A targeting ligand

Abstract

The present disclosure relates to the field of genetic engineering technology, and more specifically, to a targeting ligand. The targeting ligand provided herein forms a siRNA conjugate with a specific small interfering RNA sequence, which targets ANGPTL3 and degrades ANGPTL3 gene transcripts in cells, thereby reducing ANGPTL3 protein expression. Therefore, the siRNA conjugate formed with the targeting ligand provided herein can be used to prevent and / or treat dyslipidemia.

Description

[0001] This application is a divisional application. The parent application is a PCT application entering the Chinese national phase. The parent application number is 2021800229023, the PCT international application date is September 30, 2021, and the date of entry into the Chinese national phase is October 18, 2022. The name of the parent invention is “Angiopoietin-like 3 siRNA and its uses”. Technical Field

[0002] The present disclosure relates to the field of genetic engineering technology, and in particular, to a novel compound that can serve as a targeting ligand. Background Art

[0003] Hyperlipidemia, also known as dyslipidemia, is a systemic disease characterized by abnormal fat metabolism or transport, resulting in elevated plasma lipids above normal levels. The clinical manifestations of dyslipidemia include two main aspects: (1) xanthomas caused by lipid deposition in the dermis; and (2) atherosclerosis caused by lipid deposition in the vascular endothelium, leading to coronary heart disease and peripheral vascular disease.

[0004] Angiopoietin-like protein 3 (ANGPTL3, NM_014495.4) is a secreted protein primarily expressed in hepatocytes. Studies have shown that ANGPTL3 is a key regulator of LDL-C, HDL-C, and triglyceride metabolism, with multiple potential pathways of action. Loss-of-function mutations in ANGPTL3 can lead to decreased LDL-C, VLDL-C, HDL-C, and triglycerides (TG), resulting in a reduced risk of cardiovascular disease based on GWAS data, without the adverse phenotypes associated with known genetic defects. Therefore, inhibiting ANGPTL3 activity could be an effective approach for preventing or treating dyslipidemia.

[0005] In the prior art, ANGPTL3 antibodies are often used to inhibit its activity.

[0006] Chinese patent application number 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 diseases or disorders associated with ANGPTL3, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including hypertriglyceridemia, hypercholesterolemia, and chylomicronemia.

[0007] 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.

[0008] Compared to antibodies, siRNA drugs have advantages such as abundant candidate targets, short R&D cycles, and high clinical development success rates. Therefore, they are particularly advantageous in the treatment of chronic diseases. However, inherent defects of siRNA drugs (such as poor stability and difficulty in transmembrane transport) limit their clinical application. Structural modification of siRNA or construction of conjugates can improve the drugability of siRNA to a certain extent. Exploring the constituent molecules for constructing conjugates is a necessary step in the development of siRNA drugs for dyslipidemia and a problem that needs to be solved urgently. Summary of the Invention

[0009] The present disclosure aims to solve at least one of the technical problems in the related art to a certain extent. To this end, one purpose of the present disclosure is to provide an siRNA for inhibiting the expression of ANGPTL3. The inventors of the present disclosure designed a suitable specific small interfering RNA sequence and combined it with a targeting ligand to form an siRNA conjugate, targeting ANGPTL3 , by degrading ANGPTL3 The transcripts of the gene can be inhibited, thereby reducing the expression of ANGPTL3 protein. Therefore, the siRNA provided by the present disclosure can be used to prevent and / or treat dyslipidemia diseases.

[0010] 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.

[0011] The inventors of the present disclosure designed an appropriate small interfering RNA (siRNA) sequence to specifically reduce the synthesis of ANGPTL3 in hepatocytes while avoiding off-target effects. siRNA forms a silencing complex (RISC) that binds to the target gene ( ANGPTL3 The mRNA of the target gene is degraded by the complementary pairing with the target gene, thereby inhibiting the expression of the target gene and reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG).

[0012] The siRNA according to the embodiments of the present disclosure may also have at least one of the following additional technical features:

[0013] The present disclosure also provides an siRNA, wherein the siRNA is selected from any pair of siRNAs in any of the following groups:

[0014] (1) It can specifically target nucleotides 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;

[0015] (2) It can specifically target nucleotides 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;

[0016] (3) It can specifically target nucleotides 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;

[0017] (4) It can specifically target nucleotides 304-388 of the angiopoietin-like protein 3 gene sequence, preferably, it can specifically target 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,

[0018] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 29, and the antisense strand is selected from SEQ ID NO: 183,

[0019] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 31, and the antisense strand is selected from SEQ ID NO: 185,

[0020] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 32, and the antisense strand is selected from SEQ ID NO: 186,

[0021] Alternatively, the sense strand of the siRNA is selected from SEQ ID NO: 35, and the antisense strand is selected from SEQ ID NO: 189,

[0022] 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;

[0023] (5) It can specifically target 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,

[0024] 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;

[0025] (6) It is capable of specifically targeting nucleotides 1360-1430 of the angiopoietin-like protein 3 gene sequence, preferably, it is 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,

[0026] 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,

[0027] 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,

[0028] 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,

[0029] 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.

[0030] According to an embodiment of the present disclosure, the siRNA includes at least one modified nucleotide;

[0031] Optionally, the modified nucleotide is selected from at least one of the following:

[0032] 5'-phosphorothioate-based nucleotides, 5-methylated cytosine 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, polypeptide nucleotides, phosphoramidates, and nucleotides including non-natural bases.

[0033] According to an embodiment of the present disclosure, the length of the complementary region is at least 17 bp;

[0034] Optionally, the length of the complementary region is 18-21 bp;

[0035] Optionally, the length of the region of complementarity is 19 bp.

[0036] According to an embodiment of the present disclosure, the length of the sense strand and the antisense strand in the siRNA is no more than 25 bp;

[0037] Optionally, the length of the sense strand and the antisense strand in the siRNA is 18-25 bp;

[0038] Optionally, the length of the sense strand and the antisense strand in the siRNA is 21 bp.

[0039] According to the embodiments of the present disclosure, the bases in the sense strand and the antisense strand of the siRNA can be one-to-one complementary pairings, or can be misplaced by several bases, but have a complementary region of at least 17 bp.

[0040] Another aspect of the present disclosure provides an siRNA conjugate, comprising the aforementioned siRNA and a targeting ligand, wherein the siRNA is covalently linked to the targeting ligand;

[0041] Preferably, the targeting ligand is linked to the sense strand of the siRNA;

[0042] More preferably, the targeting ligand is linked to the 5' end of the sense strand in the siRNA via a phosphorothioate bond.

[0043] According to an embodiment of the present disclosure, the targeting ligand includes at least one N-acetyl-galactosamine.

[0044] According to an embodiment of the present disclosure, the targeting ligand is a GalNAC target head compound.

[0045] According to an embodiment of the present disclosure, the GalNAC target head compounds are 1043, 1046, and 1048, and their structures are shown in the following formula:

[0046] GalNAC Target Head 1043

[0047]

[0048] GalNAC Target Head 1046

[0049]

[0050] GalNAC Target Head 1048

[0051] According to an embodiment of the present disclosure, the targeting ligand is linked to the sense strand of the siRNA.

[0052] 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.

[0053] Therefore, 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 prevent and / or treat hyperlipidemia and hypertriglyceridemia.

[0054] Yet another aspect of the present disclosure provides a compound.

[0055] The compound has any of the following structures:

[0056]

[0057]

[0058]

[0059] TO26.

[0060] The present disclosure also provides a compound, the compound structure of which is as follows:

[0061] GalNAC Target Head 1043

[0062]

[0063] GalNAC Target Head 1046

[0064] GalNAC target head 1048.

[0065] Another aspect of the present invention provides use of the aforementioned compound in preparing siRNA conjugates.

[0066] TP23, TP25, TP26 are selected from:

[0067]

[0068] TP-23

[0069]

[0070] TP-25

[0071]

[0072] TP-26.

[0073] The present disclosure also provides the above intermediates.

[0074] In another aspect, the present disclosure provides the use of the aforementioned compound TO-23 and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to prepare TP-23, and its target head 1043-siRNA conjugate in the preparation of a drug or a kit; the aforementioned compound TO-25 and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to prepare TP-25, and its target head 1046-siRNA conjugate in the preparation of a drug or a kit; the aforementioned compound TO-26 and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to prepare TP-26, and its target head 1048-siRNA conjugate in the preparation of a drug or a kit.

[0075] The drug or kit is used to inhibit ANGPTL3 Gene expression.

[0076] Preferably, the medicine or kit is used to prevent and / or treat dyslipidemia; further preferably, the dyslipidemia includes hyperlipidemia and hypertriglyceridemia.

[0077] 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.

[0078] Thus, the kit according to the embodiment of the present disclosure can be used to inhibit ANGPTL3 Gene expression, thereby reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG) to treat, prevent and / or treat hyperlipidemia and hypertriglyceridemia.

[0079] Another aspect of the present disclosure provides a method for inhibiting a subject ANGPTL3 A method for gene expression, comprising administering the aforementioned siRNA and / or the aforementioned siRNA conjugate to a subject to inhibit ANGPTL3 Gene expression.

[0080] In another aspect, the present disclosure provides a method for inhibiting cell ANGPTL3 According to an embodiment of the present disclosure, the method comprises: transfecting the cell with the siRNA and / or the siRNA conjugate to inhibit the expression of the gene in the cell. ANGPTL3 Gene expression.

[0081] According to the embodiments of the present disclosure, the cell ANGPTL3 Gene expression method, using siRNA to form a silencing complex with the target gene ANGPTL3 The complementary sequence pairing of the target gene's mRNA causes the degradation of the target gene's mRNA, thereby inhibiting the expression of the target gene, and then reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG).

[0082] According to an embodiment of the present disclosure, the cell is derived from a mammal;

[0083] Optionally, the cell is of human origin;

[0084] Optionally, the cell is a liver cell.

[0085] The siRNA provided by the present disclosure is used to form a silencing complex in human liver cells. ANGPTL3 The sequences of the mRNA of the gene are complementary, so ANGPTL3 The mRNA of the gene is degraded, thereby inhibiting its expression, and then reducing the levels of LDL-C, VLDL-C, HDL-C and triglycerides (TG).

[0086] Another aspect of the present disclosure provides the use of the siRNA and / or the siRNA conjugate in preparing a drug or a kit. According to an embodiment of the present disclosure, the drug or kit is used to inhibit ANGPTL3 Gene expression.

[0087] The siRNA provided by the present disclosure is used to prepare a drug or a kit, wherein the drug or the kit reduces the expression of siRNA in cells. ANGPTL3 The expression level of the gene can be controlled to prevent and / or treat dyslipidemia.

[0088] According to an embodiment of the present disclosure, the medicine or kit is used to prevent and / or treat dyslipidemia;

[0089] Optionally, the dyslipidemia disease includes hyperlipidemia and hypertriglyceridemia;

[0090] Optionally, the drug or kit is used to inhibit ANGPTL3 Gene expression.

[0091] Another aspect of the present disclosure provides a method for preventing and / or treating dyslipidemia. According to an embodiment of the present disclosure, the method comprises: administering the siRNA and / or the siRNA conjugate to a subject.

[0092] According to an embodiment of the present disclosure, the dyslipidemia disease includes hyperlipidemia and hypertriglyceridemia.

[0093] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure.

[0094] Beneficial effects of the present invention:

[0095] After the targeting ligand provided by the present invention forms a conjugate with siRNA, the conjugate can reduce ANGPTL3 expression, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0097] Figure 1 The results show that after some siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 0.1 nM, the expression of siRNAs in cells was detected by real-time quantitative PCR. ANGPTL3 The expression results of the gene (abbreviated as ANL3 in the figure);

[0098] Figure 2 The results show that after some siRNAs in Table 2 were transfected into Hep 3B cells at a concentration of 10 nM, the expression of siRNAs in cells was detected by real-time quantitative PCR. ANGPTL3 The expression results of the gene (abbreviated as ANL3 in the figure);

[0099] Figure 3 shows the GalNAc-siRNA conjugate synthesized in Example 3;

[0100] Figure 4 The activity test results of each conjugate in Example 4 are shown (EC 50 value). DETAILED DESCRIPTION

[0101] The 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 understood as limiting the present disclosure.

[0102] "Pharmaceutically acceptable carriers" are generally recognized in the art and include pharmaceutically acceptable materials, compositions, or vehicles suitable for administering the compounds of the present disclosure to mammals. Such carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that participate in carrying the subject substance or transferring it from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can be used as pharmaceutically 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, powdered gum tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene 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 buffered saline; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0103] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0104] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dose form is generally the amount of the compound that produces the therapeutic effect. Generally speaking, in percent units, this amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10 to about 30%.

[0105] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or 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, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0106] The present disclosure provides an siRNA for inhibiting ANGPTL3 expression. According to an embodiment of the present disclosure, the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region that pairs 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.

[0107] According to an embodiment of the present disclosure, the sense strand includes, in addition to SEQ ID NO: 1-SEQ ID NO: 154 shown in Table 2, a continuous nucleotide sequence that differs from the sense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0108] According to an embodiment of the present disclosure, the antisense strand includes, in addition to SEQ ID NO: 155-SEQ ID NO: 308 shown in Table 2, a continuous nucleotide sequence that differs from the antisense strand shown in Table 2 by 1, 2, 3, 4, or 5 nucleotides.

[0109] According to an embodiment of the present disclosure, the siRNA includes at least one modified nucleotide;

[0110] The modified nucleotide is selected from at least one of the following:

[0111] 5'-phosphorothioate-based nucleotides, 5-methylated cytosine 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, polypeptide nucleotides, phosphoramidates, and nucleotides including non-natural bases.

[0112] According to an embodiment of the present disclosure, the length of the complementary region is 18-21 bp, for example, 19 bp.

[0113] According to an embodiment of the present disclosure, the length of the sense strand and the antisense strand in the siRNA is 18-25 bp, for example, 21 bp.

[0114] According to a specific embodiment of the present disclosure, the length of the sense chain and the antisense chain in the siRNA is 21 bp, and the bases in the sense chain and the antisense chain are complementary one to one, or the sense chain and the antisense chain in the siRNA have 19 consecutive base complementarities, that is, the length of the complementary region is 19 bp.

[0115] According to an embodiment of the present disclosure, liver cells are transfected with the siRNA to inhibit the expression of the ANGPTL3 gene in the cells.

[0116] Targeting the angiopoietin-like 3 (ANGPTL3) gene, the inventors of the present disclosure designed a suitable small interfering nucleic acid (siRNA) sequence, synthesized siRNA, and introduced the siRNA into cells using a transfection reagent to form an RNA-induce siliencing complex (RISC), which specifically recognizes and targets the mRNA sequence of the target gene and cleaves the mRNA between positions 10-11 from the 5' end, thereby leading to post-transcriptional gene silencing and regulating the expression of the angiopoietin-like 3 secretory protein.

[0117] According to an embodiment of the present disclosure, the siRNA is linked to the targeting ligand via a covalent bond.

[0118] According to an embodiment of the present disclosure, the targeting ligand includes at least one N-acetyl-galactosamine.

[0119] According to an embodiment of the present disclosure, the targeting ligand is linked to the sense strand of the siRNA.

[0120] The following embodiments of the present disclosure are described in detail. The embodiments described below are exemplary and are intended only to explain the present disclosure and are not to be construed as limiting the present disclosure. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or in the product instructions were used. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products.

[0121] Part of the synthetic route of this embodiment can be referred to CN202110397429.9 and CN202110008013.3; the embodiments of this application incorporate the above two patent applications in the form of source citations.

[0122] Example 1 In vitro cell model (Hep 3B cells) testing the activity of small interfering nucleic acids (siRNA)

[0123] 1) Prepare suspension transfection reagent: Dilute siRNA stock solution to 50 μM in DEPC water to obtain a 10 μM siRNA system. Dilute it with 50 μL of Opti-MEM to obtain a 0.2 μM siRNA system. Pipet and mix 3-5 times (final concentration 10 nM). Dilute 0.5 μL of 0.2 μM siRNA with 50 μL of Opti-MEM to obtain a 0.002 μM siRNA system. Pipet and mix 3-5 times (final concentration 0.1 nM). Dilute 2 μL of RNAiMAX with 50 μL of Opti-MEM. Pipet and mix 3-5 times. Separately, mix the transfection reagent and small interfering RNA diluent, pipette and mix 3-5 times, and let stand at room temperature for 10 minutes.

[0124] 2) Cell treatment: Hep 3B cell line confluence was >70% under microscope, and cells were plated at 2x10 5 Cells / well were plated in a 12-well plate, 900 μL of DMEM medium containing 10% FBS was added to each well, the transfection complex was added to the 12-well plate, and the plate was cultured in a 37°C, 5% CO2 incubator.

[0125] 3) 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:

[0126] Table 1: PCR primer sequences used to amplify the internal reference genes PPIB and ANGPTL3

[0127]

[0128] 4) The inhibitory rate of ANGPTL3 expression by siRNA was calculated according to the following formula: Inhibition rate = [1-(ANGPTL3 mRNA expression in the experimental group / PPIB mRNA expression in the experimental group) / (ANGPTL3 mRNA expression in the negative control group / PPIB mRNA expression in the negative control group)] × 100%. Each experimental group consisted of cells treated with siRNA; the negative control group (denoted as Blank) consisted of cells not treated with any siRNA.

[0129] The 154 pairs of siRNAs in Table 2 were obtained by the above method and transfected into Hep 3B cells at concentrations of 0.1 nM and 10 nM respectively. ANGPTL3 The inhibition rate results of gene (NM_014495.4) expression.

[0130] Table 2: 154 pairs of targeted ANGPTL3 siRNA sequences

[0131]

[0132] Attachment Figure 1 and 2 The results show that the expression of some siRNAs in Table 2 in Hep 3B cells was detected by real-time quantitative PCR after transfection of Hep 3B cells with 0.1 nM or 10 nM concentration. ANGPTL3 The results of gene expression showed that the siRNA shown in the figure could significantly reduce the expression of Hep 3B cells whether it was transfected at a concentration of 0.1 nM or 10 nM. ANGPTL3 Gene expression.

[0133] Example 2 Synthesis of GalNAc-linked Target Head

[0134] 1. Synthesis of GalNAc Target Head 1043

[0135] TO-23 and a diastereomer of TP-23 (a precursor of 1043 target-linked siRNA) were synthesized according to the following method.

[0136] 1. Synthesis of intermediate GN-17-01

[0137]

[0138] (1) Under N2 atmosphere, GC-1 (12 g, 25.89 mmol) was dissolved in DCM (200 mL), cooled 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 stirred for 10 min;

[0139] (2) Then, N-tert-butyloxycarbonyl-1,4-butanediamine (4.87 g, 25.89 mmol) was added, and the temperature was raised to 25°C and stirred for 16 hours. TLC showed that the starting material had basically disappeared.

[0140] (3) Add saturated ammonium chloride solution (100 mL) to quench the mixture, separate the liquids, and extract with DCM (100 mL × 2);

[0141] (4) The organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated. Purification by column chromatography (DCM / MeOH = 20 / 1) afforded compound GN-17-01 (15 g, 91% yield) as a white solid.

[0142] 2. Synthesis of intermediate GN-17

[0143]

[0144] (1) Dissolve GN-17-01 (15 g, 23.67 mmol) in DCM (150 mL), add TFA (50 mL), and stir at 25 °C for 1 hour. TLC shows that the starting material has basically disappeared, and concentrate;

[0145] (2) Excess TFA was removed by azeotropic distillation with acetonitrile (100 mL × 3) to obtain GN-17 (TFA salt, 12.6 g) as a foamy solid.

[0146] 3. Synthesis of intermediate TO-23-01

[0147]

[0148] (1) Under N2 atmosphere, NC-4 (2.6 g, 4.7 mmol) was dissolved in DCM (200 mL), cooled to 0-5°C in an ice-water bath, and HATU (5.6 g, 14.83 mmol) and DIEA (4.85 g, 37.6 mmol) were added and stirred for 20 min;

[0149] (2) GN-17 (8.45 g, 15.5 mmol) was then added, and the temperature was raised to 25°C with stirring for 4 hours. TLC analysis showed that the starting material had essentially disappeared.

[0150] (3) Add saturated ammonium chloride solution (50 mL) to quench, separate the liquids, and extract with DCM (100 mL × 2);

[0151] (4) Combine the organic phases, wash with saturated brine (100 mL), and dry over anhydrous Na2SO4;

[0152] (5) Filtration and concentration gave the crude product, which was purified by column chromatography (DCM / MeOH = 10 / 1) to give TO-23-01 as a white solid (6.3 g, yield 63.1%).

[0153] 4. Synthesis of compound TO-23

[0154]

[0155] (1) To a solution of TO-23-01 (6.3 g, 3.0 mmol) in MeOH (100 mL) were added 10% Pd / C (600 mg) and Pd(OH)2 / C (600 mg). The mixture was replaced with H2 three times and stirred at 25 °C for 3 h. TLC (DCM / MeOH = 8 / 1) showed that the starting material had essentially disappeared.

[0156] (2) Filtration and concentration gave the crude product, which was purified by column chromatography (DCM / MeOH / TEA = 10 / 1 / 0.1) to give TO-23 (4.5 g, 75% yield) as a white solid.

[0157] 1 H NMR (400 MHz, DMSO- d 6) 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).

[0158] MS (ESI): m / z [1 / 2M + H] + Theoretical value is 1000.5, measured value is 1000.3.

[0159] 5. Synthesis of compound TP-23 (precursor of siRNA linked to the 1043 target head)

[0160]

[0161] (1) Under 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 incubated at 25°C for 1 hour. TLC analysis showed that the starting material had essentially disappeared.

[0162] (2) Saturated NaHCO₃ (20 mL) was added to quench the reaction mixture. The organic phase was washed with saturated NaHCO₃ (20 mL) solution and saturated brine (20 mL), dried over anhydrous MgSO₄, filtered, and concentrated to obtain the crude product. Column chromatography (silica gel column pre-alkalinized with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) afforded TP-23 as a white solid (1.8 g, yield 71.1%).

[0163] 1 H NMR (400 MHz, DMSO- d 6) 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);

[0164] 31 P NMR (162 MHz, DMSO- d 6) δ 147.81;

[0165] MS (ESI): m / z [1 / 2M + Na] + Theoretical value is 1122.5, measured value is 1122.4.

[0166] 2. Synthesis of GalNAc Target Head 1046

[0167] TO25 and a diastereomer of TP-25 (a precursor of 1046 target-linked siRNA) were synthesized according to the following method.

[0168] 1. Synthesis of intermediate NC-6-01

[0169]

[0170] (1) Under N2 atmosphere, add dry THF (300 mL) to a 1000 mL three-necked flask, cool to 0-5°C in an ice bath and stir, add 60% NaH (14 g, 354.8 mmol) in batches, then slowly add 2-chloroethoxyethanol (40 g, 322.5 mmol) in THF (200 mL) dropwise, keep warm and react for 30 minutes, then add benzyl bromide (60.3 g, 354.8 mmol) dropwise to the reaction flask, raise the temperature to 25°C and stir for 16 hours. TLC monitoring shows that the raw material is basically consumed.

[0171] (2) Saturated ammonium chloride solution (150 mL) was slowly added dropwise to quench the reaction mixture. The mixture was separated and the aqueous phase was extracted with EtOAc (100 mL × 2). The organic phases were combined and washed with saturated brine (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / EtOAc = 5 / 1) to obtain compound NC-6-01 (53 g, yield 78%) as a light yellow oil.

[0172] MS (ESI): m / z [M + H] + Theoretical value is 215.1, measured value is 215.1.

[0173] 2. Synthesis of intermediate NC-6-02

[0174]

[0175] (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) dropwise to the reaction flask, heat to 60°C, and stir for 16 hours. TLC analysis shows that the starting material is almost consumed.

[0176] (2) Stop the reaction, concentrate, add purified water (300 mL), adjust the pH to 4-5 with concentrated hydrochloric acid, extract three times with EtOAc (200 mL×3), add solid sodium hydroxide to the aqueous phase to adjust the pH to 13-14, extract three times with DCM (200 mL×3), combine the organic phases, wash with saturated brine (300 mL), dry with anhydrous Na2SO4, filter and concentrate to obtain a light yellow oily substance NC-6-02 (69.5 g, 87%).

[0177] MS (ESI): m / z [M + H] + Theoretical value is 239.2, measured value is 239.1.

[0178] 3. Synthesis of intermediate NC-6-03

[0179]

[0180] (1) Add NC-6-02 (69.5 g, 0.29 mol) and tert-butyl bromoacetate (187 g, 0.96 mol) to tetrahydrofuran (700 mL) and purified water (350 mL). Stir and cool the mixture to below 5°C in an ice-water bath. Add potassium carbonate (322 g, 2.34 mol). Stir and react at 25°C for 14 hours. Complete conversion of the starting material was confirmed by TLC.

[0181] (2) Purified water (300 mL) was added to the reaction solution, and the mixture was allowed to stand for stratification. The organic phase was separated, and 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 light yellow oily substance NC-6-03 (201 g).

[0182] MS (ESI): m / z [M + H] + Theoretical value is 581.4, measured value is 581.3.

[0183] 4. Synthesis of intermediate NC-6

[0184]

[0185] (1) Dissolve NC-6-03 (23 g, 39.6 mmol) in 1,4-dioxane (200 mL), add concentrated hydrochloric acid (40 mL), and heat to 60 °C for 2 hours. TLC analysis shows that the raw material is almost consumed.

[0186] (2) Concentrate, add 1,4-dioxane (200 mL) again and concentrate to obtain a white solid crude product. Add the crude product into ethyl acetate (200 mL) and beat for 2 hours. Filter and collect the filter cake. Dry under vacuum at 50 °C to obtain a white solid compound NC-6 (22.6 g, 96.9%).

[0187] (3) MS (ESI): m / z [M + H] + Theoretical value 413.2, measured value 413.1.

[0188] 5. Synthesis of intermediate TO-25-01

[0189]

[0190] (1) Under 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 min. 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 h. LCMS detection showed that the raw materials were basically consumed.

[0191] (2) DCM (100 mL) was added for dilution. 1 N hydrochloric acid solution (80 mL × 2) was added to the reaction solution for washing. The organic phases were combined, washed with saturated sodium bicarbonate (100 mL), washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 7 / 1) to obtain a white solid compound TO-25-01 (4.3 g, yield 60%).

[0192] (3) MS (ESI): m / z [M / 2 + H] + Theoretical value is 980.0, measured value is 979.9.

[0193] 6. Synthesis of intermediate TO-25

[0194]

[0195] (1) TO-25-01 (4.3 g, 2.2 mmol) was dissolved in methanol (80 mL), and 10% palladium carbon (1.0 g) was added. H2 was replaced three times, and the mixture was stirred at 25 °C for 2 h. LCMS detection showed that the starting material had basically disappeared.

[0196] (2) Filter, concentrate, add DCM (20 mL) to dissolve, slowly add dropwise to MTBE (300 mL), stir and crystallize for 30 minutes, and filter to obtain white solid compound TO-25 (3.7 g, yield 90%).

[0197] 1 H NMR (400 MHz, DMSO- d 6) 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);

[0198] MS (ESI): m / z [1 / 2M + H] + Theoretical value is 934.9, measured value is 934.8.

[0199] 7. Synthesis of TP-25 (1046 target head linked to siRNA precursor)

[0200]

[0201] (1) Under 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. The reaction was carried out at 25°C for 30 min. TLC detection showed that the starting material had basically disappeared.

[0202] (2) The mixture was quenched by adding saturated NaHCO₃ (10 mL), diluted with DCM (10 mL), and separated. The organic phase was washed with saturated NaHCO₃ (10 mL) solution 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-alkalinized with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) to obtain TP-25 as a white solid (405 mg, 53% yield).

[0203] 1 H NMR (400 MHz, DMSO- d 6) 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);

[0204] 31 P NMR (162 MHz, DMSO- d 6) δ 147.97;

[0205] MS (ESI): m / z [1 / 2M + Na] + Theoretical value is 1057.0, measured value is 1057.4.

[0206] 3. Synthesis of GalNAc Target Head 1048

[0207] TO26 and a diastereomer of TP-26 (a precursor of 1048 target-linked siRNA) were synthesized according to the following method.

[0208] 1. Synthesis of intermediate GN-18-01

[0209]

[0210] (1) Under 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 h. Then, N-Boc-ethylenediamine (7.0 g, 43.7 mmol) and triethylamine (12.05 g, 119.1 mmol) were added to the reaction solution. The reaction was continued for 16 h. LCMS detection showed that the starting material disappeared.

[0211] (2) Add saturated sodium bicarbonate solution (200 mL) to quench the reaction, separate the liquids, extract the aqueous phase with DCM (100 mL × 3), combine the organic phases and wash with saturated ammonium chloride solution (200 mL) and saturated sodium chloride solution (200 mL), dry over anhydrous Na2SO4, filter and concentrate 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 the white solid compound GN-18-01 (24.43 g, yield 95.1%).

[0212] MS (ESI): m / z [M + H] + Theoretical value is 650.3, measured value is 650.5.

[0213] 2. Synthesis of intermediate GN-18

[0214]

[0215] (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 h. LCMS detection showed that the starting material disappeared.

[0216] (2) The solvent was decanted and the solid was concentrated to obtain the crude product. The crude product was purified by slurrying with methyl tert-butyl ether (200 mL), filtered, and the filter cake was dried under vacuum at 40°C to obtain a white solid GN-18 (49.6 g).

[0217] MS (ESI): m / z [M + H] + Theoretical value is 550.3, measured value is 550.5.

[0218] 3. Synthesis of intermediate TO-26-01

[0219]

[0220] (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 min. 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 h. LCMS detection showed that the raw materials were basically consumed.

[0221] (2) DCM (100 mL) was added for dilution. 1N hydrochloric acid solution (80 mL × 2) was added to the reaction solution for washing. The organic phases were combined, washed with saturated sodium bicarbonate (100 mL) and saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 7 / 1) to obtain a white solid compound TO-26-01 (4.7 g, yield 65%).

[0222] MS (ESI): m / z [M / 2 + H] + Theoretical value is 1004.0, measured value is 1004.2.

[0223] 4. Synthesis of intermediate TO-26

[0224]

[0225] (1) TO-26-01 (4.0 g, 2.0 mmol) was dissolved in methanol (80 mL), and 10% palladium carbon (1.0 g) was added. H2 was replaced three times, and the mixture was stirred at 25 °C for 2 h. LCMS detection showed that the starting material had basically disappeared.

[0226] (2) Filter, concentrate, add DCM (20 mL) to dissolve, slowly add dropwise to MTBE (200 mL), stir and crystallize for 30 minutes, and filter to obtain white solid compound TO-26 (3.5 g, yield 91%).

[0227] 1 H NMR (400 MHz, DMSO- d 6) 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, 3.4Hz, 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).

[0228] MS (ESI): m / z [1 / 2M + H] + Theoretical value is 958.9, measured value is 959.1.

[0229] 5. Synthesis of TP-26 (1048 target head linked to siRNA precursor)

[0230]

[0231] (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 reaction was carried out at 25°C for 30 min. TLC detection showed that the starting material had basically disappeared.

[0232] (2) The mixture was quenched by adding saturated NaHCO₃ (10 mL), diluted with DCM (10 mL), and separated. The organic phase was washed with saturated NaHCO₃ (10 mL) solution 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-alkalinized with 1.5% TEA / DCM, DCM / MeOH / TEA = 15 / 1 / 0.1) to obtain TP-26 as a white solid (600 mg, 60% yield).

[0233] 1 H NMR (400 MHz, DMSO-d 6 ) 1 H NMR (400 MHz, DMSO- d 6) 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 = 5.9Hz, 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).

[0234] 31 P NMR (162 MHz, DMSO- d 6) δ 147.89;

[0235] MS (ESI): m / z 1 / 2[M- i- Pr2N] Theoretical value is 1007.9, measured value is 1008.2.

[0236] Example 3 In vitro construction of GalNAc target head coupled (modified) siRNA conjugates

[0237] The oligonucleotide sequences for the antisense and sense strands of the following RNAi agent duplexes, as well as the targeting ligands linked 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 linked to the 5' end of the siRNA sense strand via a phosphorothioate bond.

[0238]

[0239]

[0240]

[0241] GalNAc-siRNA conjugates were synthesized as Figure 3The 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:

[0242] In the nucleic acid sequence, Ao represents adenine nucleoside, Uo represents uridine nucleoside, Go represents guanine nucleoside, and Co represents cytosine nucleoside. There is no symbol between directly adjacent nucleotides, indicating that they are connected by normal phosphate bonds.

[0243] DNA: AGCT (A represents 2'-deoxyadenosine, T represents 2'-deoxythymidine, G represents 2'-deoxyguanosine, and C represents 2'-deoxycytidine);

[0244] 2'-F: aF gF cF uF (aF represents 2'-fluoroadenosine, uF represents 2'-fluorouridine, gF represents 2'-fluoroguanosine, and cF represents 2'-fluorocytidine);

[0245] 2'-OMe: aM gM cM uM (aM represents 2'-O-methyladenosine, uM represents 2'-O-methyluridine, gM represents 2'-O-methylguanosine, and cM represents 2'-O-methylcytosine);

[0246] *: indicates connection with phosphorothioate bond;

[0247] The y and z in the sequence represent the position of the target head.

[0248] Example 4: In vitro cell model (Hep 3B cells) testing the activity of the conjugate

[0249] Human hepatocellular carcinoma 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, 5% CO2 (il60, Thermo Fisher). On the day of transfection, cells were digested with 0.25% Trypsin (Gibco, US), counted, and seeded in 24-well plates at a density of 50,000 per well using 450 μL / well. Subsequently, the test samples were added using Lipofectmine 2000 (Thermo Fisher). Transfection was performed according to the standard protocol in the RNAiMAX reagent manual, with final siRNA concentrations of 10 nM / 1 nM / 0.5 nM / 0.25 nM / 0.1 nM / 0.05 nM / 0.01 nM. A negative control siNC was used in the transfection group; its sequence is as follows:

[0250] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0251] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0252] 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:

[0253] Activity test results of each conjugate (EC 50 Value) see Figure 4 .

[0254] EC 50 The values ​​were calculated using nonlinear regression in GraphPad Prism and represent the amount of conjugate required to inhibit the expression of the target mRNA (ANGPTL3) by half.

[0255] 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.

[0256] Example 5: Construction and drug administration test of AAV-hANGPTL3 mouse model

[0257] Basic information of experimental animals:

[0258] 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.

[0259]

[0260] 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.

[0261] 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.

[0262] The results for each conjugate are shown in Table 3.

[0263] Table 3. Mouse model administration test results of each conjugate

[0264]

[0265] The results show that the selected conjugate also showed good results in reducing the relative expression level of ANGPTL3 in the in vivo activity test experiment.

[0266] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0267] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A compound, characterized in that Has any of the following structures:

2. A compound, characterized in that Has one of the following structures: GalNAC target head 1048.

3. Use of the compound according to claim 2 in the preparation of an siRNA conjugate, wherein the compound is connected to the siRNA as a targeting ligand, and the siRNA is covalently linked to the targeting ligand; the targeting ligand is connected to the sense strand in the siRNA; and the targeting ligand is connected to the 5' end of the sense strand in the siRNA via a phosphorothioate bond.

4. The use according to claim 3, characterized in that The siRNA in the siRNA conjugate includes a sense strand and an antisense strand, wherein the antisense strand includes 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 in 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 strand in SEQ ID NO: 155 to SEQ ID NO: 308 by no more than 5 nucleotides.

5. The use according to claim 4, characterized in that The siRNA comprises at least one modified nucleotide; The modified nucleotide is selected from at least one of the following: 5'-phosphorothioate-based 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, polypeptide nucleotides, and nucleotides comprising non-natural bases.

6. An intermediate for preparing a siRNA conjugate, characterized in that: Select one of the following structures:

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