Nucleic acid molecule inhibiting angptl3 gene expression

Abstract

The present application relates to a nucleic acid molecule inhibiting ANGPTL3 gene expression. The nucleic acid molecule contains or consists of substantially complementary sense and antisense sequences, wherein the sense sequence and / or antisense sequence has a nucleotide length of 14-30 nt. The nucleic acid molecule can better specifically silence an ANGPTL3 gene.

Description

A nucleic acid molecule that inhibits ANGPTL3 gene expression

[0001] Cross-references to related applications

[0002] This invention claims priority to two earlier applications: Patent Application No. 202410568807.9, filed with the China National Intellectual Property Administration on May 9, 2024, entitled "GalNAc Derivatives and Oligonucleotide Conjugates Thereof"; and Patent Application No. 202410848656.2, filed with the China National Intellectual Property Administration on June 27, 2024, entitled "A Nucleic Acid Molecule for Inhibiting ANGPTL3 Gene Expression". The full text of these two earlier applications is incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, specifically to a nucleic acid molecule that inhibits the expression of the ANGPTL3 gene, and more specifically to a nucleic acid molecule that can inhibit the expression of the ANGPTL3 gene via RNAi and its applications. Background Technology

[0004] RNA interference (RNAi) is a widespread phenomenon in natural species. It refers to the highly efficient and specific degradation of targeted mRNA induced by double-stranded RNA (dsRNA). dsRNA is a crucial tool in RNAi technology. In natural organisms, longer dsRNAs, once inside a cell, are specifically recognized and cleaved by the Dicer enzyme into shorter siRNAs (approximately 21-23 nucleotides long). The resulting siRNAs form a complex (RISC) with certain proteins, which then cleaves into single strands. RISCs bind to mRNAs complementary to the antisense strand of the siRNA within the cell, cleaving and degrading the mRNA, thus preventing protein synthesis and resulting in gene silencing. In industrial production, the chemical synthesis and modification of siRNAs are preferred to further improve the stability and efficacy of siRNA drugs.

[0005] Angiopoietin-like protein 3 (ANGPTL3) is a member of the angiopoietin-like protein family involved in regulating lipid metabolism. This family also includes ANGPTL4 and ANGPTL8. The protein encoded by the ANGPTL3 gene is produced exclusively in the liver. It plays a crucial role in regulating lipid metabolism by interacting with lipoprotein lipase (LPL) and endothelial lipase (EL) to inhibit their catalytic activity and the lipolysis of triglycerides (TG). Human genetic studies have shown that loss-of-function mutations in ANGPTL3 can effectively reduce plasma levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides, thereby reducing the risk of cardiovascular disease. ANGPTL3 has become a therapeutic target for dyslipidemia and cardiovascular disease. Evinacumab, developed by Regeneron, is a fully human monoclonal antibody targeting the IgG4 subtype of angiopoietin-like protein 3 (ANGPTL3). It was approved by the U.S. Food and Drug Administration (FDA) on February 11, 2021, for the treatment of familial homozygous hypercholesterolemia (HoFH) in children aged 12 years and older or adults. However, monoclonal antibody drugs have relatively high production costs and require frequent administration. To address the diverse and unmet treatment needs of patients, developing other inhibitors targeting the ANGPTL3 gene, such as nucleic acid inhibitors, is clinically significant.

[0006] Application Overview

[0007] This application provides a nucleic acid molecule that inhibits ANGPTL3 gene expression. The nucleic acid molecule screened by the inventors can more specifically silence the ANGPTL3 gene, inhibit ANGPTL3 protein expression, reduce serum ANGPTL3 protein levels, and thus reduce plasma TG and LDL-C levels. Furthermore, the nucleic acid molecule of this application has the advantage of relatively long-lasting therapeutic effect.

[0008] This application provides a nucleic acid molecule that inhibits the expression of angiopoietin-like protein 3 (ANGPTL3) gene, comprising substantially complementary sense and antisense sequences or consisting of substantially complementary sense and antisense sequences, wherein the antisense sequence comprises a polynucleotide sequence complementary to the ANGPTL3 gene mRNA, and the nucleotide length of the sense and / or antisense sequences is 14-30 nt.

[0009] The antisense sequence comprises at least 14 consecutive nucleotides, not more than 3 nucleotides, from the first to the 21st nucleotides starting from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186.

[0010] In some embodiments, the antisense sequence of the above-mentioned nucleic acid molecule comprises 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinguishable from the first to 21 nucleotides starting from the 5' end, of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186.

[0011] Optionally, the antisense sequence comprises 18, 19, 20, or 21 consecutive nucleotides, consisting of no more than 3 nucleotides distinguishable from the first to 21 nucleotides from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, and 186.

[0012] In some embodiments, the aforementioned nucleic acid molecule contains at least 14 consecutive nucleotides that are distinguishable by no more than 3 nucleotides from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, and 185.

[0013] In some embodiments, the aforementioned nucleic acid molecule contains 18-21 consecutive nucleotides that are no more than 3 nucleotides different from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, 185;

[0014] Optionally, the positive sequence comprises 18, 19, 20, or 21 consecutive nucleotides that are no more than 3 nucleotides different from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, and 185.

[0015] In some embodiments of the above-mentioned nucleic acid molecules, the difference of no more than 3 nucleotides can mean a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness.

[0016] In some embodiments, the nucleotide length of the aforementioned nucleic acid molecule is 14-30 nt, 17-30 nt, 17-23 nt, 17-21 nt, 17-19 nt, 19-25 nt, 19-23 nt, 19-21 nt, 21-25 nt, 21-23 nt, 25-30 nt, or 27-30 nt; optionally, it is 21-23 nt.

[0017] In some embodiments, the aforementioned nucleic acid molecule is dsRNA or shRNA.

[0018] In some embodiments, the aforementioned nucleic acid molecule comprises a sense sequence, an antisense sequence, and a linker strand (or shRNA loop), wherein the linker strand connects the 3' nucleotide of the sense sequence and the 5' nucleotide of the antisense sequence. The choice of the linker strand is known in the art.

[0019] In some embodiments of the above-mentioned nucleic acid molecules, the dsRNA is siRNA, and the antisense sequence and sense sequence are the corresponding antisense strand and sense strand, respectively.

[0020] In some embodiments of the above-mentioned nucleic acid molecule, the positive strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may have 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides or 1-2 nucleotides;

[0021] And / or, wherein the antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may have 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides or 1-2 nucleotides.

[0022] In some embodiments, the antisense strand of the aforementioned nucleic acid molecule comprises or is any one of the polynucleotide sequences selected from SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, and 186; optionally, the antisense sequence comprises or is any one of the polynucleotide sequences selected from SEQ ID NO: 104, 20, 24, 66, 68, 166, 170, 178, and 180; further optionally, the antisense strand comprises or is the polynucleotide sequence shown in SEQ ID NO: 104.

[0023] In some implementation schemes, the aforementioned nucleic acid molecules include:

[0024] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 104;

[0025] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 20;

[0026] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 21, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 22;

[0027] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 23, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 24;

[0028] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 35, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 36;

[0029] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 37, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 38;

[0030] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 39, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 40;

[0031] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 51, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 52;

[0032] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 55, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 56;

[0033] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 65, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 66;

[0034] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 67, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 68;

[0035] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 79, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 80;

[0036] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 101, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 102;

[0037] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 123, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 124;

[0038] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 133, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 134;

[0039] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 135, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 136;

[0040] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 137, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 138;

[0041] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 147, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 148;

[0042] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 149, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 150;

[0043] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 161, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 162;

[0044] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 165, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 166;

[0045] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 167, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 168;

[0046] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 169, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 170;

[0047] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 177, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 178;

[0048] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 179, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 180;

[0049] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 181, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 182;

[0050] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 183, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 184;

[0051] Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 185, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 186;

[0052] Optionally, the sense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 20;

[0053] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 23, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 24;

[0054] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 65, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 66;

[0055] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 67, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 68;

[0056] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 104;

[0057] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 165, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 166;

[0058] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 169, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 170;

[0059] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 177, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 178;

[0060] Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 179, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 180;

[0061] Further optionally, the sense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 104.

[0062] In some embodiments of the above-described nucleic acid molecule, one or more nucleotides of the antisense strand are chemically modified; and / or, one or more nucleotides of the sense strand are chemically modified;

[0063] Optionally, all nucleotides of the antisense strand and / or the sense strand are chemically modified.

[0064] In some embodiments of the above-described nucleic acid molecule, the chemical modification is selected from one or more of the following:

[0065] Locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification, 2'-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluorinated modification, 2'-deoxy modification, 2'-hydroxyl modification, thiophosphate bond modification, 2'-amino-modification, aminophosphate modification, methylphosphate modification, 5'-vinylphosphate modification, DNA modification, fluorescent probe modification.

[0066] In some embodiments, the aforementioned nucleic acid molecules contain a 2'-O-methyl modification, a 2'-fluoro modification, and / or a thiophosphate bond; and / or, the antisense strand contains a 2'-O-methyl modification, a 2'-fluoro modification, and / or a thiophosphate bond.

[0067] In some embodiments of the above-mentioned nucleic acid molecules, the chemical modification is selected from one, two, three, or four of the following:

[0068] (1) The antisense strand has at least the 2nd, 14th and 16th nucleotides starting from the 5' end that are 2'-fluorinated nucleotides;

[0069] (2) The nucleotides at least the 7th, 9th, 10th and 11th positions of the positive strand starting from the 5' end are 2'-fluorinated nucleotides;

[0070] (3) At least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the other nucleotides in the sense strand and / or antisense strand that are not 2'-fluorinated are modified with 2'-O-methyl; optionally, all the other nucleotides in the sense strand and / or antisense strand that are not 2'-fluorinated are modified with 2'-O-methyl.

[0071] (4) The first and second phosphate bonds of the antisense chain starting from its 5' end and the first and second phosphate bonds starting from its 3' end form thiophosphate bonds through thiolation; the first and second phosphate bonds of the sense chain starting from its 5' end form thiophosphate bonds through thiolation.

[0072] In some embodiments of the aforementioned nucleic acid molecule, the 7th and 9th-11th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorine modification, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorine modification; optionally, the 7th and 9th-11th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorine modification, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorine modification, and the remaining nucleotides not modified with 2′-fluorine contain a 2′-O-methyl modification; further optionally, the 7th and 9th-11th nucleotides of the sense strand starting from the 5' end are modified with 2′-fluorine, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end are modified with 2′-fluorine, the remaining nucleotides not modified with 2′-fluorine are modified with 2′-O-methyl, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

[0073] In some embodiments of the aforementioned nucleic acid molecule, the 7th and 9th-12th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorine modification, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorine modification; optionally, the 7th and 9th-12th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorine modification, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorine modification, and the remaining nucleotides not modified with 2′-fluorine contain a 2′-O-methyl modification; further optionally, the 7th and 9th-12th nucleotides of the sense strand starting from the 5' end of the nucleic acid molecule are modified with 2′-fluorine, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end are modified with 2′-fluorine, and the remaining nucleotides not modified with 2′-fluorine are modified with 2′-O-methyl, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

[0074] In some embodiments, the nucleic acid molecule described above contains a motif selected from (1) or (2) below:

[0075] (1) Sensitive strand: The nucleotides at positions 7 and 9-11 of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification;

[0076] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain a 2′-O-methyl modification;

[0077] Optionally, the positive strand: the 7th and 9th-11th nucleotides of the positive strand starting from the 5' end are 2′-fluorinated nucleotides, the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates;

[0078] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluoro-modified nucleotides, and the remaining nucleotides that are not 2′-fluoro-modified are 2′-O-methyl-modified nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates.

[0079] (2) Sensitive strand: The nucleotides at positions 7 and 9-12 of the positive strand, starting from the 5' end, contain 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain 2′-O-methyl modification;

[0080] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain a 2′-O-methyl modification;

[0081] Optionally, the positive strand: the 7th and 9th-12th nucleotides of the positive strand starting from the 5' end are 2′-fluorinated nucleotides, the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates;

[0082] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, and the remaining nucleotides that are not 2′-fluorinated are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

[0083] In some embodiments, the aforementioned nucleic acid molecules are further linked by phosphate thioester bonds between the first and second nucleotides at the 5' end of the sense strand, and between the second and third nucleotides; and by phosphate thioester bonds between the first and second nucleotides at the 5' end of the antisense strand, between the second and third nucleotides, and between the first and second nucleotides at the 3' end of the antisense strand, and between the second and third nucleotides.

[0084] In some embodiments, the nucleic acid molecule described above contains a motif selected from (1) or (2) below:

[0085] (1) Sensitive strand: The 7th and 9th-11th nucleotides of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification; the first and second nucleotides at the 5' end of the positive strand are connected by phosphate thioester bonds, as are the second and third nucleotides.

[0086] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain a 2′-O-methyl modification. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by a thiophosphate bond, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides.

[0087] Optionally, the sense strand: the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides; the first and second nucleotides at the 5' end of the sense strand are linked by phosphate thioester bonds, as are the second and third nucleotides.

[0088] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by thiophosphate bonds, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides.

[0089] When the length of the sense strand is 21 nt, the optional modification scheme in the sense strand is motif 1 modification; when the length of the antisense strand is 23 nt, the optional modification scheme in the antisense strand is motif 2 modification. Motif 1 modification of the sense strand + motif 2 modification of the antisense strand constitutes a nucleic acid molecule modification method.

[0090] (2) Sensitive strand: The 7th and 9th-12th nucleotides of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification; the first and second nucleotides at the 5' end of the positive strand are connected by phosphate thioester bonds, as are the second and third nucleotides.

[0091] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain a 2′-O-methyl modification. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by thiophosphate bonds, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides.

[0092] Optionally, the sense strand: the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides; the first and second nucleotides at the 5' end of the sense strand are linked by phosphate thioester bonds, as are the second and third nucleotides.

[0093] Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, while the remaining nucleotides that are not 2′-fluorinated are 2′-O-methyl nucleotides. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by thiophosphate bonds, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides.

[0094] When the length of the sense strand is 21 nt, the optional modification scheme in the sense strand is motif 3 modification; when the length of the antisense strand is 23 nt, the optional modification scheme in the antisense strand is motif 2 modification. Motif 3 modification of the sense strand + motif 2 modification of the antisense strand constitutes a nucleic acid molecule modification method.

[0095] In some implementation schemes, the aforementioned nucleic acid molecules include:

[0096] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 10; the polynucleotide sequence shown in SEQ ID NO: 9 is SEQ ID NO: 103 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 10 is SEQ ID NO: 104 with motif 2.

[0097] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 407, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 10; the polynucleotide sequence shown in SEQ ID NO: 407 is SEQ ID NO: 103 with motif 3, and the polynucleotide sequence shown in SEQ ID NO: 10 is SEQ ID NO: 104 with motif 2.

[0098] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 1, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 2; the polynucleotide sequence shown in SEQ ID NO: 1 is SEQ ID NO: 19 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 2 is SEQ ID NO: 20 with motif 2;

[0099] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 3, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 4; the polynucleotide sequence shown in SEQ ID NO: 3 is SEQ ID NO: 23 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 4 is SEQ ID NO: 24 with motif 2;

[0100] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 6; the polynucleotide sequence shown in SEQ ID NO: 5 is SEQ ID NO: 65 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 6 is SEQ ID NO: 66 with motif 2.

[0101] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 7, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 8; the polynucleotide sequence shown in SEQ ID NO: 7 is SEQ ID NO: 67 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 8 is SEQ ID NO: 68 with motif 2.

[0102] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 11, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 12; the polynucleotide sequence shown in SEQ ID NO: 11 is SEQ ID NO: 165 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 12 is SEQ ID NO: 146 with motif 2.

[0103] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 13, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 14; the polynucleotide sequence shown in SEQ ID NO: 13 is SEQ ID NO: 169 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 14 is SEQ ID NO: 170 with motif 2.

[0104] The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 15, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 16; the polynucleotide sequence shown in SEQ ID NO: 15 is SEQ ID NO: 177 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 16 is SEQ ID NO: 178 with motif 2.

[0105] Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 17, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 18; the polynucleotide sequence shown in SEQ ID NO: 17 is SEQ ID NO: 179 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 18 is SEQ ID NO: 180 with motif 2.

[0106] Optionally, the sense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 10;

[0107] Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 407, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 10.

[0108] In some embodiments, the aforementioned nucleic acid molecule is linked to an organ-targeting ligand, such as a liver-targeting ligand.

[0109] In some embodiments, the aforementioned nucleic acid molecule is linked to at least one desialyl glycoprotein receptor (ASGPR) ligand.

[0110] In some embodiments, the organ-targeting ligand of the aforementioned nucleic acid molecule is attached to the 5' or 3' end of the sense sequence.

[0111] In some embodiments of the aforementioned nucleic acid molecules, the ASGPR ligand is one or more GalNAc derivatives conventional in the art that are linked by a divalent or trivalent branched structure.

[0112] In some embodiments of the above-described nucleic acid molecule, the GalNAc ligand is selected from one of the following structures:

[0113] In this case, when one of X or Y is an oligonucleotide, the other is hydrogen.

[0114] In some embodiments of the above-described nucleic acid molecule, the GalNac ligand is selected from one of the following structures:

[0115] Where X is O or S.

[0116] in It represents the connection to the 3' end of the positive strand of the nucleic acid molecule; optionally, the GalNac ligand is connected via a phosphate ester bond or a thiophosphate ester bond;

[0117] Further, optionally, the nucleic acid molecule has any one of the structures of (1)-(8):

[0118] (1) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to L96 via a phosphate ester bond; Antisense chain: The antisense chain modified as motif 2;

[0119] (2) Sensitive chain: The 3' end of the positive chain modified as motif 3 is connected to L96 via a phosphate ester bond; Antisense chain: The antisense chain modified as motif 2;

[0120] (3) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to ligand 1 via a thiophosphate bond; Antisense chain: The antisense chain modified as motif 2;

[0121] (4) Sensitive chain: The 3' end of the positive chain modified as motif 3 is connected to ligand 1 via a thiophosphate bond; Antisense chain: The antisense chain modified as motif 2;

[0122] (5) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to ligand 2 via a thiophosphate bond; Antisense chain: The antisense chain modified as motif 2;

[0123] (6) Sensitive chain: The 3' end of the positive chain modified as motif 3 is connected to ligand 2 via a thiophosphate bond; Antisense chain: The antisense chain modified as motif 2;

[0124] (7) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to ligand 3 via a thiophosphate bond; Antisense chain: The antisense chain modified as motif 2;

[0125] Or, (8) sense chain: the 3' end of the sense chain modified as motif 3 is connected to ligand 3 via a thiophosphate bond; antisense chain: the antisense chain modified as motif 2.

[0126] In some embodiments, the nucleic acid molecules described above are selected from the following nucleic acid molecules:

[0127] Wherein: Am, Um, Cm, and Gm represent ribonucleotides A, U, C, and G modified with 2′-O-methyl, respectively; Af, Uf, Cf, and Gf represent ribonucleotides A, U, C, and G modified with 2′-fluoro, respectively. The 's' between adjacent nucleotides indicates that the adjacent nucleotides are linked by a phosphate thioester bond, and the 's' at the 3' end of the positive strand indicates that the 3' end of the positive strand is linked to the ligand via a phosphate thioester bond.

[0128] This application provides a nucleic acid molecule selected from nucleic acid molecules PC07, PC08, PC09, PC10 or ANG639-E20-PC08.

[0129] The second aspect of this application provides a second nucleic acid molecule that can be transcribed in a cell into the nucleic acid molecule of the first aspect, such as dsRNA or shRNA as described in the first aspect. In some embodiments, the second nucleic acid molecule is a circular or linear nucleic acid molecule. In some embodiments, the second nucleic acid molecule is a circular or linear plasmid. In some embodiments, the nucleic acid molecule belongs to an artificially constructed viral genome, selected from, but not limited to, lentiviral vectors or other retroviral vectors, adenovirus vectors, AAV vectors, poxvirus vectors, baculovirus vectors, and herpes simplex virus vectors. In some embodiments, the nucleic acid molecule belongs to a cellular genome, such as a nuclear genome, mitochondrial nucleic acid, or cytoplasmic free nucleic acid.

[0130] A third aspect of this application also provides a nucleic acid delivery device comprising the nucleic acid molecule of the first aspect or the second nucleic acid molecule of the second aspect. In some embodiments, the nucleic acid delivery device is a liposome, lipid nanoparticles or other polymers, endosomes, exosomes, or vesicles.

[0131] A fourth aspect of this application also provides a viral particle comprising the nucleic acid molecule of the first aspect or the second nucleic acid molecule of the second aspect. In some embodiments, the viral particle is an enveloped virus or a capped virus particle. In some embodiments, the viral particle is a pseudovirus particle. In some embodiments, the viral particle belongs to AAV, baculovirus, poxvirus, herpesvirus, alphavirus, lentivirus, or other retrovirus.

[0132] A fifth aspect of this application provides a cell comprising the nucleic acid molecule of the first aspect or the second nucleic acid molecule of the second aspect. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as a stem cell, like a hematopoietic stem cell, mesenchymal stem cell, etc.

[0133] A sixth aspect of this application provides a pharmaceutical composition comprising the nucleic acid molecule of the first aspect or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0134] The seventh aspect of this application also provides the use of the nucleic acid molecule of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery body of the third aspect, the viral particle of the fourth aspect, the cell of the fifth aspect, and the pharmaceutical composition of the sixth aspect for preparing a drug that inhibits the expression of the ANGPTL3 gene in a subject. In some embodiments, the drug that inhibits the expression of the ANGPTL3 gene is a drug that lowers triglyceride levels and / or lowers LDL-C levels. In some embodiments, the drug that lowers triglyceride levels and / or lowers LDL-C levels is a drug for the prevention or treatment of dyslipidemia and / or cardiovascular disease. In some embodiments, the drug that inhibits the expression of the ANGPTL3 gene in a subject is a drug for the prevention or treatment of hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0135] The eighth aspect of this application also provides a method for inhibiting the expression of the ANGPTL3 gene in a subject, comprising the following steps:

[0136] The effective dose of the aforementioned first-aspect nucleic acid, the aforementioned second-aspect nucleic acid, the aforementioned third-aspect nucleic acid delivery body, the aforementioned fourth-aspect viral particles, the aforementioned fifth-aspect cells, or the aforementioned sixth-aspect pharmaceutical composition is administered to a subject who requires inhibition of ANGPTL3 gene expression. In some embodiments, the subject requiring inhibition of ANGPTL3 gene expression is a subject who requires reduction of triglyceride levels and / or reduction of LDL-C levels. In some embodiments, the subject requiring reduction of triglyceride levels and / or reduction of LDL-C levels is a subject who requires prevention or treatment of dyslipidemia and / or cardiovascular disease. In some embodiments, the subject requiring inhibition of ANGPTL3 gene expression is a subject who requires prevention or treatment of hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia.

[0137] The ninth aspect of this application also provides the nucleic acid molecule of the first aspect, the second nucleic acid molecule of the second aspect, the nucleic acid delivery body of the third aspect, the viral particle of the fourth aspect, the cell of the fifth aspect, and the pharmaceutical composition of the sixth aspect, for inhibiting ANGPTL3 gene expression. In some embodiments, inhibiting ANGPTL3 gene expression reduces triglyceride levels and / or LDL-C levels. In some embodiments, reducing triglyceride levels and / or LDL-C levels is for the prevention or treatment of dyslipidemia and / or cardiovascular disease. In some embodiments, inhibiting ANGPTL3 expression is for the prevention or treatment of hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0138] The optional embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application. The aspects and embodiments of this application described herein include aspects and embodiments described as "comprising," "forming," and "substantially consisting of." Attached Figure Description

[0139] Figure 1 shows the results of DLR high-throughput screening of candidate siRNA sequence gene expression in Example 1 (293T cells).

[0140] Figure 2 shows the relative expression levels of ANGPTL3 mRNA in Hep3B cells from the Top 28 candidate modified sequences in Example 2.

[0141] Figure 3 shows the effect of the Top9 candidate modification sequence in Example 3 on the expression level of ANGPTL3 protein in humanized mouse serum.

[0142] Figure 4 shows the effect of the candidate molecule modification sequence on serum TG in cynomolgus monkeys in Example 4.

[0143] Figure 5 shows the effect of the candidate molecular modification sequence in Example 4 on the expression level of ANGPTL3 protein in cynomolgus monkey serum.

[0144] Figure 6 shows the effect of candidate molecule modification sequences paired with different ligands on the expression level of ANGPTL3 protein in mouse serum when used in Example 6.

[0145] Figure 7 shows the effect of candidate molecule modification sequences paired with different ligands on the expression level of cynomolgus monkey proteins in Example 7.

[0146] Figure 8 shows the effect of candidate molecular sequences combined with different motif modifications on the expression level of ANGPTL3 protein in humanized mouse serum.

[0147] Application details

[0148] This application provides a nucleic acid molecule that inhibits ANGPTL3 gene expression. Compared with the positive control nucleic acid molecule, the nucleic acid molecule screened by the inventors can more specifically silence the ANGPTL3 gene, inhibit ANGPTL3 protein expression, reduce serum ANGPTL3 protein levels, and thus reduce plasma TG and LDL-C levels. At the same time, the nucleic acid molecule of this application has the advantage of relatively long-lasting therapeutic effect.

[0149] the term

[0150] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which the described technology pertains. All technical and patent disclosures referenced in this application are incorporated herein by reference in their entirety.

[0151] As used in this application, the term "nucleic acid molecule" may refer to any molecule having a nucleotide sequence consisting of two or more nucleotides linked together, including linkages via phosphate ester bonds, modified phosphate ester bonds (e.g., thiophosphate ester bonds), or other linkages.

[0152] As used in this application, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a specific order. This polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain within a nucleic acid molecule. Therefore, "nucleotide sequence" can be represented as a precise polynucleotide sequence composed of various nucleotides (e.g., ATCG) (e.g., any one of SEQ ID NO: 1 to 100), or as nucleotides at specific positions within a sequence, such as "nucleotides at positions 1 to 10 in the ANGPTL3 gene mRNA sequence." In this application, unless otherwise specified, in sequences or nucleic acid molecules containing "nucleotides at specific positions within a sequence," the order of the nucleotides at those specific positions is consistent with the order of the aforementioned nucleotides within the sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid molecule of RNA and DNA, and may also contain non-natural nucleotides or artificially modified nucleotides. For example, in this application, the antisense sequence comprises at least 14 consecutive nucleotide sequences that differ by no more than 3 nucleotides from the nucleotide sequence shown in positions 1-21 from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186. NO:104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186. The nucleotide sequence from position 1 to 21 starting from the 5' end of any one of these sequences can be an unmodified naked nucleotide sequence or a nucleotide sequence in which at least one nucleotide has been modified.

[0153] The term "nucleotide" in this application, besides referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, should also be understood to refer to their associated structural variants, including derivatives and analogs, which are functionally equivalent in relation to the specific context of use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino oligonucleotides. In some embodiments, the term "nucleotide" in this application does not include non-natural nucleotides with modified bases. In some embodiments, the term "nucleotide" in this application does not include nucleotides with modified bases. In this application, "G," "C," "A," "T," and "U" generally represent nucleotides with guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, in the context of RNA and in RNA sequences, unless otherwise specified, "T" refers to uridine or uracil. It should be understood that in the context of nucleotide sequences in this application, "nucleotide," "nucleotide residue," and "base" are used interchangeably. The number of nucleotide pairs and base pairs is measured in bp, where one bp represents one nucleotide pair or one base pair. The number of nucleotides is measured in nt, where one nt represents one nucleotide. In this application, polynucleotides are DNA or RNA nucleotides. Polynucleotides typically contain an unlimited number of nucleotides. Oligonucleotides are shorter polynucleotides, such as those consisting of 10 to 100 nucleotides.

[0154] As used in this application, the formation of hydrogen bonds between bases or nucleotides according to the Watson-Crick base pairing principle is referred to as complementarity, pairing, or matching, such as A pairing with T or U, C pairing with G or I. All other base pairings are referred to as non-complementarity. When referring to "substantially complementary" between polynucleotide sequences, nucleic acid chains, or between polynucleotide sequences and nucleic acid chains, it means complete complementarity or complementarity of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In complementarity, one or more mismatches are permissible, and one or more bulges are permissible in the polynucleotide sequence or nucleic acid chain; therefore, the formed complementary regions can be continuous or spaced. If one of the two complementary polynucleotide sequences is shorter than the other, the calculation is based on the number of nucleotides in the shorter sequence. For example, if the sense sequence is 21 nt and the antisense sequence is 23 nt, the length of the complementary region is 21 bp, which is calculated as 21 / 21, meaning it is 100% complementary, or completely complementary.

[0155] As used in this application, antisense sequence and sense sequence have the common meaning in the art, specifically relative to the target sequence ANGPTL3 gene mRNA sequence of this application. That is, antisense sequence refers to a sequence in the nucleic acid molecule that is substantially complementary to the ANGPTL3 gene mRNA sequence, and sense sequence refers to a sequence in the nucleic acid molecule that has at least 10 consecutive identical nucleotides to the ANGPTL3 gene mRNA sequence.

[0156] As used in this application, "at least 14 consecutive nucleotide sequences that differ by no more than 3 nucleotides" means that when compared with at least 14 consecutive nucleotide sequences (a specific reference range in the reference sequence) from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186, the corresponding nucleotide sequences in the nucleotide sequence used for comparison (i.e., the nucleic acid molecule of this application) that differ from the specific reference range in the reference sequence by no more than 3 nucleotides. Different nucleotides can be located either in the middle or at both ends of the nucleotide sequence to be compared in the nucleic acid molecule of this application.

[0157] As used in this application, "ANGPTL3" refers to angiopoietin-like protein 3, also known as ANG-5, ANGPT5, ANL3, or FHBL2. Its sources include, but are not limited to, any vertebrate or mammalian origin, specifically such as primates (e.g., humans, monkeys, and further, cynomolgus monkeys), cattle, chickens, rodents (e.g., mice, rats, guinea pigs), pigs, sheep, etc.

[0158] As used in this application, “ANGPTL3 gene” is the angiopoietin-like protein 3 gene.

[0159] As used herein, "ANGPTL3 gene mRNA" refers to the mRNA encoding angiopoietin-like protein 3, which can be transcribed from ANGPTL3 gene DNA and can be mature mRNA or pre-mRNA, thus it may or may not contain introns. Because the ANGPTL3 gene may have a few nucleotide mutations in different individuals, unless otherwise specified, the ANGPTL3 gene mRNA sequence in this application is intended to include all mRNA sequences transcribed from ANGPTL3 gene mutants. Human ANGPTL3 gene mRNA sequences can be found, for example, in GenBank accession number GI:27329 (NM_014495.4). Other examples of ANGPTL3 gene mRNA sequences are readily available using publicly available databases, such as GenBank. In this application, ANGPTL3 gene mRNA is the target sequence.

[0160] As used in this application, "dsRNA" refers to double-stranded RNA, which contains two antiparallel and substantially complementary nucleic acid strands. Unless otherwise specified, the length of double-stranded RNA is not specifically defined in this application. Since siRNA (small interfering RNA) is a double-stranded RNA, the term "dsRNA" encompasses siRNA, which can perform RNAi and is the primary tool for achieving RNAi. dsRNA also includes double-stranded RNA longer than siRNA, where "longer than siRNA" can mean that its sense strand is longer than siRNA, or its antisense strand is longer than siRNA, or both its sense and antisense strands are longer than siRNA. Typically, double-stranded RNA longer than the siRNA sequence it contains, upon entering the cell, is broken down into siRNA by a type III endonuclease called Dicer. When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in the RISC unwind the siRNA double helix. When it binds to a target mRNA complementary to the antisense strand of the siRNA, one or more endonucleases in the RISC cleave the target, inducing gene silencing. Typically, most or all of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but this does not preclude the inclusion of one or two non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or two strands. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, the dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0161] As used in this application, "protruding nucleotide" is used in contrast to "flat-ended" or "flat-ended," which means that there are no unpaired nucleotides at the 3' and 5' ends of a double-stranded nucleotide, i.e., no protruding nucleotides; "protruding nucleotide" means that in a double-stranded nucleotide, one strand has more nucleotides than the other strand, resulting in unpaired nucleotides at the 3' and 5' ends of the two strands.

[0162] As used in this application, the terms "3' end" and "5' end" have the common meaning in the art. Where no specific direction is explicitly indicated (i.e., "3' end direction" and "5' end direction"), they encompass the meanings of "3' end" and "5' end," with "3' end" and "5' end" emphasizing the specific end position. In this application, unless it would cause ambiguity to those skilled in the art, no distinction is made between "5' end" and "5' end," nor between "3' end" and "3' end."

[0163] As used in this application, the terms “close,” “near,” or “far” when describing nucleotide positions on the same sequence or nucleoside chain refer to the number of nucleotides separating the two nucleotide positions.

[0164] As used in this application, based on the location of the chemical modification, nucleotide chemical modification methods are divided into phosphate ester modification, base modification, and ribose modification. Chemical modification methods have accelerated the development of siRNA drugs. Due to the uniqueness of drugs, the specific application of chemical modification methods still needs to be explored in different specific scenarios. "Ribose modification" refers to chemical modification at various positions of the sugar ring of ribonucleic acid, including substituent modification, such as 2'-modification (e.g., 2'-O-methyl, 2'-fluoro, 2'-MOE), 5' modification (e.g., 5'-Mo), and isomerization modification (e.g., LNA, GNA modification, etc.). "Phosphate ester" modification mainly refers to the modification of the phosphodiester backbone between ribose on siRNA, mainly including thiophosphate (PS), dithiophosphate (PS2), methylphosphate (MP), methoxypropyl phosphate (MOP), and peptide nucleic acid (PNA), etc. In this field and in this application, phosphate ester bond and phosphodiester bond have the same meaning, and thiophosphate bond and thiophosphate diester bond have the same meaning and can be used interchangeably. Base modification refers to chemical modifications made to the bases of siRNA, mainly divided into three forms: purine modification (such as N6-methyladenosine), pyrimidine modification (such as 5-methylcytidine), and base substitution. When applying specific modifications to a particular siRNA, it is necessary to consider the impact of modifications at different positions and in different quantities on the overall function of the siRNA. These different overall modification rules are referred to as "motifs" in this application. For example, the following sequence in the antisense strand of siRNA, AmsGfsAmGmUmAmUmAmAmCmCmUmUmCfCmAfUmUmUmUmGmsAmsGm, can be extracted as NmsNfsNm ... In this application, the overall modification rule is referred to as motif 2. The following sequence in the siRNA positive strand, CmsAmsAmAmAmUmGfGmAfAfGfGmUmUmAmUmAmCmUmCmUmNm, has its overall modification rule extracted as NmsNmsNmNmNmNmNfNmNfNfNfNfNm ...

[0165] As used herein, “GalNAc” or “N-acetylgalactosamine” refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term “GalNAc” or “N-acetylgalactosamine” includes both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. Optionally, the GalNAc compound of this application is in the β-form, namely 2-(acetylamino)-2-deoxy-β-D-galactopyranose. Accordingly, GalNAc derivatives refer to chemical derivatives containing the above-described GalNAc structure.

[0166] As used in this application, "inhibition of ANGPTL3 gene expression" includes inhibition of the ANGPTL3 gene at any level, such as at least partial inhibition of ANGPTL3 gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0167] "Inhibition of ANGPTL3 gene expression" includes inhibiting the expression of any ANGPTL3 gene (e.g., mouse ANGPTL3 gene, rat ANGPTL3 gene, monkey ANGPTL3 gene, or human ANGPTL3 gene) as well as variants (e.g., naturally occurring variants) or mutants of the ANGPTL3 gene. Therefore, the ANGPTL3 gene can be the wild-type ANGPTL3 gene, the mutant ANGPTL3 gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic ANGPTL3 gene.

[0168] [This application also provides the following independent technical solutions. If the descriptions and explanations in these independent technical solutions are inconsistent with other explanations in this application, they are only used to explain these independent technical solutions and do not affect the explanations of other parts of this application.]

[0169] [Independent Technical Solution Introduction: This independent technical solution provides an RNA molecule or its stereoisomer, solvate, isotopic derivative or pharmaceutically acceptable salt thereof targeting ANGPTL3 (angiopoietin-like 3), comprising a sense strand and an antisense strand complementary to form a double-stranded region, wherein the antisense strand contains nucleotides differing from a complementary sequence of the target gene sequence by 0, 1, 2 or 3 bases, and the sense strand contains a sequence sufficiently complementary to the antisense strand sequence to form a double-stranded region, wherein the double-stranded region is 15-25 bp in length, and the sense strand of the RNA molecule contains at least 19 consecutive nucleotides of the following structure: NmNmNm] The antisense strand of the RNA molecule comprises at least 21 consecutive nucleotides in the following structure: NmNfNm ...

[0170] According to the independent technical solution, the RNA molecule targeting ANGPTL3 has, in some embodiments, a sense strand length of 19-25 nt (19, 20, 21, 22, 23, 24, or 25 nt) and an antisense strand length of 21-27 nt (21, 22, 23, 24, 25, 26, or 27 nt). In some embodiments, the sense strand length of the RNA molecule is 19-23 nt. In some embodiments, the sense strand length of the RNA molecule is 21-23 nt. In some embodiments, the antisense strand length of the RNA molecule is 21-25 nt. In some embodiments, the antisense strand length of the RNA molecule is 23-27 nt. In some embodiments, the sense strand length of the RNA molecule is 19-23 nt, and the antisense strand length is 21-25 nt. In some embodiments, the sense strand length of the RNA molecule is 21-25 nt, and the antisense strand length is 23-27 nt. In some embodiments, the sense strand of the RNA molecule is 21 nt long, and the antisense strand is 23 nt long. In some embodiments, the double-stranded region of the RNA molecule is 19-23 nt long. In some embodiments, the double-stranded region of the RNA molecule is 21-23 nt long. In some embodiments, the double-stranded region of the RNA molecule is 21 nt long. In some embodiments, the double-stranded region of the RNA molecule is 23 nt long. In some embodiments, the sense strand of the RNA molecule is 21 nt long, the antisense strand is 23 nt long, and the double-stranded region of the RNA molecule is 19 nt, 20 nt, or 21 nt long. In some embodiments, the sense strand of the RNA molecule is 21 nt long, the antisense strand is 23 nt long, and the double-stranded region of the RNA molecule is 21 nt long.

[0171] According to the RNA molecule targeting ANGPTL3 of this independent technical solution, in some embodiments, it has blunt ends at both the 3' and 5' ends of the antisense strand. In some embodiments, it may have nucleotide overhangs (i.e., protruding ends) at one or both of the 3' ends of the sense and antisense strands. In some embodiments, it has a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In any embodiment in which one or both strands contain an overhang, the overhang consists of 1-5 (1, 2, 3, 4, or 5) nucleotides. In any embodiment in which one or both strands contain an overhang, the overhang consists of 1, 2, or 3 nucleotides. In any embodiment in which one or both strands contain an overhang, the overhang consists of 2 nucleotides.

[0172] According to the RNA molecule targeting ANGPTL3 of this independent technical solution, in some embodiments, the modification motifs of its sense strand and / or antisense strand further include one or more phosphate-thioester bonds. In some embodiments, its antisense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at the 5' end. In some embodiments, its antisense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at the 3' end. In some embodiments, its antisense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at both the 3' and 5' ends. In some embodiments, its sense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at the 5' end. In some embodiments, its sense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at the 3' end. In some embodiments, its antisense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at both the 3' and 5' ends, and its sense strand includes two consecutive phosphate-thioester bonds between the terminal nucleotides at the 5' end. In some embodiments, the antisense strand contains two consecutive phosphate-thioester bonds between the terminal nucleotides at both the 3' and 5' ends, and the sense strand contains two consecutive phosphate-thioester bonds between the terminal nucleotides at both the 3' and 5' ends. In some embodiments, the antisense strand contains two consecutive phosphate-thioester bonds between the terminal nucleotides at both the 3' and 5' ends, and the sense strand contains two consecutive phosphate-thioester bonds between the terminal nucleotides at the 5' end, and the sense strand contains one phosphate-thioester bond between the terminal nucleotide at the 3' end and the ligand. In any embodiment in which one or both strands contain one or more phosphate-thioester bonds, the bonds between the nucleotides within the strand can be native 3' to 5' phosphodiester bonds.

[0173] According to the independent technical solution, the ANGPTL3-targeting RNA molecule further comprises a 5'-vinyl phosphate (5'-vp) modification at the 5' end of its antisense strand. This 5'-vp can be a 5'-E-VP isomer (i.e., trans-vinyl phosphate), a 5'-Z-VP isomer (i.e., cis-vinyl phosphate), or a mixture thereof. In some embodiments, the ANGPTL3-targeting RNA molecule according to the independent technical solution further comprises a 5'-E-VP modification at the 5' end of its antisense strand.

[0174] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution may further include ligand modification. The ligand may be a portion taken up by host cells. Ligand modification can improve properties of the RNA molecule such as cellular uptake, intracellular targeting, half-life, or drug metabolism or pharmacokinetics. In some embodiments, compared to unmodified RNA molecules, ligand-modified RNA molecules exhibit enhanced affinity or cellular uptake against selected targets (such as specific tissue types, cell types, organelles, etc.), such as hepatocytes. Ligand modification does not interfere with the activity of the RNA molecule.

[0175] In some embodiments, the ligand modification is one or more ligand modifications to the 3' end, 5' end, and / or middle of the sequence of the RNA molecule targeting ANGPTL3.

[0176] In some embodiments, the ligand is selected from the following: cholesterol, C14-22 saturated or unsaturated hydrocarbon groups, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylgalactosamine derivatives or analogs, and N-acetylglucosamine derivatives or analogs. In some embodiments, the ligand targets cell surface receptors, including galactose, galactosamine, lactose, or N-acetylgalactosamine / glucosamine moieties. In some embodiments, the ligand preferably targets the liver, particularly hepatic parenchymal cells. In some preferred embodiments, the ligand may also be human serum albumin (HSA), hyaluronic acid, peptides, etc. In some preferred embodiments, the ligand targets the ASGPR receptor.

[0177] In some embodiments, the ligand is linked to the 5' or 3' end nucleotide of the sense or antisense strand of an RNA molecule via a phosphodiester bond or a thiophosphodiester bond. In some embodiments, the ASGPR ligand is a GalNAc polymer formed from one, two, three, or more GalNAc derivatives (referred to as a monovalent GalNAc conjugate, a divalent GalNAc conjugate, a trivalent GalNAc conjugate, or a polyvalent GalNAc conjugate, respectively).

[0178] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution further comprises at least one desialylate glycoprotein receptor (ASGPR) ligand.

[0179] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution, wherein the ligand is linked to the 5' or 3' end nucleotide of the sense or antisense strand nucleotide sequence via a phosphodiester bond or a thiophosphodiester bond.

[0180] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution, wherein the ASGPR ligand is a GalNAc polymer formed from two or three GalNAc derivatives.

[0181] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution, or its stereoisomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, has its 3' end of the sense strand linked to an ASGPR ligand, the ASGPR ligand having a structure of L96 as shown in Formula I or ligand 1 as shown in Formula II.

[0182] Where X is OH or SH (or when the RNA molecule forms a salt, X is O). - or S - Both forms are within the scope of this application; among which This means that it is linked to the rest of the RNA molecule via a phosphodiester bond or a thiophosphate diester bond.

[0183] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution has a nucleotide sequence comprising or being as shown in (C):

[0184] (C) Sense strand: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense strand: NmsNfsNmNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmsNmsNm.

[0185] It should be understood that in the ANGPTL3-targeting RNA molecule of this independent technical solution, Nm represents a ribonucleotide modified with 2'-O-methyl, Nf represents a ribonucleotide modified with 2'-fluoro, and vp- indicates that the nucleotide to its right is modified with 5'-vinyl phosphate. It should also be understood that in the ANGPTL3-targeting RNA molecule of this independent technical solution, when the nucleotide sequence is marked with 's', 's' indicates that the two nucleotides before and after it are linked by a phosphate thioester bond; two nucleotides without 's' are linked by a phosphate ester bond. However, when the nucleotide sequence is not marked with 's', any two nucleotides can be linked by a phosphate thioester bond, a phosphate dithioester bond, or a derivative of another phosphate dithioester bond.

[0186] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution has a nucleotide sequence comprising or being as shown in (C):

[0187] (C) Sense strand: NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm, antisense strand: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNfNmNmNmNmNmsNmsNm.

[0188] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution has a nucleotide sequence comprising or being as shown in (C):

[0189] (C) Sense strand: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand, antisense strand: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0190] In this case, the 3' position of the "-ligand" nucleotide is connected to the ligand via a phosphodiester bond or a thiophosphate diester bond.

[0191] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution has a nucleotide sequence comprising or being as shown in (C):

[0192] (C) Sense strand: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm-L96, antisense strand: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0193] Wherein, "-L96" indicates that the 3' position of the nucleotide is connected to L96 via a phosphodiester bond or a thiophosphate diester bond, preferably via a phosphodiester bond.

[0194] In some embodiments, the RNA molecule targeting ANGPTL3 according to this independent technical solution has a nucleotide sequence comprising or being as shown in (C):

[0195] (C) Sense strand: NmsNmsNmNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNm-ligand 1, antisense strand: vp-NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm;

[0196] Wherein, "-ligand 1" indicates that the 3' position of the nucleotide is connected to ligand 1 via a phosphodiester bond or a thiophosphate diester bond, preferably via a thiophosphate diester bond.

[0197] In some embodiments, the nucleotide sequences shown in (C) above do not contain any of the chemical modifications not indicated in any of the nucleotide sequences shown in (C) above, compared to the natural nucleotides. For example, Nm does not contain any chemical modification other than 2'-OMe, Nms does not contain any chemical modification other than 2'-OMe and thiophosphate, Nf does not contain any chemical modification other than 2'-F, Nfs does not contain any chemical modification other than 2'-f and thiophosphate, and vp-Nms does not contain any chemical modification other than 5'-VP, 2'-OMe, and thiophosphate. [The independent technical solution is terminated.]

[0198] The term "solid support" refers to a matrix of inorganic particles, polymers, or other solid materials that can be linked to target compounds by surface modification with active groups (such as NH2), thereby enabling the synthesis of oligonucleotides. Examples of solid supports include, but are not limited to, controllable microporous glass (CPG) and polystyrene (PS).

[0199] The term "conjugate" or "conjugate molecule" refers to a compound formed by the covalent linkage between its individual chemical parts.

[0200] As used in this application, the following abbreviations have the following corresponding meanings:

[0201] In this application, the above abbreviations represent specific nucleosides.

[0202] As used in this application, the term "about" refers to the general range of error for various values ​​that is readily known to those skilled in the art. References to "about" values ​​or parameters in this application include (and describe) embodiments for that value or parameter itself. As used in this application, when the term "about" precedes a numerical value, it indicates a range of 10% above or below that value. For example, "about 100" encompasses both 90 and 110.

[0203] As used in this application, unless otherwise indicated, the singular forms “a,” “an,” and “the” include the plural forms.

[0204] Note: This is merely a schematic diagram of a thiophosphate diester bond; other possible structures include the following: The above structures are interchangeable in this application; This also illustrates a phosphodiester bond, which also includes... The above structures are interchangeable in this application.

[0205] Unless otherwise defined in this application, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0206] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following embodiments are intended to illustrate, not limit, the scope of this application. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0207] Unless otherwise stated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These conventional techniques are described in existing literature. All cells were obtained from the Chinese Academy of Sciences Type Culture Collection Committee, or from other publicly available sources; other reagents are commercially available. In this application, humanized ANGPTL3 mice from Shanghai Southern Model Biotechnology Co., Ltd. and Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. were used. The ANGPTL3 expression level of mice from Shanghai Southern Model Biotechnology Co., Ltd. was several times higher than that from Biocytogen.

[0208] It should be understood that this application includes the various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art to which this application pertains. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments. Example

[0209] Example 1: Sequence design and synthesis of ANGPTL3 siRNA

[0210] 1. siRNA design

[0211] Based on the mRNA sequence (NM_014495.4) of the human ANGPTL3 gene (as shown in Table 1), multiple ANGPTL3 siRNAs were designed at different sites (as shown in Table 2). All designed individual siRNAs can target all transcripts of the target gene. The sequences were compared with all other non-target gene sequences by sequence similarity software and showed the lowest homology.

[0212] Table 1 Target Genes

[0213] Table 2 Sequence Design

[0214] 2. siRNA synthesis (natural RNA / 2'-methoxy or 2'-fluorine modified RNA / RNA containing GalNac ligand)

[0215] The siRNA actually used in this application embodiment contains unmodified ribonucleotides, 2'-methoxy or 2'-fluorine modified ribonucleotides, and ribonucleotides containing the corresponding GalNAc ligands. The siRNA is synthesized according to the theoretical yield of 1 μmol, using a 1 μmol universal Frit solid support. Alternatively, all oligonucleotides were prepared on a solid-phase support with corresponding ligands on an LK-192X synthesizer. According to sequence requirements, all phosphoramidite monomers corresponding to the nucleosides were diluted 1:20 (g / mL) with anhydrous acetonitrile, and coupled twice for 3 min. Activation was performed using 0.3 M benzylthiotetrazole acetonitrile solution, followed by deprotection with 3% TCA, and capping and oxidation with CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid-phase support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65 °C oven for 3 h to remove the protecting group. The solution was then cooled to room temperature and concentrated under vacuum for 30 min. It was filtered through a 0.22 μm filter into a sample vial and purified using a semi-preparative reversed-phase purification system for single-strand purification. The elution gradient was 7%–30% (ACN: 100 mM TEAA) for 10 min at a flow rate of 5 mL / min. After purification, the solution was concentrated under vacuum and evaporated to dryness at room temperature. The sample was then dissolved in water, and each solution was desalted on a GE Hi-Trap desalting column to elute the final oligonucleotide product. All characteristics and purities were confirmed using ESI-MS and IEX HPLC. The final product was obtained by UV concentration determination using a microplate reader, mixing equimolar amounts of the sense and antisense strands, heating at 95°C for 5 min, slowly annealing to room temperature, and finally evaporating to dryness at room temperature using a vacuum concentrator.

[0216] In this application, both unmodified naked siRNA sequences and modified siRNAs were prepared according to this method. The difference lies in the type of phosphoramide monomer corresponding to the added nucleotides. The phosphoramide monomers corresponding to the nucleotides used in this application are all prior art. In addition, appropriate solid-phase carriers are used depending on whether a ligand is liganded and the type of ligand used: when no ligand is required, a general Frit solid-phase carrier is used; when a ligand is required, a solid-phase carrier containing the corresponding GalNAc ligand is prepared according to the general method in the art for ligand-solid-phase carrier connection or the method described in this application (e.g., a solid-phase carrier connected to L96, a solid-phase carrier connected to ligand 1, a solid-phase carrier connected to ligand 2).

[0217] Of course, when linking nucleotide sequences to ligands, one can either sequentially link nucleotides to a solid support linked to ligands as described above, or use a common Frit solid support to complete the sequence synthesis (without removing the protecting group and solid support) and then stepwise conjugate the corresponding GalNAc phosphoridamide monomer.

[0218] In this embodiment, the inhibitory activity of ANGPTL3siRNA in the in vitro psicheck-2 system was detected using a naked sequence without ligands.

[0219] 3. Detection of the inhibitory activity of ANGPTL3 siRNA in the in vitro psicheck-2 system

[0220] 1) Construct detection plasmid

[0221] Recombinant plasmids were constructed using the psicheck-2 plasmid (purchased from Shanghai Jereh Biotechnology Co., Ltd., a dual-luciferase reporter gene plasmid that can express firefly luciferase and kidney luciferase). The recombinant plasmids contained the full-length target mRNAs of all the ANGPTL3 siRNAs to be tested, and the cloning sites were the 5'XhoI and 3'NotI sites of the psicheck-2 plasmid.

[0222] 2) ANGPTL3 siRNA and recombinant plasmid were co-transfected into different cells.

[0223] 293T cells (renal epithelial cell line) were placed in DMEM medium containing 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. After digestion, the cell density was adjusted to 1.5 × 10⁶ cells per well. 5Cells were seeded into 24-well plates. Transfection complex preparation: 250 μL of Opti-MEM, 40 ng of recombinant plasmid, and 5 μL of 10 nM siRNA were mixed to form a mixture. 250 μL of Opti-MEM and 2.5 μL of Lipofectamine 2000 transfection reagent were mixed to form another mixture. The mixtures were allowed to stand for 5 min. The two mixtures were then combined and allowed to stand for 20 min to form the transfection complex. The transfection complex was added to the 24-well plates and incubated at 37°C with 5% CO2 for 6 h. The supernatant was aspirated, and 500 μL of complete culture medium was added to each well. The plates were then incubated for another 24 h.

[0224] In addition to the experimental group, an NC group (without associated siRNA) was set up as a negative control for each cell transfection. The sequence of NC is as follows:

[0225] SS:UUCUCCGAACGUGUCACGUTT(SEQ ID NO:405)

[0226] AS: ACGUGACACGUUCGGAGAATT(SEQ ID NO:406)

[0227] Both the experimental and control groups had three independent replicates.

[0228] 3.3 DLR Detection and Analysis

[0229] The Dual-Luciferase Reporter Assay System (purchased from Promega) was used for detection. Cells were lysed and collected according to the kit instructions. The fluorescence intensity of firefly (Photinus pyralis) luciferase and renal (Renilla reniformis) luciferase was detected sequentially using an Infinite Eplex microplate reader (TECAN). The ratio of the fluorescence intensity of renal (Renilla reniformis) luciferase to that of firefly (Photinus pyralis) luciferase was calculated, and the NC group was used as a control for normalization.

[0230] First, DLR screening of ANGPTL3 siRNA was performed in 293T cells. Based on the relative activity of luciferase, the top 60 siRNA molecules with strong RNA interference capabilities were identified, as shown in Figure 1. Then, siRNA molecules with sequences different from those of humans and cynomolgus monkeys were excluded, resulting in the top 28 siRNA molecules with strong RNA interference capabilities (as shown in Table 3). Table 3 shows the average dual-luciferase reporter gene expression levels of the experimental group relative to the NC group of the top 28 siRNA molecules with strong interference capabilities shown in Figure 1.

[0231] Table 3. Relative luciferase activities of the top 28 siRNAs with strong interference capabilities obtained from DLR detection analysis.

[0232] Example 2: In vitro detection of the inhibitory activity of modified siRNA against the ANGPTL3 gene.

[0233] 1. Modified siRNA

[0234] Modification strategies are important, as certain modifications can reduce the repressive activity of siRNA against the ANGPTL3 gene. The following modification strategies yield siRNAs with high repressive activity:

[0235] (1) The 3' end of the antisense strand has a 2-nucleotide protrusion relative to the 3' end of the sense strand. This structure is beneficial for loading the antisense strand into RISC, enhancing the interference activity of RNAi without affecting stability.

[0236] (2) Starting from the 5' end, the 2nd, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluorine, and other positions are modified with 2'-methoxy as much as possible. This structure is conducive to RISC binding to mRNA and is protected from ribonuclease attack.

[0237] (3) Starting from the 5' end, the 7th and 9th-11th nucleotides of the positive strand are modified with 2'-fluorine, and other positions are modified with 2'-methoxy as much as possible. This structure is conducive to RISC binding to mRNA.

[0238] (4) The first and second phosphate ester bonds of the antisense strand starting from its 5' end and the first and second phosphate ester bonds starting from its 3' end are thiophosphate bonds formed by thiolation. That is, the phosphate ester bonds between the first and second nucleotides at either end and between the second and third nucleotides are thiophosphate bonds formed by thiolation. This structure is beneficial for maintaining the stability of nucleic acids.

[0239] (5) The first and second phosphate ester bonds of the positive sequence starting from its 5' end are thiophosphate bonds formed by thiolation. That is, the phosphate ester bonds between the first and second nucleotides at the 5' end and between the second and third nucleotides are thiophosphate bonds formed by thiolation. This structure is beneficial for maintaining the stability of nucleic acids.

[0240] We modified the 28 candidate sequences and positive control AG05488 obtained from the previous screening in Table 1 according to the above modification scheme. The positive sequence is:

[0241] NmsNmsNmNmNmNmNmNfNmNfNfNm ...

[0242] Table 4. Combinations of siRNA modification strategies for candidate sequences

[0243] In this application and Table 4, Nm represents ribonucleotide N with 2'-O-methyl modification, and Nf represents nucleotide N with 2'-fluorine modification. Specifically, Am, Um, Cm, and Gm represent ribonucleotides A, U, C, and G modified with 2'-O-methyl, respectively; Af, Uf, Cf, and Gf represent ribonucleotides A, U, C, and G modified with 2'-fluorine, respectively. 's' indicates that adjacent nucleotides are linked by a phosphate thioester bond. If both nucleotide modification and phosphate ester bond modification exist simultaneously, they are combined. For example, when three nucleotides UGA are represented as UmsGfsAm, it means that nucleotide U is modified with 2'-O-methyl, nucleotide G is modified with 2'-fluorine, and nucleotide A is modified with 2'-O-methyl. Simultaneously, U and G are linked by a phosphate thioester bond, G and A are linked by a phosphate thioester bond, and L96 is linked to the 3' end of the positive strand of the RNA molecule by a phosphate ester bond. In this application, in molecules appearing in sequence form, nucleotides without the 's' label are linked by conventional phosphate ester bonds.

[0244] 2. Transfection of Hep3B cells with ANGPTL3 siRNA

[0245] The activity of the modified siRNA was further verified in Hep3B cells.

[0246] Hep3B cells (human liver cancer cells) were placed in DMEM medium containing 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were seeded and transfected. The cultured cells were digested and the cell density was adjusted to 1.0 × 10⁶ cells per well. 5Cells were seeded into 24-well plates. The transfection complex was prepared by mixing 250 μL of Opti-MEM and 5 μL of 10 nM siRNA, and then mixing 250 μL of Opti-MEM and 2.5 μL of Lipofectamine 2000 transfection reagent. The mixtures were allowed to stand for 5 min, and then combined and allowed to stand for 20 min to form the transfection complex. The transfection complex was added to the 24-well plates and incubated at 37°C with 5% CO2 for 6 h. The supernatant was aspirated, and 500 μL of complete culture medium was added to each well. The plates were then incubated for another 24 h.

[0247] In addition to the experimental group, an NC negative control group (without siRNA) was also set up for each cell transfection.

[0248] 3. Real-time quantitative PCR analysis:

[0249] Cells were lysed 48 hours after transfection, and total RNA was extracted using a column extraction kit (Novizan). Real-time quantitative PCR was performed using a CFX96 real-time PCR instrument (Bio-Rad) with GAPDH as an internal control gene. Primers used were:

[0250] Table 5 Primer sequence information

[0251] 4. Data Analysis

[0252] After the PCR reaction, relative quantification was performed using the 2–ΔΔCt (Livak) method with the reference gene GAPDH as the standard. The ANGPTL3 mRNA expression level in the NC group was 1. Figure 2 shows that among the 28 candidate siRNA sequences transfected at a final concentration of 0.1 nM, the siRNA molecules with higher inhibitory activity than the positive control AG05488 were ANG50-EG, ANG65-EG, ANG338-EG, ANG339-EG, ANG639-EG, ANG1356-EG, ANG1384-EG, ANG1404-EG, and ANG1408-EG.

[0253] Example 3: Detection of the effectiveness of modified siRNA molecules in mice

[0254] The efficacy of the nine modified siRNA molecules with high inhibitory activity obtained in Example 2 and the positive control AG05488 in mice was tested. Sixty-six 6-8 week old humanized ANGPTL3 mice (Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.), half male and half female, were used in the experiment. They were randomly divided into groups of six mice according to body weight and administered a single subcutaneous injection of 3 mg / kg (dosage grouping is shown in Table 6). Blood samples were collected on days -3, 7, 14, 21, 35, 49, and 70 (after fasting for 4 hours before blood collection; the first dose was on day 0). Serum was separated, and the expression level of ANGPTL3 protein in the serum was detected using the Human ANGPTL3 ELISA Kit (R&D), and inter-group comparisons were performed.

[0255] Table 6. Experimental protocol for subcutaneous siRNA drug injection in mice.

[0256] The experimental results (Figure 3, Table 7) showed that, compared with before administration, the serum ANGPTL3 protein level in the ANG50-EG, ANG65-EG, ANG338-EG, ANG339-EG and ANG639-EG groups decreased to the lowest point on day 7 after administration, with reductions of 93%, 93%, 92%, 91% and 86% respectively, and remained at a low level, with significantly better effects than Yangshen AG05488-EG.

[0257] Table 7. Expression levels of ANGPTL3 protein in serum.

[0258] Example 4: Detection of the effectiveness of modified siRNA molecules in cynomolgus monkeys

[0259] Based on the results in Table 7, ANG639-EG, ANG50-EG, and ANG65-EG were selected. Pre-toxicology experiments were conducted on these three sequences. Compared with ANG639-EG, the other two sequences were more toxic. ANG639-EG was used for further efficacy testing.

[0260] Six cynomolgus monkeys (Beijing Zhaoyan Biotechnology Co., Ltd.) were used in the experiment and randomly assigned according to body weight to a positive control group and an experimental test group (two test compounds, ANG639-EG and AG05488-EG, both siRNAs modified with GalNAc derivative L96), with three animals in each group. Each animal received a single subcutaneous injection of 3 mg / kg of the drug. Blood was collected from the cephalic vein of the forelimb on days -2, 4, 7, 14, 21, 28, 42, 56, 70, and 84 (after fasting overnight, with the first dose on day 1). Serum was separated for the detection of ANGPTL3 protein and TG.

[0261] I. TG Detection

[0262] 1. Method

[0263] The concentration of TG in serum was measured using a Toshiba TBA120 blood biochemistry analyzer, and intergroup comparisons were performed.

[0264] 2. Results

[0265] Table 8 shows the normalized to post-treatment mean relative TG content from Example 4.

[0266] The experimental results (Figure 4, Table 8) showed that, compared with before administration, serum TG levels in both the AG05488-EG and ANG639-EG groups were significantly reduced after administration and remained at a low level, with the lowest reductions being 44% and 66%, respectively. Among them, the ANG639-EG molecule had a more significant effect on reducing TG than the AG05488-EG molecule.

[0267] II. ANGPTL3 protein detection

[0268] 1. Method

[0269] The expression level of ANGPTL3 protein in serum was detected using the Human Angiopoietin-like 3Immunoassay ELISA kit (R&D Systems), and intergroup comparisons were performed.

[0270] 2. Results

[0271] Table 9 shows the normalized to post-treatment mean relative ANGPTL3 protein content from Example 4.

[0272] The results (Figure 5, Table 9) showed that, compared with pre-administration levels, serum ANGPTL3 protein levels in the AG05488-EG group decreased to their lowest point (74%) 21 days after administration and remained at a low level until day 84, when serum ANGPTL3 protein levels increased slightly. In the ANG639-EG group, serum ANGPTL3 protein levels decreased by 90% on day 21 after administration and remained at a low level until day 84. Therefore, the ANG639-EG molecule was significantly more effective than AG05488-EG in reducing ANGPTL3 protein levels.

[0273] Example 5: Rat Toxicity Experiment

[0274] Twelve SPF-grade Sprague-Dawley (SD) rats (Wuhan Branch of Hubei Tianqin Biotechnology Co., Ltd.) were randomly divided into two groups according to body weight: a solvent control group and an ANG639-EG group, with six rats in each group (half male and half female). A single subcutaneous injection of the drug (dose: 250 mg / kg) was administered, and the rats were observed for two weeks. During the experiment, animals in each group were routinely observed twice daily. Local observation of the drug administration site, body weight, and clinical pathology (blood biochemistry) tests were performed regularly on surviving animals in each group. At the end of the observation period (day 15), the animals in each group were dissected as planned. During dissection, gross lesions of the organs were observed, the liver and kidneys were weighed, organ coefficients were calculated, and histopathological examinations of the liver, kidneys, axillary lymph nodes, inguinal lymph nodes, and mesenteric lymph nodes were performed.

[0275] Table 10 shows the blood biochemical results of each group on day 15 after drug administration in Example 5.

[0276] Table 11 Summary of organ weights and coefficients from Example 5

[0277] The results showed that all animals were in good condition during the experiment, with no significant changes in body weight and no abnormalities caused by the test substance. Blood biochemical changes were minor, and related pathological changes were mild. No gross abnormalities were observed in any of the experimental animals, and no significant changes in liver and kidney weight or organ system numbers were observed in any group.

[0278] In summary, the combined results of in vitro activity experiments, in vivo experiments in cynomolgus monkeys, and in vivo experiments in humanized mice confirm that ANG639 has a more effective and longer-lasting inhibitory activity than ginseng molecules, with a more significant and sustained reduction in ANGPTL3 protein and TG levels, while no obvious toxic side effects were observed, achieving unexpected technical results.

[0279] The modified double-stranded RNAi in this application exhibits high stability while maintaining high inhibitory activity, achieving unexpected technical results.

[0280] Example 6: Detection of the effectiveness of siRNAs linked with different ligands in mice

[0281] The molecular structures of siRNAs linked with different ligands are shown in Table 12. Thirty-two 6-8 week old humanized ANGPTL3 male and female mice (Shanghai Southern Model Biotechnology Co., Ltd.) were used in the experiment. They were randomly divided into groups of eight mice each, half male and half female, based on body weight. A single subcutaneous injection of 3 mg / kg was administered (dosage groupings are shown in Table 13). Blood samples were collected on days -3, 7, 21, and 35 (after fasting for 4 hours prior to blood collection; the first dose was taken on day 0). Serum was separated, and the expression level of ANGPTL3 protein in the serum was detected using the Human ANGPTL3 ELISA Kit (R&D), with comparisons made between groups.

[0282] Table 12. Molecular Structure

[0283] In the table above, ligands are linked to the 3' end of the positive strand of the ANG639-EG molecule. Specifically, L96 is linked to the 3' end of the positive strand of the ANG639-EG molecule via a phosphate ester bond, and ligands 1-3 are linked to the 3' end of the positive strand of the ANG639-EG molecule via a thiophosphate ester bond. In this application, in molecules appearing in sequence form, nucleotides without an 's' symbol are linked by conventional phosphate ester bonds. The specific details of each molecule are as follows:

[0284] (1) PC07:

[0285] Chain of Justice: GmsAmsAmAmUmAmGfAmAfAfAfUmCmAmGmCmUmCmAmGmCmUmCmAmGmAm(SEQ ID NO:9)-L96;

[0286] Antisense strand: UmsCfsUmGmAmGmCmUmGmAmUmUmUmUfCmUfAmUmUmUmCmsUmsUm (SEQ ID NO:10), wherein each nucleotide does not contain any modifications other than L96, 2'-F, 2'-OMe and thiophosphate, and the 3' end of SEQ ID NO:9 is linked to L96 via a phosphate ester bond;

[0287] (2) PC08:

[0288] Justice Chain: GmsAmsAmAmUmAmGfAmAfAfAfUmCmAmGmCmUmCmAmGmCmUmCmAmGmAms(SEQ ID NO:412)-ligand 1;

[0289] Antisense strand: UmsCfsUmGmAmGmCmUmGmAmUmUmUmUfCmUfAmUmUmUmCmsUmsUm (SEQ ID NO:10), wherein each nucleotide does not contain any modifications other than ligands 1, 2'-F, 2'-OMe and thiophosphate, and the 3' end of SEQ ID NO:9 is linked to ligand 1 via a thiophosphate bond;

[0290] (3) PC09:

[0291] Justice Chain: GmsAmsAmAmUmAmGfAmAfAfAfUmCmAmGmCmUmCmAmGmCmUmCmAmGmAms(SEQ ID NO:412)-ligand 2;

[0292] Antisense strand: UmsCfsUmGmAmGmCmUmGmAmUmUmUmUfCmUfAmUmUmUmCmsUmsUm (SEQ ID NO:10), wherein each nucleotide does not contain any modifications other than ligand 2, 2'-F, 2'-OMe and thiophosphate, and the 3' end of SEQ ID NO:9 is linked to ligand 2 via a thiophosphate bond;

[0293] (4) PC10:

[0294] Justice Chain: GmsAmsAmAmUmAmGfAmAfAfAfUmCmAmGmCmUmCmAmGmCmUmCmAmGmAms(SEQ ID NO:412)-ligand 3;

[0295] Antisense strand: UmsCfsUmGmAmGmCmUmGmAmUmUmUmUfCmUfAmUmUmUmCmsUmsUm (SEQ ID NO:10), wherein each nucleotide does not contain any modifications other than ligand 3, 2'-F, 2'-OMe and thiophosphate, and the 3' end of SEQ ID NO:9 is linked to ligand 3 via a thiophosphate bond;

[0296] Table 13. Inhibition rate of ANGPTL3 protein in serum after a single 3mpk dose.

[0297] The experimental results (Table 13, Figure 6) showed that, compared with before drug administration, the serum ANGPTL3 protein levels in the PC07 (L96), PC08 (ligand 1), PC09 (ligand 2), and PC10 (ligand 3) groups decreased to their lowest point on day 7 after drug administration, decreasing by 71%, 78%, 73%, and 73%, respectively, and then began to rebound. Among them, PC08 had the best effect, and its effect was significantly better than that of PC07, PC09, and PC10 throughout the course of drug administration.

[0298] The structures of L96, ligand 1, ligand 2, and ligand 3 are shown in Table 14 below. The method of conjugating the ligands to the oligonucleotide has been described in "2. siRNA Synthesis" of Example 1. The monomers of ligand 1 and ligand 2 and the preparation of ligand 1 and ligand 2 are shown in Preparation Example 1 and Preparation Example 2. The method of preparing ligand 1 and ligand 2 using the above monomers and conjugating them to the oligonucleotide is conventional in the art. For example, the preparation method disclosed in CN114585633A can be referred to.

[0299] Table 14. Structures of different ligands

[0300] Example 7: Detection of the effectiveness of siRNAs linked with different ligands in cynomolgus monkeys.

[0301] Four cynomolgus monkeys (Beijing Zhaoyan Biotechnology Co., Ltd.) were used in the experiment and randomly assigned to two experimental test groups according to their body weight (the drug administration groups are shown in Table 15, where the drug administration molecules for PC07 and PC08 groups are the same as those in the corresponding groups in Example 6), with two animals in each group. Each animal was given a single subcutaneous injection of 3 mg / kg of the drug. Blood was collected from the cephalic vein of the forelimb on days -2, 4, 7, 14, 21, 28, 42, 56, 70, and 84 (the animals were fasted overnight before blood collection, and the first dose was on day 0). Serum was separated for ANGPTL3 protein detection.

[0302] Table 15 Percentage reduction in serum ANGPTL3 protein after a single 3mpk dose

[0303] The experimental results (Table 15, Figure 7) showed that, compared with before administration, both PC07 (L96) and PC08 (ligand 1) groups could reduce the serum ANGPTL3 protein level in cynomolgus monkeys after administration, and the level dropped to the lowest point on day 28, with reductions of 89% and 91%, respectively, before rebounding. Among them, the effect of PC08 was better than that of PC07 throughout the process.

[0304] Example 8: Detection of the effectiveness of different modified siRNAs in humanized mice

[0305] Eighteen humanized ANGPTL3 mice (male and female, 6-8 weeks old, Shanghai Southern Model Biotechnology Co., Ltd.) were used in the experiment, with six mice in each group. A single subcutaneous injection of 3 mg / kg was administered. Blood samples were collected on days 3, 7, 21, 35, and 42 (after fasting for 4 hours prior to blood collection; the first dose was taken on day 0). Serum was separated, and the expression level of ANGPTL3 protein in the serum was detected using the Human ANGPTL3 ELISA Kit (R&D). Intergroup comparisons were also performed (see Table 16 for drug administration groups).

[0306] Table 16 Inhibition rate of serum ANGPTL3 protein after a single 3mpk dose

[0307] Experimental results (Table 16, Figure 8) showed that, compared with pre-drug administration, both PC08 and the novel motif-modified ANG639-E20-PC08 groups reduced serum ANGPTL3 protein levels in humanized mice after administration. The novel motif-modified ANG639-E20-PC08 molecule was modified as follows, with specific sequence examples shown in Table 17. In Table 17, the 3' end of the positive strand is linked to ligand 1 via a phosphate thioester bond for siRNA delivery:

[0308] The sequence of justice:

[0309] NmsNmsNmNmNmNmNfNmNfNfNfNfNmNmNmNmNmNmNmNmNmNm (motif 3);

[0310] Antonym sequences:

[0311] NmsNfsNmNmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (motif 2)

[0312] Table 17 Information on New Sequence Modifications

[0313] The molecular details of ANG639-E20-PC08 in the table above are as follows:

[0314] Justice Chain: GmsAmsAmAmUmAmGfAmAfAfAfUfCmAmGmCmUmCmAmGmCmUmCmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAmGmAms(SEQ ID NO:407)-ligand 1;

[0315] Antisense strand: UmsCfsUmGmAmGmCmUmGmAmUmUmUmUfCmUfAmUmUmUmCmsUmsUm (SEQ ID NO:10), wherein each nucleotide does not contain any modifications other than ligand 1, 2'-F, 2'-OMe and thiophosphate, and the 3' end of SEQ ID NO:407 is linked to ligand 1 via a thiophosphate bond.

[0316] In this application, in molecules appearing in sequence form, unless otherwise specified, nucleotides not marked with 's' are linked by conventional phosphate ester bonds.

[0317] Preparation Example 1: Preparation of ligand 1 phosphoridamide monomer, ligand 1 monomer linked to a solid support, and ligand 1 linked to a solid support.

[0318] I. Synthesis of Compound 1

[0319] ① Synthesis of compound 16:

[0320] 10 g of compound 15 and 100 mL of pyridine were added to a flask. After stirring to dissolve, the mixture was cooled in an ice bath. 20 g of B2Cl was carefully added dropwise, and the mixture was stirred in an ice bath for 10 min under nitrogen protection, followed by stirring at room temperature for 16 hours. A saturated aqueous solution of sodium bicarbonate was slowly added to the reaction mixture in an ice bath, and after stirring for 1 hour, the mixture was concentrated to remove pyridine. DCM was added to the residue, and after stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 18 g of a white solid powder, with a yield of 85.3%. ESI+MS: m / z 594.3 [M+H] + .

[0321] ② Synthesis of compound 17:

[0322] 10 g of compound 16 and HOAc / Ac2O (50 mL / 50 mL) were added to a flask. After stirring to dissolve, the mixture was heated to 100 °C and stirred at 100 °C for 2 hours. The reaction solution was cooled to room temperature and carefully poured into 200 mL of ice water. DCM was then added, and the mixture was stirred for 15 min. After standing, the liquid was separated, and the organic phase was washed successively with water, 5% sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to give 5 g of white solid powder, with a yield of 71.4%. ESI+MS: m / z 415.2 [M+H] + .

[0323] ③ Synthesis of compound 19:

[0324] 5 g of compound 17 and 40 mL of DCM were added to a three-necked flask. After stirring to dissolve, the mixture was cooled to -30 °C. Under nitrogen protection, 4.02 g of TMSOTf was carefully added dropwise while stirring at -30 °C for 30 min. Subsequently, 3.17 g of a DCM solution of compound 18 was carefully added dropwise. After the addition was complete, stirring was continued at -30 °C for 1 hour. The reaction solution was heated to 0–5 °C, and 5% sodium bicarbonate aqueous solution was carefully added dropwise. After stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was washed successively with 5% sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography and reversed-phase chromatography to obtain 2.8 g of white solid powder, yield 40.5%, ESI+MS: m / z 574.3 [M+H]. + .

[0325] ④ Synthesis of Compound 1:

[0326] 2.5 g of compound 19 and 25 mL of MeOH were added to a flask. The mixture was stirred thoroughly, and 320 mg of MeONa was carefully added dropwise under nitrogen protection. The mixture was stirred at room temperature for 6 hours, followed by concentration. The crude product was then converted to a white solid powder by reverse phase chromatography, yielding 1 g of powder (62.9% yield). ESI-MS: m / z 424.4 [M+OAc] - ] - .

[0327] II. Synthesis of Compound 6

[0328] ① Synthesis of compound 2:

[0329] 1.2 g of compound 1 and 12 mL of pyridine were added to a three-necked flask. After stirring to dissolve, 1.2 g of DMTrCl was added, and the mixture was stirred at room temperature for 1 hour under nitrogen protection. Excess DMTrCl was quenched by slowly adding 0.6 mL of methanol to the reaction mixture, and after stirring at room temperature for 15 min, 303.5 mg of NaHCO3 was added. The mixture was concentrated to obtain a crude product, which was dissolved by adding DCM / H2O and washing with water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product of compound 2, which was used directly in the next reaction step.

[0330] ② Synthesis of compound 3:

[0331] Ethanol (24 mL) was added to the crude compound 2, and the temperature was slowly raised to 50 °C. After stirring to dissolve, hydrazine hydrate (0.75 mL) was added, and the mixture was stirred overnight at 50 °C. Then, the temperature was lowered to room temperature, and stirring was continued at room temperature for half an hour. At this point, a large amount of white solid gradually precipitated. The mixture was filtered, and the residue was washed with ethanol. The filtrate was concentrated under reduced pressure, then redissolved in DCM / H2O. The liquid was separated, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain the crude compound 3, which was directly used in the next reaction.

[0332] ③ Synthesis of compound 5 (i.e., ligand 1 phosphoridamide monomer):

[0333] 0.9 g of compound 4, 24 mL of DCM, 0.84 mL of Et3N, and 2.3 g of HBTU were added sequentially to the crude compound 3. The mixture was stirred at room temperature for 2 hours under nitrogen protection. The reaction was quenched with 9 mL of water, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum and purified by reverse-phase preparation (75% ACN-H2O) to give 1.4 g of compound 5, with a three-step yield of 44.2%. ESI-MS: m / z 1025.6 [M+OAc] - ] - .

[0334] ④ Synthesis of compound 6:

[0335] 1.4 g of compound 5 was dissolved in 14 mL of DCM and cooled at 0 ± 2 °C. 524.7 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 81.3 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another 2 hours. The reaction mixture was cooled at 0 ± 2 °C, quenched with 14 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with saturated brine (1 x 14 mL) at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum, and the crude product was redissolved in DCM / MTBE and carefully added dropwise to a vigorously stirred heptane solution. A large amount of viscous oil gradually precipitated from the walls and bottom of the flask. After standing for 10 min, the supernatant was decanted. The crude product was chromatographically analyzed using EA:hept. = 10:1 to 2:1 as eluent. After concentration and drying, 560 mg of a white solid powder was obtained, with a yield of 32% and a phosphorus spectrum purity of 98.19%. ESI-MS: m / z 1225.4 [M+OAc] - ] - .

[0336] III. Synthesis of Compound 8

[0337] ① Synthesis of compound 7:

[0338] 2g of compound 5 was dissolved in 20mL of DCM, and 248.4mg of succinic anhydride and 0.69mL of Et3N were added sequentially. Finally, 25.3mg of DMAP was added, and the mixture was stirred at room temperature for 16 hours. HPLC monitoring showed a significant amount of reactant remaining, so 310.4mg of succinic anhydride and 0.69mL of Et3N were added. Finally, 25.3mg of DMAP was added, and the mixture was stirred at room temperature for another 36 hours. The reaction solution was cooled at 0±2℃, ice water was added, followed by DCM, and finally 40mL of 1% HOAc aqueous solution was added. After stirring for 10 minutes, the organic phase was separated and washed sequentially with 1% HOAc aqueous solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product. The crude product was chromatographically analyzed using DCM:MeOH = 70:1–20:1 eluent. After concentration and drying, 0.9g of white solid powder was obtained, with a yield of 40.7%. No succinic acid residue was detected, and the succinic anhydride residue was 0.26%. ESI-MS: m / z 1065.7 [MH] - .

[0339] ② Synthesis of compound 8 (i.e., monomer of ligand 1 attached to the solid support):

[0340] 300 mg of compound 7, 72.7 mg of DIPEA, 106 mg of HBTU, and 10 mL of acetonitrile were added to a three-necked flask. The mixture was stirred at 25 °C for 10 minutes, followed by the addition of 650 mg of solid support PS. Stirring continued at 25 °C for 24 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed with acetonitrile. The collected filter cake was concentrated under vacuum to remove the solvent, yielding 850 mg of solid. 700 mg of this solid was added to a three-necked flask, along with 1.74 g of acetic anhydride, 4.15 mg of DMAP, 103 mg of triethylamine, and 10 mL of pyridine. The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed successively with acetonitrile, methanol, and acetonitrile. The collected filter cake was concentrated under vacuum to remove the solvent, yielding 750 mg of compound 8. The loading was determined to be 254.82 μmol / g.

[0341] IV. Preparation of Ligand 1 Attached to the Solid Support

[0342] Two phosphorus amide monomers (compound 6) for ligand 1 are reacted sequentially with compound 8. The phosphorus amide group reacts with the DMTlorO- group to obtain the precursor of ligand 1 linked to a solid-phase support. Alternatively, two compounds 6 are reacted sequentially with compound 8 through conventional solid-phase synthesis cycles (deprotection, coupling, oxidation, and capping) to obtain the precursor (or intermediate) of ligand 1 linked to a solid-phase support. Further solid-phase synthesis cycles are then performed using the corresponding 2'-modified monomers. After the reaction is complete, the synthesized molecule is dissociated from the solid-phase support by ammonolysis to obtain the oligonucleotide sequence linked to ligand 1.

[0343] Preparation Example 2: Preparation of ligand 2 phosphoridamide monomer, ligand 2 monomer linked to a solid support, and ligand 2 linked to a solid support.

[0344] I. Synthesis of Compound 9

[0345] ① Synthesis of compound 21:

[0346] 10 g of compound 20 was dissolved in DCM (90 mL). While cooling in an ice bath, a solution of 17.53 g of ethyl trifluoroacetate in DCM (10 mL) was carefully added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred for 1 hour. The reaction solution was then concentrated to obtain crude compound 21, which was used directly in the next step. ESI-MS: m / z 184.0 [MH] - .

[0347] ② Synthesis of compound 23:

[0348] 25.2 g of compound 22 and 90 mL of DCM were added to a three-necked flask. After stirring to dissolve, the mixture was cooled to -30 °C, and 18.01 g of TMSOTf was carefully added dropwise under nitrogen protection while stirring at -30 °C for 30 min. Subsequently, 10 g of a DCM solution of compound 21 (10 mL) was carefully added dropwise, and stirring was continued at -30 °C for 1 hour. The reaction mixture was heated to 0–5 °C, and 5% sodium bicarbonate aqueous solution was carefully added dropwise. After stirring for 15 min, the mixture was allowed to stand and separated. The organic phase was washed successively with 5% sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to obtain 8 g of white solid powder, yield 27.6%, ESI-MS: m / z 536.2 [MH]. - .

[0349] ③ Synthesis of compound 24:

[0350] 8 g of compound 23 and 25 mL of MeOH were added to a flask. The mixture was stirred thoroughly, and 1.07 g of MeONa was carefully added dropwise under nitrogen protection, with stirring at room temperature for 4 hours. The mixture was quenched with 1 mL of 10% NH4Cl aqueous solution and then concentrated. The crude product was then subjected to reverse-phase chromatography to obtain 3 g of a white solid powder, yield 67%, ESI-MS: m / z 302.1 [M+H]. + .

[0351] ④ Synthesis of compound 9:

[0352] 3 g of compound 24 and 30 mL of pyridine were added to a flask. After stirring and dissolving, 3.71 g of DMTrCl was added in two portions under ice bath and nitrogen protection. After the addition was complete, the mixture was stirred at room temperature for 1 hour. Methanol (3 mL) was slowly added to the reaction mixture to quench the reaction. After stirring at room temperature for 15 min, 1 g of NaHCO3 was added. Stirring was continued for 15 min, and the mixture was concentrated to obtain a crude product. DCM / H2O (30 mL / 30 mL) was added, stirred and dissolved, and washed with brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to column chromatography to obtain 4.5 g of white solid powder, yield 75%, ESI-MS: m / z 662.2 [M+OAc] - ] - .

[0353] II. Synthesis of Compound 12

[0354] ① Synthesis of compound 10:

[0355] 5 g of compound 9 was dissolved in 15 mL of ACN, and NaOH solution (662.6 mg NaOH dissolved in 7.5 mL of water) was added. The mixture was stirred at room temperature for 2 hours. EA was added to the reaction solution, and after stirring for 10 min, the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum to obtain crude compound 10, which was used directly in the next step. ESI-MS: m / z 566.4 [M+OAc] - ] - .

[0356] ② Synthesis of compound 11:

[0357] 3.5 g of compound 4, 35 mL of DCM, 3.26 mL of Et3N, and 8.9 g of HBTU were added sequentially to the crude compound 10. The mixture was stirred at room temperature for 2 hours under nitrogen protection. Insoluble matter was removed by filtration, and the residue was washed with DCM (2 x 3.5 mL). The filtrates were combined. The filtrate was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain the crude product. The crude product was chromatographically analyzed with eluents of EA:hept. = 10:1 to 1:5 and EA:ACN = 10:1. After concentration and drying, 3.6 g of white solid powder was obtained, with a two-step yield of 49%. ESI-MS: m / z 995.7 [M+OAc] - ] - .

[0358] ③ Synthesis of compound 12 (i.e., ligand 2 phosphoramide monomer):

[0359] 1.6 g of compound 11 was dissolved in 16 mL of DCM and cooled at 0 ± 2 °C. 618.9 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine was added, followed by 95.9 mg of 1H-tetrazole. The reaction mixture was stirred at 0 ± 2 °C for 15 min, then brought to room temperature and stirred for another hour. The reaction mixture was cooled at 0 ± 2 °C, quenched with 16 mL of 5% NaHCO3, and the organic phase was separated. The organic phase was washed with saturated brine at 0 ± 2 °C, dried over anhydrous sodium sulfate, and filtered. The collected organic phase was concentrated under vacuum. The crude product was redissolved in EA (32 mL) and carefully added dropwise to a vigorously stirred hept. (320 mL) solution. At this time, a large amount of viscous oily substance gradually precipitated on the walls and bottom of the flask. After standing for 10 min, the supernatant was decanted. The residue was redissolved in EA (32 mL) and carefully added dropwise to a vigorously stirred hept. (320 mL) solution. At this time, a large amount of viscous oily substance gradually precipitated on the walls and bottom of the flask. After standing for 10 min, the supernatant was decanted to obtain the crude product. After alkalizing the column, the crude product was eluted with EA:hept. = 10:1 to 1:5. After concentration and drying, 900 mg of white solid powder was obtained, with a yield of 46.4% and a phosphine purity of 94.55%. ESI-MS: m / z 1195.8 [M+OAc] - ] - .

[0360] III. Synthesis of Compound 14

[0361] ① Synthesis of compound 13:

[0362] 1 g of compound 11 was dissolved in 10 mL of DCM, followed by the addition of 128.4 mg of succinic anhydride and 0.36 mL of Et3N. Finally, 13.1 mg of DMAP was added, and the mixture was stirred at room temperature for 36 hours. The reaction solution was cooled at 0 ± 2 °C, and ice water was added to the reaction mixture, followed by DCM. Finally, 20 mL of 1% HOAc aqueous solution was added, and the mixture was stirred for 10 min. The organic phase was separated and washed successively with 1% HOAc aqueous solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The collected organic phase was concentrated under vacuum to obtain a crude product. The crude product was chromatographically analyzed using a DCM:MeOH eluent of 50:1 to 10:1. After concentration and drying, 0.8 g of a white solid powder was obtained, with a yield of 72.1%. No residues of succinic acid and succinic anhydride were detected. ESI-MS: m / z 1035.7 [MH] - .

[0363] ② Synthesis of compound 14 (i.e., monomer of ligand 2 attached to the solid support):

[0364] In a three-necked flask, 300 mg of compound 13, 87.4 mg of DIPEA, 128 mg of HBTU, and 11 mL of acetonitrile were added. The mixture was stirred at 25 °C for 10 minutes, followed by the addition of 730 mg of an amine solid support. Stirring continued at 25 °C for 24 hours. After the reaction was complete, the mixture was filtered, and the filter cake was concentrated under vacuum to remove the solvent, yielding 900 mg of solid. 800 mg of this solid was added to a three-necked flask, along with 817 mg of acetic anhydride, 1.95 mg of DMAP, 48.6 mg of triethylamine, and 10 mL of pyridine. The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed successively with acetonitrile, methanol, and acetonitrile. The filter cake was concentrated under vacuum to remove the solvent, yielding 700 mg of compound 14. The loading was determined to be 330 μmol / g.

[0365] IV. Preparation of ligand 2 attached to the solid support

[0366] Two phosphorus amide monomers (compound 12) are sequentially reacted with compound 14, and the phosphorus amide group reacts with the DMTlorO- group to obtain the precursor of ligand 2 linked to a solid-phase support. Alternatively, two compounds 12 are sequentially reacted with compound 14, and through conventional solid-phase synthesis cycles (deprotection, coupling, oxidative introduction of thiophosphate modification, capping), the precursor (or intermediate) of ligand 2 linked to a solid-phase support is obtained. Further solid-phase synthesis cycles are performed using the corresponding 2'-modified monomers. After the reaction is complete, the entire synthesized molecule is dissociated from the solid-phase support by ammonolysis, yielding the oligonucleotide sequence linked to ligand 2.

[0367] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When they represent RNA sequences, "T" represents uridine. Furthermore, in the context of RNA, unless otherwise specified, "T" and "U" refer to uracil or uridine.

Claims

1. A nucleic acid molecule that inhibits the expression of the ANGPTL3 gene, comprising substantially complementary sense and antisense sequences or consisting of substantially complementary sense and antisense sequences, wherein the nucleotide length of the sense and / or antisense sequences is 14-30 nt; The antisense sequence comprises at least 14 consecutive nucleotide sequences, distinct by no more than 3 nucleotides, from the first to the 21st nucleotides starting from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186.

2. The nucleic acid molecule according to claim 1, wherein the antisense sequence comprises 18-21 consecutive nucleotides, consisting of no more than 3 nucleotides distinguishable from the first to 21 nucleotides starting from the 5' end, of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186; Optionally, the antisense sequence comprises 18, 19, 20, or 21 consecutive nucleotides, consisting of no more than 3 nucleotides distinguishable from the first to 21 nucleotides from the 5' end of any one of the sequences SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, and 186.

3. The nucleic acid molecule according to claim 1 or 2, wherein the positive sequence comprises at least 14 consecutive nucleotides that are distinguishable by no more than 3 nucleotides from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, 185.

4. The nucleic acid molecule according to claim 3, wherein the positive sequence strand comprises 18-21 consecutive nucleotides that are no more than 3 nucleotides different from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, 185; Optionally, the positive sequence comprises 18, 19, 20, or 21 consecutive nucleotides that are no more than 3 nucleotides different from any one of the sequences SEQ ID NO: 103, 19, 21, 23, 35, 37, 39, 51, 55, 65, 67, 79, 101, 123, 133, 135, 137, 147, 149, 161, 165, 167, 169, 177, 179, 181, 183, and 185.

5. The nucleic acid molecule according to any one of claims 1-4, wherein the difference of no more than 3 nucleotides is a difference of 3 nucleotides, a difference of 2 nucleotides, a difference of 1 nucleotide, or complete sameness.

6. The nucleic acid molecule according to any one of claims 1-5, wherein the nucleotide length of the sense sequence and / or antisense sequence is 14-30 nt, 17-30 nt, 17-23 nt, 17-21 nt, 17-19 nt, 19-25 nt, 19-23 nt, 19-21 nt, 21-25 nt, 21-23 nt, 25-30 nt, or 27-30 nt; optionally, it is 21-23 nt.

7. The nucleic acid molecule according to any one of claims 1-6, wherein the nucleic acid molecule is dsRNA or shRNA.

8. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule comprises a sense sequence, an antisense sequence, and a linker strand (or shRNA loop), wherein the linker strand connects the 3' nucleotide of the sense sequence and the 5' nucleotide of the antisense sequence.

9. The nucleic acid molecule according to claim 7, wherein the dsRNA is siRNA, and the antisense sequence and sense sequence are the corresponding antisense strand and sense strand, respectively.

10. The nucleic acid molecule according to any one of claims 1-7 or 9, wherein the positive strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may have 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides or 1-2 nucleotides; And / or, wherein the antisense strand further comprises 1-6 overhanging nucleotides at its 3' end and / or 5' end; optionally, the overhangs may have 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides.

11. The nucleic acid molecule according to claim 10, wherein the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 104, 20, 22, 24, 36, 38, 40, 52, 56, 66, 68, 80, 102, 124, 134, 136, 138, 148, 150, 162, 166, 168, 170, 178, 180, 182, 184, 186; optionally, the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 104, 20, 24, 66, 68, 166, 170, 178, 180; further optionally, the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO:

104.

12. The nucleic acid molecule according to claim 11, wherein: The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 104; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 20; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 21, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 22; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 23, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 24; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 75, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 36; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 37, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 38; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 39, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 40; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 51, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 52; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 55, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 56; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 65, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 66; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 67, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 68; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 79, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 80; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 101, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 102; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 123, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 124; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 133, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 134; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 135, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 136; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 137, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 138; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 147, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 148; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 149, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 150; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 161, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 162; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 165, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 166; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 167, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 168; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 169, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 170; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 177, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 178; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 179, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 180; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 181, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 182; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 183, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 184; Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 185, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 186; Optionally, The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 104; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 20; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 23, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 24; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 65, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 66; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 67, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 68; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 165, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 166; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 169, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 170; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 177, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 178; Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 179, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 180; Further optionally, the sense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 103, and the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO:

104.

13. The nucleic acid molecule according to any one of claims 1-12, wherein one or more nucleotides of the antisense strand are chemically modified; and / or, wherein one or more nucleotides of the sense strand are chemically modified; Optionally, all nucleotides of the antisense strand and / or the sense strand are chemically modified.

14. The nucleic acid molecule according to claim 13, wherein the chemical modification is selected from any one or more of the following: Locked nucleic acid modification, ring-opening or non-locked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification, 2'-O-allyl modification, 2'-C-alkyl modification, 2'-C-allyl modification, 2'-fluorinated modification, 2'-deoxy modification, 2'-hydroxyl modification, thiophosphate bond modification, 2'-amino-modification, aminophosphate modification, methylphosphate modification, 5'-vinylphosphate modification, DNA modification, fluorescent probe modification.

15. The nucleic acid molecule according to claim 14, wherein the positive strand contains a 2'-O-methyl modification, a 2'-fluoro modification, and / or a thiophosphate bond; And / or, wherein the antisense chain contains a 2'-O-methyl modification, a 2'-fluoro modification, and / or a thiophosphate bond.

16. The nucleic acid molecule according to claim 13, wherein the chemical modification is selected from one, two, three, or four of the following: (1) The antisense strand has at least the 2nd, 14th and 16th nucleotides starting from the 5' end that are 2'-fluorinated nucleotides; (2) The nucleotides at least the 7th, 9th, 10th and 11th positions of the positive strand starting from the 5' end are 2'-fluorinated nucleotides; (3) At least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the other nucleotides in the sense strand and / or antisense strand that are not 2'-fluorinated are modified with 2'-O-methyl; optionally, all the other nucleotides in the sense strand and / or antisense strand that are not 2'-fluorinated are modified with 2'-O-methyl. (4) The first and second phosphate bonds of the antisense chain starting from its 5' end and the first and second phosphate bonds starting from its 3' end form thiophosphate bonds through thiolation; the first and second phosphate bonds of the sense chain starting from its 5' end form thiophosphate bonds through thiolation.

17. The nucleic acid molecule according to claim 16, wherein the 7th, 9th-11th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorinated modification and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorinated modification; Optionally, the nucleotides at positions 7 and 9-11 of the sense strand of the nucleic acid molecule starting from the 5' end contain a 2′-fluorinated modification, and / or the nucleotides at positions 2, 14, and 16 of the antisense strand starting from the 5' end contain a 2′-fluorinated modification, and the remaining nucleotides not modified with 2′-fluorinated modification contain a 2′-O-methyl modification; Further optionally, the nucleotides at positions 7 and 9-11 of the sense strand of the nucleic acid molecule, starting from the 5' end, are modified with 2′-fluorination, and / or the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are modified with 2′-fluorination, and the remaining nucleotides not modified with 2′-fluorination are modified with 2′-O-methyl, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

18. The nucleic acid molecule according to claim 16, wherein the 7th, 9th-12th nucleotides of the sense strand starting from the 5' end contain a 2′-fluorinated modification and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorinated modification; Optionally, the 7th and 9th-12th nucleotides of the sense strand of the nucleic acid molecule starting from the 5' end contain a 2′-fluorinated modification, and / or the 2nd, 14th, and 16th nucleotides of the antisense strand starting from the 5' end contain a 2′-fluorinated modification, and the remaining nucleotides not modified with 2′-fluorinated modification contain a 2′-O-methyl modification; Further optionally, the nucleotides at positions 7 and 9-12 of the sense strand of the nucleic acid molecule, starting from the 5' end, are modified with 2′-fluorination, and / or the nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are modified with 2′-fluorination, and the remaining nucleotides not modified with 2′-fluorination are modified with 2′-O-methyl, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

19. The nucleic acid molecule according to any one of claims 16-18, wherein the nucleic acid molecule comprises a motif selected from (1) or (2): (1) Sensitive strand: The nucleotides at positions 7 and 9-11 of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification; Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain a 2′-O-methyl modification; Optionally, the positive strand: the 7th and 9th-11th nucleotides of the positive strand starting from the 5' end are 2′-fluorinated nucleotides, the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates; Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluoro-modified nucleotides, and the remaining nucleotides that are not 2′-fluoro-modified are 2′-O-methyl-modified nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates. (2) The positive strand: The nucleotides at positions 7 and 9-12 of the positive strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain a 2′-O-methyl modification, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate; Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides that are not 2′-fluorinated contain a 2′-O-methyl modification; Optionally, the positive strand: the 7th and 9th-12th nucleotides of the positive strand starting from the 5' end are 2′-fluorinated nucleotides, the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphates; Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, and the remaining nucleotides that are not 2′-fluorinated are 2′-O-methyl nucleotides, and the nucleic acid molecule does not further contain any other modifications other than thiophosphate.

20. The nucleic acid molecule according to any one of claims 16-19, wherein the nucleic acid molecule is further linked by phosphate thioester bonds between the first and second nucleotides at the 5' end of the sense strand, and between the second and third nucleotides; and by phosphate thioester bonds between the first and second nucleotides at the 5' end of the antisense strand, between the second and third nucleotides, between the penultimate first and second nucleotides, and between the first and second nucleotides at the 3' end of the antisense strand, and between the second and third nucleotides.

21. The nucleic acid molecule according to claim 20, wherein the nucleic acid molecule comprises a motif selected from (1) or (2): (1) Sensitive strand: The 7th and 9th-11th nucleotides of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification; the first and second nucleotides at the 5' end of the positive strand are connected by phosphate thioester bonds, as are the second and third nucleotides. Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain a 2′-O-methyl modification. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by a thiophosphate bond, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides. Optionally, the sense strand: the 7th and 9th-11th nucleotides starting from the 5' end of the sense strand are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides; the first and second nucleotides at the 5' end of the sense strand are linked by phosphate thioester bonds, as are the second and third nucleotides. Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by thiophosphate bonds, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides. When the length of the sense strand nucleotide is 21 nt, the optional modification scheme in the sense strand is motif 1 modification; when the length of the antisense strand nucleotide is 23 nt, the optional modification scheme in the antisense strand is motif 2 modification. Motif 1 modification of the sense strand + motif 2 modification of the antisense strand constitutes a nucleic acid molecule modification scheme. (2) Sensitive strand: The 7th and 9th-12th nucleotides of the positive strand starting from the 5' end contain 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain 2′-O-methyl modification; the first and second nucleotides at the 5' end of the positive strand are connected by phosphate thioester bonds, as are the second and third nucleotides. Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, contain a 2′-fluorinated modification, and the remaining nucleotides without 2′-fluorinated modification contain a 2′-O-methyl modification. The first and second nucleotides at the 5' end of the antisense strand are linked by a thiophosphate bond, as are the first and second nucleotides at the 3' end of the antisense strand, as are the second and third nucleotides; Optionally, the sense strand: the 7th and 9th-12th nucleotides starting from the 5' end of the sense strand are 2′-fluorinated nucleotides, and the remaining nucleotides without 2′-fluorination are 2′-O-methyl nucleotides; the first and second nucleotides at the 5' end of the sense strand are linked by phosphate thioester bonds, as are the second and third nucleotides. Antisense strand: The nucleotides at positions 2, 14, and 16 of the antisense strand, starting from the 5' end, are 2′-fluorinated nucleotides, while the remaining nucleotides that are not 2′-fluorinated are 2′-O-methyl nucleotides. The first and second nucleotides at the 5' end of the antisense strand, as well as the second and third nucleotides, are linked by thiophosphate bonds, as are the first and second nucleotides at the 3' end of the antisense strand, as well as the second and third nucleotides. When the length of the sense strand is 21 nt, the optional modification scheme in the sense strand is motif 3 modification; when the length of the antisense strand is 23 nt, the optional modification scheme in the antisense strand is motif 2 modification. Motif 3 modification of the sense strand + motif 2 modification of the antisense strand constitutes a nucleic acid molecule modification method.

22. The nucleic acid molecule according to claim 21, wherein: The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 10; the polynucleotide sequence shown in SEQ ID NO: 9 is SEQ ID NO: 103 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 10 is SEQ ID NO: 104 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 407, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 10; the polynucleotide sequence shown in SEQ ID NO: 407 is SEQ ID NO: 103 with motif 3, and the polynucleotide sequence shown in SEQ ID NO: 10 is SEQ ID NO: 104 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 1, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 2; the polynucleotide sequence shown in SEQ ID NO: 1 is SEQ ID NO: 19 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 2 is SEQ ID NO: 20 with motif 2; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 3, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 4; the polynucleotide sequence shown in SEQ ID NO: 3 is SEQ ID NO: 43 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 4 is SEQ ID NO: 24 with motif 2; The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 6; the polynucleotide sequence shown in SEQ ID NO: 5 is SEQ ID NO: 65 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 6 is SEQ ID NO: 66 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 7, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 8; the polynucleotide sequence shown in SEQ ID NO: 7 is SEQ ID NO: 67 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 8 is SEQ ID NO: 68 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 11, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 12; the polynucleotide sequence shown in SEQ ID NO: 11 is SEQ ID NO: 165 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 12 is SEQ ID NO: 166 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 15, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 16; the polynucleotide sequence shown in SEQ ID NO: 15 is SEQ ID NO: 169 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 16 is SEQ ID NO: 170 with motif 2. The sense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 17, and the antisense strand contains or is a polynucleotide sequence as shown in SEQ ID NO: 18; the polynucleotide sequence shown in SEQ ID NO: 17 is SEQ ID NO: 177 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 18 is SEQ ID NO: 178 with motif 2. Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 19, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 20; the polynucleotide sequence shown in SEQ ID NO: 19 is SEQ ID NO: 179 with motif 1, and the polynucleotide sequence shown in SEQ ID NO: 20 is SEQ ID NO: 180 with motif 2; Further optionally, the sense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 9, and the antisense strand comprises or is a polynucleotide sequence as shown in SEQ ID NO: 10; Alternatively, the sense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO: 407, and the antisense strand may contain or be a polynucleotide sequence as shown in SEQ ID NO:

10.

23. The nucleic acid molecule according to any one of claims 1-22, wherein the nucleic acid molecule is linked to an organ-targeting ligand; optionally, the ligand is a liver-targeting ligand.

24. The nucleic acid molecule according to any one of claims 1-23, wherein the nucleic acid molecule is linked to at least one desialylate glycoprotein receptor (ASGPR) ligand.

25. The nucleic acid molecule according to any one of claims 1-24, wherein the organ-targeting ligand is attached to the 5' end or 3' end of the sense sequence.

26. The nucleic acid molecule according to claim 24 or 25, wherein the ASGPR ligand is one or more GalNAc derivatives linked by a divalent or trivalent branched structure.

27. The nucleic acid molecule according to claim 26, wherein the GalNAc derivative comprises the following structure: in, When one of X or Y is an oligonucleotide, the other is hydrogen.

28. The nucleic acid molecule of claim 27, wherein the GalNac ligand is selected from one of the following structures: Where X is O or S. Where in equations I and II The terminator represents the connection to the 3' end of the positive sequence of the nucleic acid molecule; preferably, the connection is made via a phosphate ester bond or a thiophosphate ester bond.

29. The nucleic acid molecule according to claim 28, wherein the nucleic acid molecule has any one of the structures of (1)-(8): (1) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to L96 via a phosphate ester bond; Antisense chains: Modifications such as antisense chains of motif 2; (2) Sensitive chain: The 3' end of the positive chain modified such as motif 3 is connected to L96 via a phosphate ester bond; Antisense chains: Modifications such as antisense chains of motif 2; (3) Sensitive chain: The 3' end of the positive chain modified such as motif 1 is connected to ligand 1 via a thiophosphate bond; Antisense chains: Modifications such as antisense chains of motif 2; (4) Sensitive chain: The 3' end of the positive chain modified such as motif 3 is connected to ligand 1 via a thiophosphate bond; Antisense chains: Modifications such as antisense chains of motif 2; (5) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to ligand 2 via a thiophosphate bond; Antisense chains: Modifications such as antisense chains of motif 2; (6) Sensitive chain: The 3' end of the positive chain modified such as motif 3 is connected to ligand 2 via a thiophosphate bond; Antisense chains: Modifications such as antisense chains of motif 2; (7) Sensitive chain: The 3' end of the positive chain modified as motif 1 is connected to ligand 3 via a thiophosphate bond; Antisense chains: Modifications such as antisense chains of motif 2; Or, (8) the positive chain: the 3' end of the positive chain modified such as motif 3 is connected to ligand 3 via a thiophosphate bond; Antisense chain: Modifies antisense chains such as motif 2.

30. The nucleic acid molecule according to claim 29, wherein the nucleic acid molecule is selected from the following nucleic acid molecules: in: Am, Um, Cm, and Gm represent ribonucleotides A, U, C, and G modified with 2′-O-methyl, respectively; Af, Uf, Cf, and Gf represent ribonucleotides A, U, C, and G modified with 2′-fluoro, respectively. The 's' between adjacent nucleotides indicates that the adjacent nucleotides are linked by a phosphate thioester bond, and the 's' at the 3' end of the positive strand indicates that the 3' end of the positive strand is linked to a ligand via a phosphate thioester bond.

31. A second nucleic acid molecule, which can be transcribed in a cell into a dsRNA or shRNA precursor, wherein the dsRNA or shRNA is a nucleic acid molecule according to any one of claims 1-30.

32. A nucleic acid delivery system comprising a nucleic acid molecule as described in any one of claims 1-30 or a second nucleic acid molecule as described in claim 31.

33. A viral particle comprising the second nucleic acid molecule of claim 31.

34. A cell comprising the second nucleic acid molecule of claim 31.

35. A pharmaceutical composition comprising a nucleic acid molecule or a pharmaceutically acceptable salt thereof as described in any one of claims 1-30, a second nucleic acid molecule as described in claim 31, a nucleic acid delivery body as described in claim 32, a viral particle as described in claim 33, a cell as described in claim 34, and a pharmaceutically acceptable excipient.

36. The use of the nucleic acid molecule of any one of claims 1-30, the second nucleic acid molecule of claim 31, the nucleic acid delivery body of claim 32, the viral particle of claim 33, the cell of claim 34, and the pharmaceutical composition of claim 35 in the preparation of a medicament for inhibiting the expression of the ANGPTL3 gene in a subject.

37. The use according to claim 36, wherein the drug for inhibiting ANGPTL3 gene expression is a drug for reducing triglyceride levels and / or reducing LDL-C levels.

38. The use according to claim 37, the drug for lowering triglyceride levels and / or lowering LDL-C levels is a drug for the prevention or treatment of dyslipidemia and / or cardiovascular disease.

39. The use according to claim 36, wherein the drug for inhibiting ANGPTL3 gene expression is a drug for the prevention or treatment of hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

40. A method for inhibiting ANGPTL3 gene expression in a subject, comprising the following steps: The administration of an effective dose of any one of claims 1-30, the second nucleic acid molecule of claim 31, the nucleic acid delivery body of claim 32, the viral particle of claim 33, the cell of claim 34, and the pharmaceutical composition of claim 35 to a subject who requires inhibition of ANGPTL3 gene expression.

41. The method of claim 40, wherein the subject with a need to inhibit ANGPTL3 gene expression is a subject with a need to reduce triglyceride levels and / or reduce LDL-C levels.

42. The method according to claim 41, wherein the subject with a need to lower triglyceride levels and / or lower LDL-C levels is a subject with a need to prevent or treat dyslipidemia and / or cardiovascular disease.

43. The method according to claim 40, wherein the subject requiring inhibition of ANGPTL3 gene expression is a subject requiring prevention or treatment of hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia.

44. The nucleic acid molecule according to any one of claims 1-30, the second nucleic acid molecule according to claim 31, the nucleic acid delivery body according to claim 32, the virus particle according to claim 33, the cell according to claim 34, and the pharmaceutical composition according to claim 35, for inhibiting ANGPTL3 gene expression; Optionally, inhibiting ANGPTL3 gene expression in the subject can reduce triglyceride levels and / or LDL-C levels in the subject; further optionally, reducing triglyceride levels and / or LDL-C levels in the subject can prevent or treat dyslipidemia and / or cardiovascular disease. Optionally, inhibiting ANGPTL3 expression in subjects can be used to prevent or treat hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, pancreatitis, homozygous and heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

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