravastatin (Pravachol), fluvastatin (Lescol), atorvastatin (Lipitor), and cerivastatin (Baycol), etc. If taken for a long time, there may be abdominal distension, diarrhea, constipation, headache, insomnia, rash, thrombotic thrombocytopenic purpura (manifested as diffuse ecchymosis on the face, chest, and extremities, accompanied by a decrease in platelet count). In addition, there may also be mental depression, paresthesia, mostly occurring on the face, scalp, tongue, and extremities, manifested as numbness, burning sensation, skin allergy or pain. And it can cause skin peeling and elevated serum transaminase. The most serious adverse reaction is rhabdomyolysis, manifested as muscle weakness, muscle pain, anuria, elevated serum creatine kinase level, etc., with an incidence rate of about 1‰. If not discovered and stopped taking the drug in time, it will produce severe myopathy and even cause renal failure.

[0006] Therefore, there is an urgent need to develop a drug for treating dyslipidemia that can be taken for a long time and has few side effects.

[0007] Angiopoietin-like protein 3 (ANGPTL3, NM_014495.4) is a secreted protein mainly expressed in liver cells. Existing studies have shown that angiopoietin-like protein 3 (ANGPTL3) is a key regulator of LDL-C, HDL-C, and triglyceride metabolism, with multiple potential action nodes. Loss-of-function mutations in ANGPTL3 can lead to a decrease in LDL-C, VLDL-C, HDL-C, and triglyceride (TG), thereby reducing the risk of cardiovascular diseases based on GWAS, and there are no adverse phenotypes of known genetic defects. Therefore, inhibiting the activity of ANGPTL3 can effectively prevent or treat dyslipidemia.

[0008] In the prior art, ANGPTL3 antibodies are mostly used to inhibit its activity.

[0009] Chinese Patent with Application No. CN201280038908.0 discloses a fully humanized antibody or antigen-binding fragment of a human antibody that specifically binds to human angiopoietin-like protein 3 (hANGPTL3) and inhibits or interferes with at least one of its activities. This human anti-hANGPTL3 antibody can be used to treat ANGPTL3-related diseases or disorders, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including hypertriglyceridemia, hypercholesterolemia, chylomicronemia, etc.

[0010] Chinese patent application number CN201780026147.X discloses a method for treating patients with familial hypercholesterolemia, including HeFH and HoFH. The method involves administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3, in combination with other agents, to reduce at least one lipid parameter in the patient. The method can be used to treat hypercholesterolemia, as well as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including hypertriglyceridemia and chylomicronemia.

[0011] Compared to antibodies, siRNA drugs offer advantages such as a richer pool of candidate targets, shorter development cycles, and a higher clinical development success rate. Therefore, they hold a distinct advantage in the treatment of chronic diseases. The development of siRNA drugs for dyslipidemia is urgent. Summary of the Invention

[0012] The present disclosure aims to at least partially address one of the technical problems in the related art. To this end, one object of the present disclosure is to provide an siRNA for inhibiting ANGPTL3 expression. The inventors of the present disclosure designed appropriate specific small interfering RNA sequences and siRNA conjugates to target ANGPTL3. This siRNA degrades ANGPTL3 gene transcripts in cells, thereby reducing ANGPTL3 protein expression. Therefore, the siRNA provided herein can be used to prevent and / or treat dyslipidemia.

[0013] To this end, the present disclosure provides, in one aspect, an siRNA. According to an embodiment of the present disclosure, the siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region that pairs complementary with the sense strand, wherein the sense strand is selected from a nucleotide sequence that differs from the nucleotide sequence of each of SEQ ID NO: 1 to SEQ ID NO: 154 by no more than 5 nucleotides, and the antisense strand is selected from a nucleotide sequence that differs from the nucleotide sequence of each of SEQ ID NO: 155 to SEQ ID NO: 308 by no more than 5 nucleotides.

[0014] The inventors of the present disclosure designed an appropriate small interfering RNA (siRNA) sequence to specifically reduce ANGPTL3 synthesis in hepatocytes while avoiding off-target effects. The siRNA forms an RNA-induced silencing complex (RISC), which pairs with the target gene (ANGPTL3) mRNA sequence, causing mRNA degradation and inhibiting target gene expression, thereby reducing LDL-C, VLDL-C, HDL-C, and triglyceride (TG) levels.

[0015] The siRNA according to the embodiments of the present disclosure may further have at least one of the following additional technical features:

[0016] The present disclosure also provides an siRNA, and the siRNA is selected from any pair of siRNAs in any of the following groups:

[0017] (1) It can specifically target the nucleotides at positions 60-80 of the angiopoietin-like protein 3 gene 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;

[0018] (2) It can specifically target the nucleotides at positions 107-133 of the angiopoietin-like protein 3 gene sequence; 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;

[0019] (3) It can specifically target the nucleotides at positions 163-187 of the angiopoietin-like protein 3 gene 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;

[0020] (4) It can specifically target the nucleotides at positions 304-388 of the angiopoietin-like protein 3 gene sequence, preferably, it can specifically target the nucleotides at positions 304-359 of the angiopoietin-like protein 3 gene sequence; 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,

[0021] 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,

[0022] Or, the sense strand of the siRNA is selected from SEQ ID NO:31, and the antisense strand is selected from SEQ IDNO:185,

[0023] 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,

[0024] 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,

[0025] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:36, and the antisense strand is selected from SEQ ID NO:190;

[0026] (5) capable of specifically targeting nucleotides 430-459 of the angiopoietin-like protein 3 gene sequence; preferably, the sense strand of the siRNA is selected from SEQ ID NO:43, and the antisense strand is selected from SEQ ID NO:197,

[0027] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:44, and the antisense strand is selected from SEQ ID NO:198;

[0028] (6) capable of specifically targeting nucleotides 1360-1430 of the angiopoietin-like protein 3 gene sequence, preferably capable of specifically targeting nucleotides 1397-1430 of the angiopoietin-like protein 3 gene sequence; 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,

[0029] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:150, and the antisense strand is selected from SEQ ID NO:304,

[0030] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:151, and the antisense strand is selected from SEQ ID NO:305,

[0031] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO:152, and the antisense strand is selected from SEQ ID NO:306,

[0032] 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.

[0033] According to an embodiment of the present disclosure, the siRNA includes at least one modified nucleotide;

[0034] Optionally, the modified nucleotide is selected from at least one of the following:

[0035] 5'-thiophosphate nucleotides, 5-methylcytosine nucleotides, 2'-O-methyl modified nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-fluorinated modified nucleotides, 3'-nitrogen substituted modified nucleotides, 2'-deoxy-2'-fluorinated modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, morpholino nucleotides, peptide nucleotides, phosphoramidates, and nucleotides including unnatural bases.

[0036] According to an embodiment of the present disclosure, the length of the complementary region is at least 17 bp;

[0037] Optionally, the length of the complementary region is 18 - 21 bp;

[0038] Optionally, the length of the complementary region is 19 bp.

[0039] According to an embodiment of the present disclosure, the lengths of the sense strand and the antisense strand in the siRNA are not more than 25 bp;

[0040] Optionally, the lengths of the sense strand and the antisense strand in the siRNA are 18 - 25 bp;

[0041] Optionally, the lengths of the sense strand and the antisense strand in the siRNA are 21 bp.

[0042] According to an embodiment of the present disclosure, the bases in the sense strand and the antisense strand of the siRNA can be complementary base - paired one by one, or can be misaligned by several bases, but have a complementary region of at least 17 bp.

[0043] On the other hand, the present disclosure provides an siRNA conjugate, which includes the aforementioned siRNA and a targeting ligand, wherein the siRNA is covalently linked to the targeting ligand;

[0044] Preferably, the targeting ligand is linked to the sense strand of the siRNA;

[0045] More preferably, the targeting ligand is linked to the 5'-end of the sense strand of the siRNA through a thiophosphate bond.

[0046] According to an embodiment of the present disclosure, the targeting ligand includes at least one N-acetyl-galactosamine.

[0047] According to an embodiment of the present disclosure, the targeting ligand is a GalNAC targeting compound.

[0048] According to an embodiment of the present disclosure, the GalNAC targeting compounds are 1043, 1046, 1048, and their structures are shown in the following formulas 1 - 3:

[0049]

[0050] According to an embodiment of the present disclosure, the targeting ligand is linked to the sense strand of the siRNA.

[0051] Another aspect of the present disclosure provides a pharmaceutical composition. According to an embodiment of the present disclosure, the pharmaceutical composition includes the aforementioned siRNA and / or the aforementioned siRNA conjugate, and optionally, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0052] Thus, the pharmaceutical composition according to the embodiments of the present disclosure can be used to inhibit the synthesis of ANGPTL3 by cells, thereby reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG) to treat, prevent and / or treat hyperlipidemia and hypertriglyceridemia.

[0053] Another aspect of the present disclosure provides a kit. According to an embodiment of the present disclosure, the kit includes the siRNA and / or siRNA conjugate.

[0054] Thus, the kit according to the embodiments of the present disclosure can be used to inhibit the expression of the ANGPTL3 gene in cells, thereby reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG) to treat, prevent and / or treat hyperlipidemia and hypertriglyceridemia.

[0055] In another aspect, the present disclosure provides a method for inhibiting ANGPTL3 gene expression in a subject, comprising: administering the aforementioned siRNA and / or the aforementioned siRNA conjugate to the subject to inhibit ANGPTL3 gene expression.

[0056] In another aspect, the present disclosure provides a method for inhibiting ANGPTL3 gene expression in a cell. According to an embodiment of the present disclosure, the method comprises: transfecting the cell with the siRNA and / or the siRNA conjugate to inhibit ANGPTL3 gene expression in the cell.

[0057] According to the method of inhibiting the expression of the ANGPTL3 gene in cells according to the embodiments of the present disclosure, siRNA is used to form a silencing complex, which is complementary to the sequence of the mRNA of the target gene ANGPTL3 gene, thereby degrading the mRNA of the target gene and inhibiting the expression of the target gene, thereby reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG).

[0058] According to an embodiment of the present disclosure, the cell is derived from a mammal;

[0059] Optionally, the cells are of human origin;

[0060] Optionally, the cells are hepatocytes.

[0061] Using the siRNA provided by the present disclosure, a silencing complex is formed in human hepatocytes, which is complementary to the sequence of the mRNA of the ANGPTL3 gene, degrades the mRNA of the ANGPTL3 gene, thereby inhibiting its expression, and then reduces the levels of LDL-C, VLDL-C, HDL-C and triglyceride (TG).

[0062] Another aspect of the present disclosure provides the use of the siRNA and / or the siRNA conjugate described above in the preparation of a drug or a kit. According to the embodiments of the present disclosure, the drug or the kit is used to inhibit the expression of the ANGPTL3 gene.

[0063] Using the siRNA provided by the present disclosure to prepare a drug or a kit, the drug or the kit reduces the expression level of the ANGPTL3 gene in cells through the siRNA therein, thereby preventing and / or treating dyslipidemia diseases.

[0064] According to the embodiments of the present disclosure, the drug or the kit is used to prevent and / or treat dyslipidemia diseases;

[0065] Optionally, the dyslipidemia diseases include hyperlipidemia and hypertriglyceridemia;

[0066] Optionally, the drug or the kit is used to inhibit the expression of the ANGPTL3 gene in cells.

[0067] Another aspect of the present disclosure provides a method for preventing and / or treating dyslipidemia diseases. According to the embodiments of the present disclosure, the method includes: administering the siRNA and / or the siRNA conjugate to a subject.

[0068] According to the embodiments of the present disclosure, the dyslipidemia diseases include hyperlipidemia and hypertriglyceridemia.

[0069] Additional aspects and advantages of the present disclosure will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present disclosure.

[0070] Advantageous effects of the present invention:

[0071] The siRNA conjugate provided by the present invention can reduce the expression of ANGPTL3 in both cell models and mouse models, and can reduce the expression level by more than 50% compared with the control group, and can be reduced by nearly 90% at most, having good clinical application prospects. Description of the Drawings

[0072] The above and / or additional aspects and advantages of the present disclosure will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

[0073] Figure 1 The figure shows the results of the expression levels of the ANGPTL3 gene (abbreviated as ANL3 in the figure) in cells detected by real-time quantitative PCR after transfection of Hep 3B cells with some siRNAs in Table 2 at a concentration of 0.1 nM.

[0074] Figure 2 The figure shows the results of the expression levels of the ANGPTL3 gene (abbreviated as ANL3 in the figure) in cells detected by real-time quantitative PCR after transfection of Hep 3B cells with some siRNAs in Table 2 at a concentration of 10 nM.

[0075] Figure 3 The figure shows the GalNAc-siRNA conjugate synthesized in Example 3.

[0076] Figure 4 The figure shows the results of the activity tests (EC 50 value) of each conjugate in Example 4. Detailed Description of the Embodiments

[0077] Embodiments of the present disclosure will be described in detail below. The following described embodiments are exemplary and are only used to explain the present disclosure and should not be construed as a limitation to the present disclosure.

[0078] "Pharmaceutically acceptable carrier" is well recognized in the art and includes pharmaceutically acceptable materials, compositions or carriers suitable for administering the compounds of the present disclosure to mammals. The carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials involved in carrying the subject substance or transferring it from one organ or part of the body to another organ or another part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: saccharides 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; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; 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 non-toxic and compatible substances used in pharmaceutical formulations.

[0079] Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0080] The pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The formulations may conveniently be in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. The amount of active ingredient that may be combined with a carrier substance to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. Generally, on a percentage basis, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10 to about 30%.

[0081] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, e.g., arresting the development of the disease; or (c) alleviating the disease, e.g., relieving the symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug or compound to an individual to treat, cure, relieve, improve, alleviate or inhibit a disease in the individual, including but not limited to administering a drug containing the compound described herein to an individual in need thereof.

[0082] The present disclosure provides an siRNA for inhibiting ANGPTL3 expression. According to an embodiment of the present disclosure, the siRNA includes a sense strand and an antisense strand, the antisense strand including a complementary region that is complementary paired with the sense strand, wherein the sense strand is selected from nucleotide sequences that differ from the nucleotide sequences of each of SEQ ID NO: 1 - SEQ ID NO: 154 by no more than 5 nucleotides, and the antisense strand is selected from nucleotide sequences that differ from the nucleotide sequences of each of SEQ ID NO: 155 - SEQ ID NO: 308 by no more than 5 nucleotides.

[0083] According to an embodiment of the present disclosure, in addition to including SEQ ID NO: 1 - SEQ ID NO: 154 shown in Table 2, the sense strand further includes a continuous nucleotide sequence that differs from the sense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0084] According to an embodiment of the present disclosure, in addition to including SEQ ID NOs: 155-SEQ ID NO: 308 shown in Table 2, the antisense strand further includes a continuous nucleotide sequence that differs from the antisense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0085] According to an embodiment of the present disclosure, the siRNA includes at least one modified nucleotide;

[0086] The modified nucleotide is selected from at least one of the following:

[0087] 5'-thiophosphate group nucleotides, 5-methylcytosine nucleotides, 2'-O-methyl modified nucleotides, 2'-O-2-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 3'-nitrogen substituted modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, morpholino nucleotides, peptide nucleotides, phosphoramidates, and nucleotides including unnatural bases.

[0088] According to an embodiment of the present disclosure, the length of the complementary region is 18-21 bp, for example, 19 bp.

[0089] According to an embodiment of the present disclosure, the lengths of the sense strand and the antisense strand in the siRNA are 18-25 bp, for example, 21 bp.

[0090] According to a specific embodiment of the present disclosure, the lengths of the sense strand and the antisense strand in the siRNA are 21 bp, and the bases in the sense strand and the antisense strand are complementary one by one, or there are 19 consecutive bases complementary in the sense strand and the antisense strand of the siRNA, that is, the length of the complementary region is 19 bp.

[0091] According to an embodiment of the present disclosure, the liver cells are transfected with the siRNA to inhibit the expression of the ANGPTL3 gene in the cells.

[0092] For the angiopoietin-like 3 (ANGPTL3) gene target, the inventors of the present disclosure designed a suitable small interfering nucleic acid (siRNA) sequence, synthesized the siRNA, and used a transfection reagent to introduce the siRNA into cells to form a silencing complex (RNA-induce siliencing complex, RISC), which specifically recognizes and targets and binds to the mRNA sequence of the target gene, and cleaves the mRNA between the 10th and 11th positions from the 5' end, thereby causing post-transcriptional gene silencing and regulating the expression of the angiopoietin-like 3 secreted protein.

[0093] According to an embodiment of the present disclosure, the siRNA is covalently linked to a targeting ligand.

[0094] According to an embodiment of the present disclosure, the targeting ligand includes at least one N-acetyl-galactosamine.

[0095] According to an embodiment of the present disclosure, the targeting ligand is linked to the sense strand of the siRNA.

[0096] Embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be construed as a limitation of the present disclosure. For those techniques or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0097] Partial synthetic routes of this embodiment can refer to CN202110397429.9 and CN202110008013.3; the embodiments of this application are incorporated into the above two patent applications by reference.

[0098] Example 1 Testing the activity of small interfering nucleic acid (siRNA) in an in vitro cell model (Hep 3B cells)

[0099] 1) Preparation of suspension transfection reagent: The concentration of the siRNA mother liquor is 50 μM, and it is diluted with DEPC water to obtain a 10 μM siRNA system. 50 μL of Opti-MEM is used to dilute it to obtain a 0.2 μM siRNA system, and it is pipetted 3-5 times to mix evenly (final concentration 10 nM). 50 μL of Opti-MEM is used to dilute 0.5 μL of 0.2 μM SiRNA to obtain a 0.002 μM siRNA system, and it is pipetted 3-5 times to mix evenly (final concentration 0.1 nM); 50 μL of Opti-MEM is used to dilute 2 μL of RNAiMAX, and it is pipetted 3-5 times to mix evenly. The transfection reagent and the small interfering nucleic acid dilution are mixed separately, pipetted 3-5 times to mix evenly, and left to stand at room temperature for 10 min.

[0100] 2) Treating cells: Observe under the microscope that the confluence rate of the Hep 3B cell line is >70%, perform cell plating, plate 12-well plates at 2x10 5 cells / well, add 900 μL of DMEM medium containing 10% FBS to each well, add the transfection complex to the 12-well plates, and culture in an incubator at 37 °C and 5% CO2.

[0101] 3) After 24 h, extract the total RNA of the cells, and detect the expression of the ANGPTL3 mRNA sequence in the cells by Quantitative Real-Time PCR (qRT-PCR). The PCR primers for amplifying the internal reference gene PPIB and ANGPTL3 are shown in Table 1:

[0102] Table 1: PCR primer sequences for amplifying reference genes PPIB and ANGPTL3

[0103]

[0104] 4) The inhibition rate of small interfering nucleic acid on the expression level of ANGPTL3 was calculated according to the following equation: Inhibition rate = [1 - (expression level of ANGPTL3 mRNA in experimental group / expression level of PPIB mRNA in experimental group) / (expression level of ANGPTL3 mRNA in negative control group / expression level of PPIB mRNA in negative control group)] × 100%. Among them, each experimental group was cells treated with small interfering nucleic acid; the negative control group (denoted as Blank) was cells not treated with any small interfering nucleic acid.

[0105] After transfecting Hep 3B cells with 154 pairs of siRNAs in Table 2 at concentrations of 0.1 nM and 10 nM respectively using the above method, the results of the inhibition rate of the expression of ANGPTL3 gene (NM_014495.4) were obtained.

[0106] Table 2: 154 pairs of siRNA sequences targeting ANGPTL3

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Appendix Figure 1 and 2 respectively show the results of the expression levels of ANGPTL3 gene in cells detected by real-time quantitative PCR after transfecting Hep3B cells with some siRNAs in Table 2 at concentrations of 0.1 nM or 10 nM. It shows that the siRNAs shown in the attached figures can significantly reduce the expression of ANGPTL3 gene whether transfected into Hep 3B cells at a concentration of 0.1 nM or 10 nM.

[0116] Example 2 Synthesis of GalNAc-linked targeting head

[0117] I. Synthesis of GalNAc targeting head 1043

[0118] An enantiomer of TO-23 and TP-23 (precursor of 1043 targeting head linked to siRNA) was synthesized according to the following method.

[0119] 1. Synthesis of intermediate GN-17-01

[0120]

[0121] (1) Under N2 atmosphere, dissolve GC-1 (12 g, 25.89 mmol) in DCM (200 mL), cool to 0 - 5 °C in an ice-water bath, add HBTU (11.78 g, 31 mmol) and DIEA (10 g, 77.67 mmol), and stir for 10 minutes;

[0122] (2) Then add N-Boc-1,4-butanediamine (4.87 g, 25.89 mmol), warm to 25 °C and stir for 16 hours. TLC shows that the raw materials have basically disappeared;

[0123] (3) Quench with saturated ammonium chloride solution (100 mL), separate the layers, and extract with DCM (100 mL × 2);

[0124] (4) Combine the organic phases, wash with saturated brine (100 mL), dry over anhydrous Na2SO4, filter and concentrate. Purify by column chromatography (DCM / MeOH = 20 / 1) to obtain white solid compound GN-17-01 (15 g, yield 91%).

[0125] 2. Synthesis of intermediate GN-17

[0126]

[0127] (1) Dissolve GN-17-01 (15 g, 23.67 mmol) in DCM (150 mL), add TFA (50 mL), stir at 25 °C for 1 hour. TLC shows that the raw materials have basically disappeared, and concentrate;

[0128] (2) Azeotrope with acetonitrile (100 mL × 3) and TFA to remove the excess TFA to obtain a foamy solid GN-17 (TFA salt, 12.6 g).

[0129] 3. Synthesis of intermediate TO-23-01

[0130]

[0131] (1) Under N2 atmosphere, dissolve NC-4 (2.6 g, 4.7 mmol) in DCM (200 mL), cool it to 0 - 5 °C in an ice-water bath, add HATU (5.6 g, 14.83 mmol) and DIEA (4.85 g, 37.6 mmol), and stir for 20 minutes;

[0132] (2) Then add GN-17 (8.45 g, 15.5 mmol), warm it up to 25 °C and stir for 4 hours. Detect by TLC, and the raw materials basically disappear;

[0133] (3) Quench with saturated ammonium chloride solution (50 mL), separate the layers, and extract with DCM (100 mL × 2);

[0134] (4) Combine the organic phases, wash with saturated brine (100 mL), and dry with anhydrous Na2SO4;

[0135] (5) Filter and concentrate to obtain the crude product. Purify by column chromatography (DCM / MeOH = 10 / 1) to obtain white solid TO-23-01 (6.3 g, yield 63.1%).

[0136] 4. Synthesis of Compound TO-23

[0137]

[0138] (1) Add 10% Pd / C (600 mg) and Pd(OH)2 / C (600 mg) to a MeOH (100 mL) solution of TO-23-01 (6.3 g, 3.0 mmol), replace with H2 three times, stir at 25 °C for 3 hours, and detect by TLC (DCM / MeOH = 8 / 1) that the raw materials basically disappear;

[0139] (2) Filter and concentrate to obtain the crude product. Purify by column chromatography (DCM / MeOH / TEA = 10 / 1 / 0.1) to obtain white solid TO-23 (4.5 g, yield 75%).

[0140] 11H 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).

[0141] MS (ESI): m / z [1 / 2M + H] + Theoretical value: 1000.5, Measured value: 1000.3.

[0142] 5. Synthesis of Compound TP-23 (Precursor of 1043 Target Head Linked to siRNA)

[0143]

[0144] (1) Under N2 atmosphere, dissolve TO-23 (2.3 g, 1.15 mmol) in dry DCM (40 mL), add DIEA (0.86 mL, 5.2 mmol), and slowly add a dry DCM (2 mL) solution of 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (0.46 mL, 2.1 mmol) dropwise using a syringe. React at 25 °C for 1 hour. TLC detection shows that the raw materials have basically disappeared;

[0145] (2) Quench with saturated NaHCO3 (20 mL), separate the layers, wash the organic layer with saturated NaHCO3 (20 mL) solution and saturated brine (20 mL), dry over anhydrous MgSO4, filter and concentrate to obtain the crude product. Purify by column chromatography (the silica gel column is pre-alkalized with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) to obtain white solid TP-23 (1.8 g, yield 71.1%).

[0146] 11H 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 = 5.9 Hz, 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);

[0147] 31 31P NMR (162 MHz, DMSO-d6) δ 147.81;

[0148] MS (ESI): m / z [1 / 2M + Na] + Theoretical value 1122.5, measured value 1122.4.

[0149] II. Synthesis of GalNAc targeting head 1046

[0150] An epimer of TO25 and TP-25 (the precursor of the 1046 targeting head linked to siRNA) was synthesized according to the following method.

[0151] 1. Synthesis of intermediate NC-6-01

[0152]

[0153] (1) Under N2 atmosphere, dry THF (300 mL) was added to a 1000 mL three-necked flask, cooled to 0 - 5 °C in an ice bath and stirred. 60% NaH (14 g, 354.8 mmol) was added in portions, and then a THF solution (200 mL) of 2-chloroethoxyethanol (40 g, 322.5 mmol) was slowly added dropwise. The reaction was kept warm for 30 minutes, and then benzyl bromide (60.3 g, 354.8 mmol) was added dropwise to the reaction flask. The temperature was raised to 25 °C and stirred for 16 hours. TLC monitoring showed that the raw materials were almost completely consumed.

[0154] (2) Quench the reaction by slowly adding saturated ammonium chloride solution (150 mL), then perform liquid-liquid separation. Extract the aqueous phase with EtOAc (100 mL × 2). Combine the organic phases, wash with saturated brine (300 mL), dry over anhydrous Na2SO4, filter, and concentrate to obtain the crude product. Purify the crude product by silica gel column chromatography (petroleum ether / EtOAc = 5 / 1) to obtain the light yellow oily compound NC-6-01 (53 g, yield 78%).

[0155] MS(ESI): m / z [M+H] + Theoretical value: 215.1, Measured value: 215.1.

[0156] 2. Synthesis of Intermediate NC-6-02

[0157]

[0158] (1) Place ethylenediamine (196 g, 3.26 mol) in a 2000 mL three-necked flask, add acetonitrile (1000 mL), potassium carbonate (90 g, 0.65 mol), and sodium iodide (60.6 g, 0.33 mol), and stir. Then slowly add a solution of NC-6-01 (70 g, 0.33 mol) in acetonitrile (100 mL) to the reaction flask. Heat the mixture to 60 °C and stir for 16 hours. Monitor the reaction by TLC until the starting materials are almost completely consumed.

[0159] (2) Stop the reaction, concentrate the solution, add purified water (300 mL), adjust the pH to 4 - 5 with concentrated hydrochloric acid, and extract three times with EtOAc (200 mL × 3). Add solid sodium hydroxide to the aqueous phase to adjust the pH to 13 - 14, and extract three times with DCM (200 mL × 3). Combine the organic phases, wash with saturated brine (300 mL), dry over anhydrous Na2SO4, filter, and concentrate to obtain the light yellow oily compound NC-6-02 (69.5 g, 87%).

[0160] MS(ESI): m / z [M+H] + Theoretical value: 239.2, Measured value: 239.1.

[0161] 3. Synthesis of Intermediate NC-6-03

[0162]

[0163] (1) Add NC-6-02 (69.5 g, 0.29 mol) and tert-butyl bromoacetate (187 g, 0.96 mol) to a mixture of tetrahydrofuran (700 mL) and purified water (350 mL), stir, cool the mixture to below 5 °C in an ice-water bath, and add potassium carbonate (322 g, 2.34 mol). Stir the reaction mixture at 25 °C for 14 hours. Monitor the reaction by TLC until the starting materials are completely converted.

[0164] (2) The reaction solution was added with purified water (300 mL), allowed to stand for layering, and the organic phase was separated. The aqueous phase was extracted twice with EtOAc (200 mL × 2), the organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous Na2SO4, filtered and concentrated to obtain a pale yellow oily substance NC-6-03 (201 g).

[0165] MS(ESI): m / z [M+H] + The theoretical value is 581.4, and the measured value is 581.3.

[0166] 4. Synthesis of intermediate NC-6

[0167]

[0168] (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 temperature was raised to 60 °C for reaction for 2 hours. TLC detection showed that the raw materials were basically consumed.

[0169] (2) It was concentrated, 1,4-dioxane (200 mL) was added again and concentrated to obtain a crude white solid. The crude product was added to ethyl acetate (200 mL) and slurried for 2 hours, filtered by suction, the filter cake was collected, and dried in vacuo at 50 °C to obtain a white solid compound NC-6 (22.6 g, 96.9%).

[0170] (3) MS(ESI): m / z [M+H] + The theoretical value is 413.2, and the measured value is 413.1.

[0171] 5. Synthesis of intermediate TO-25-01

[0172]

[0173] (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. Subsequently, 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 showed that the raw materials were basically consumed.

[0174] (2) Dilute with DCM (100 mL), add 1N hydrochloric acid solution (80 mL × 2) to wash the reaction solution, combine the organic phases, wash with saturated sodium bicarbonate (100 mL), saturated brine (100 mL), dry over anhydrous Na2SO4, filter, and concentrate to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 7 / 1) to obtain the white solid compound TO-25-01 (4.3 g, yield 60%).

[0175] (3) MS (ESI): m / z [M / 2 + H] + Theoretical value 980.0, measured value 979.9.

[0176] 6. Synthesis of Intermediate TO-25

[0177]

[0178] (1) Dissolve TO-25-01 (4.3 g, 2.2 mmol) in methanol (80 mL), add 10% palladium on carbon (1.0 g), replace with H2 three times, stir at 25 °C for 2 hours, and detect by LCMS. The raw material has basically disappeared.

[0179] (2) Filter, concentrate, add DCM (20 mL) to dissolve, slowly dropwise add to MTBE (300 mL), stir and crystallize for 30 minutes, filter by suction to obtain the white solid compound TO-25 (3.7 g, yield 90%).

[0180] 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);

[0181] MS (ESI): m / z [1 / 2M + H] + Theoretical value 934.9, measured value 934.8.

[0182] 7. Synthesis of TP-25 (Precursor of 1046 Target Head Linked to siRNA)

[0183]

[0184] (1) Under N2 atmosphere, dissolve TO-25 (700 mg, 0.37 mmol) in dry DCM (10 mL), add DIEA (0.31 mL, 1.9 mmol), and slowly dropwise add a dry DCM (1 mL) solution of 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (0.19 mL, 0.74 mmol) using a syringe. React at 25 °C for 30 minutes. Monitor by TLC, and the raw materials basically disappear.

[0185] (2) Quench with saturated NaHCO3 (10 mL), dilute with DCM (10 mL), separate the layers, wash the organic phase with saturated NaHCO3 (10 mL) solution and saturated brine (10 mL), dry over anhydrous Na2SO4, filter and concentrate to obtain the crude product. Purify by column chromatography (the silica gel column is pre-alkalized with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) to obtain white solid TP-25 (405 mg, yield 53%).

[0186] 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);

[0187] 31 P NMR (162 MHz, DMSO-d6) δ 147.97;

[0188] MS (ESI): m / z [1 / 2M + Na] + Theoretical value 1057.0, measured value 1057.4.

[0189] III. Synthesis of GalNAc targeting head 1048

[0190] According to the following method, a diastereoisomer of TO26 and TP-26 (the precursor of the 1048 targeting head linked to siRNA) was synthesized.

[0191] 1. Synthesis of Intermediate GN-18-01

[0192]

[0193] (1) Under a N2 atmosphere, GC-2 (20.1 g, 39.7 mmol) was dissolved in DCM (200 mL), and CDI (7.09 g, 73.7 mmol) was added in batches. 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 reaction was carried out for 16 hours. LCMS detection showed that the raw materials disappeared.

[0194] (2) The reaction was quenched by adding saturated sodium bicarbonate solution (200 mL). After liquid separation, the aqueous phase was extracted with DCM (100 mL × 3). The combined organic phases were washed with saturated ammonium chloride solution (200 mL) and saturated sodium chloride solution (200 mL), dried over anhydrous Na2SO4, filtered and concentrated to obtain the crude product. The crude product was washed with methyl tert-butyl ether (100 mL), and the oily product was concentrated to obtain white solid compound GN-18-01 (24.43 g, yield 95.1%).

[0195] MS(ESI): m / z [M+H] + Theoretical value 650.3, measured value 650.5.

[0196] 2. Synthesis of Intermediate GN-18

[0197]

[0198] (1) GN-18-01 (45.52 g, 70 mmol) was added in batches to the HCl / EtOAc solution (2N, 500 mL), and the mixture was stirred at 25 °C for 2 hours. LCMS detection showed that the raw materials disappeared.

[0199] (2) The solvent was poured out, and the solid was concentrated to obtain the crude product. The crude product was purified by pulping with methyl tert-butyl ether (200 mL), filtered, and the filter cake was dried in vacuo at 40 °C to obtain white solid GN-18 (49.6 g).

[0200] MS(ESI): m / z [M+H] + Theoretical value 550.3, measured value 550.5.

[0201] 3. Synthesis of Intermediate TO-26-01

[0202]

[0203] (1) Under 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. Subsequently, 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 the mixture was stirred at 25 °C for 16 hours. LCMS detection showed that the raw materials were almost completely consumed.

[0204] (2) Add DCM (100 mL) for dilution, add 1N hydrochloric acid solution (80 mL × 2) to the reaction solution for washing. Combine the organic phases, wash with saturated sodium bicarbonate (100 mL) and saturated brine (100 mL), dry over anhydrous Na2SO4, filter and concentrate to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 7 / 1) to obtain the white solid compound TO-26-01 (4.7 g, yield 65%).

[0205] MS (ESI): m / z [M / 2 + H] + Theoretical value: 1004.0, measured value: 1004.2.

[0206] 4. Synthesis of Intermediate TO-26

[0207]

[0208] (1) Dissolve TO-26-01 (4.0 g, 2.0 mmol) in methanol (80 mL), add 10% palladium on carbon (1.0 g), replace with H2 three times, and stir at 25 °C for 2 hours. LCMS detection showed that the raw materials almost disappeared.

[0209] (2) Filter, concentrate, add DCM (20 mL) to dissolve, slowly add dropwise to MTBE (200 mL), stir for crystallization for 30 minutes, filter by suction to obtain the white solid compound TO-26 (3.5 g, yield 91%).

[0210] 1H NMR(400MHz,DMSO-d6)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).

[0211] MS (ESI): m / z [1 / 2M+H] + Theoretical value is 958.9, measured value is 959.1.

[0212] 5. Synthesis of TP-26 (1048 target head linked to siRNA precursor)

[0213]

[0214] (1) Under 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. The mixture was reacted at 25°C for 30 min. TLC detection showed that the starting material had basically disappeared.

[0215] (2) Saturated NaHCO₃ (10 mL) was added to quench the mixture, and the mixture was diluted with DCM (10 mL). The organic phase was washed with saturated NaHCO₃ (10 mL) and saturated brine (10 mL), dried over anhydrous NaSO₄, filtered, and concentrated to obtain the crude product. The product was purified by column chromatography (silica gel column pre-basified with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) to obtain TP-26 as a white solid (600 mg, 60% yield).

[0216] 1 H NMR (400 MHz, DMSO-d6) 1H NMR (400MHz, DMSO-d6) 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).

[0217] 31 P NMR (162MHz, DMSO-d6) δ147.89;

[0218] MS (ESI): m / z 1 / 2[Mi-Pr2N] calcd. 1007.9, found 1008.2.

[0219] Example 3 In vitro construction of GalNAc target head coupled (modified) siRNA conjugates

[0220] The oligonucleotide sequences of the antisense and sense strands of the following RNAi agent duplexes, as well as the linkage of the targeting ligand to the RNA, were synthesized on a solid phase for oligonucleotide synthesis using the phosphoramidite coupling technique reported in J. Org. Chem. 2012, 77, 4566-4577; Curr. Protoc. Nucleic Acid Chem., 81, e107. Targeting ligands 1046, 1048, and 1043 were all linked to the 5' end of the siRNA sense strand via a phosphorothioate bond.

[0221]

[0222]

[0223] GalNAc-siRNA conjugates were synthesized as Figure 3 The conjugate structure in the second column includes three parts. For example, the structure of G1043-S2A2-A265 is as follows: the target head 1043 is connected to the 5' end of the sense strand of the siRNA numbered A265 through a phosphorothioate bond, and S2A2 is the modification type of the siRNA A265. The specific modification groups and modification methods are as follows:

[0224] In the nucleic acid sequence, Ao represents adenosine, Uo represents uridine, Go represents guanosine, Co represents cytidine, and there is no symbol between directly adjacent nucleotides, indicating that they are connected by normal phosphodiester bonds.

[0225] DNA: A G C T (A represents 2'-deoxyadenosine, T represents 2'-deoxythymidine, G represents 2'-deoxyguanosine, C represents 2'-deoxycytidine);

[0226] 2'-F: aF gF cF uF (aF represents 2'-fluoroadenosine, uF represents 2'-fluorouridine, gF represents 2'-fluoroguanosine, cF represents 2'-fluorocytidine);

[0227] 2'-OMe: aM gM cM uM (aM represents 2'-O-methyladenosine, uM represents 2'-O-methyluridine, gM represents 2'-O-methylguanosine, cM represents 2'-O-methylcytidine);

[0228] *: represents connection by phosphorothioate bond;

[0229] In the sequence, y and z represent the positions of the target head.

[0230] Example 4: Testing the activity of the conjugate using an in vitro cell model (Hep 3B cells)

[0231] Human hepatocellular carcinoma Hep3B cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in DMEM (Gibco, US) supplemented with 10% fetal bovine serum (FBS) (Gibco, US) at 37 °C under 5% CO2 conditions (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 a density of 50,000 cells / well in 450 μL / well. Subsequently, the test samples were added by transfection with lipofectmine2000 (Thermo Fisher). Transfection was performed according to the standard procedure of the RNAiMAX reagent instructions, and the final concentration of siRNA was 10 nM / 1 nM / 0.5 nM / 0.25 nM / 0.1 nM / 0.05 nM / 0.01 nM. The transfection group used siNC as a negative control, and its sequence was:

[0232] Sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’

[0233] Antisense strand: 5’-ACGUGACACGUUCGGAGAATT-3’.

[0234] After 24 hours, total RNA was extracted from the cells, and the expression of ANGPTL3 mRNA sequences in the cells was detected by quantitative real-time PCR. The PCR primers used to amplify the internal reference genes PPIB and ANGPTL3 are shown in Table 1:

[0235] Activity test results of each conjugate (EC 50 Value) see Figure 4 .

[0236] EC 50 The values were calculated using nonlinear regression using GraphPad Prism, and represent the amount of the conjugate required to inhibit the expression of the target mRNA (ANGPTL3) by half.

[0237] The results show that the selected conjugates showed good results in reducing the relative expression level of ANGPTL3 in the in vitro activity test experiment.

[0238] Example 5: Construction and drug administration test of AAV-hANGPTL3 mouse model

[0239] Basic information of experimental animals:

[0240] Experimental animals were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and were SPF-grade animals. Before administration, the mice were weighed and observed, and animals with uniform weight and normal condition were selected for subsequent experiments.

[0241]

[0242] Animals were kept under normal conditions and had free access to food and water. Animals were acclimated for 3-7 days before the start of the experiment.

[0243] Modeling and Dosing: Each mouse was injected with 100 μL of a 2.5×10^11 titer virus solution via the tail vein. Seven days later, the animals were randomly divided into groups and each test substance was administered subcutaneously at a dose of 5 mg / kg. Seventy-two hours after dosing, the animals were sacrificed by cervical dislocation, and liver tissue was harvested for RNA extraction and quantification.

[0244] The results for each conjugate are shown in Table 3.

[0245] Table 3. Mouse model administration test results of each conjugate

[0246]

[0247]

[0248] As can be seen from the results, the selected conjugate also showed good results in reducing the relative expression level of ANGPTL3 in the in vivo activity test.

[0249] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some 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 disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0250] Although the embodiments of the present disclosure have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present disclosure. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present disclosure.

Claims

1. A siRNA, and the siRNA is 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 gene sequence; 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) capable of specifically targeting nucleotides 107-133 of the angiopoietin-like protein 3 gene sequence; 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 gene sequence; 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) capable of specifically targeting nucleotides 304-388 of the angiopoietin-like protein 3 gene sequence; capable of specifically targeting nucleotides 304-359 of the angiopoietin-like protein 3 gene sequence; 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, 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) capable of specifically targeting nucleotides 430-459 of the angiopoietin-like protein 3 gene sequence; 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) capable of specifically targeting nucleotides 1360-1430 of the angiopoietin-like protein 3 gene sequence, preferably capable of specifically targeting nucleotides 1397-1430 of the angiopoietin-like protein 3 gene sequence; 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, Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 150, and the antisense strand is selected from SEQ ID NO:

304. Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 151, and the antisense strand is selected from SEQ ID NO:

305. Alternatively, 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. It is characterized in that The siRNA includes at least one modified nucleotide. The modified nucleotide is selected from at least one of the following: 5'-thiophosphate nucleotide, 5-methylcytosine nucleotide, 2'-O-methyl modified nucleotide, 2'-O-2-methoxyethyl modified nucleotide, 2'-fluoro modified nucleotide, 3'-nitrogen substituted modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino modified nucleotide, morpholino nucleotide, peptide nucleotide, aminophosphate, and nucleotide including unnatural base.

2. The siRNA according to claim 1, wherein The length of the complementary region is at least 17 bp.

3. The siRNA according to claim 2, wherein The length of the complementary region is 18 - 21 bp.

4. The siRNA according to claim 3, wherein The length of the complementary region is 19 bp.

5. A siRNA conjugate, characterized in that, The siRNA conjugate includes the siRNA according to any one of claims 1 - 4 and a targeting ligand.

6. The siRNA conjugate according to claim 5, wherein, The siRNA is covalently linked to the targeting ligand; the targeting ligand is linked to the sense strand of the siRNA; the targeting ligand is linked to the 5' end of the sense strand of the siRNA through a thiophosphate bond.

7. The siRNA conjugate according to claim 6, characterized in that The targeting ligand includes at least one N-acetyl-galactosamine.

8. The siRNA conjugate according to claim 7, wherein The targeting ligand is a GalNAC targeting compound; the GalNAC targeting compound is 1043, 1046 or 1048.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the siRNA according to any one of claims 1 - 4 and / or the siRNA conjugate according to any one of claims 5 - 8.

10. Use of the siRNA according to any one of claims 1 - 4, and / or the siRNA conjugate according to any one of claims 5 - 8, and / or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating dyslipidemia diseases.

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