Sirna inhibiting angtpl3, and modification and use thereof

Abstract

The present application provides a double-stranded RNA molecule targeting the ANGPTL3 gene and a modification thereof. Both in vivo and in vitro experiments show that the double-stranded RNA molecule and the modification thereof provided in the present application can effectively inhibit ANGPTL3 gene expression. This indicates that the double-stranded RNA molecule and the modification thereof provided in the present application have great drug development potential and application value in ANGPTL3 metabolism-related diseases in which ANGPTL3 gene expression is abnormal, such as hypertriglyceridemia, obesity, hyperlipidemia, and lipid and / or cholesterol metabolism disorders.

Description

siRNAs that inhibit ANGTPL3 and their modifications and applications Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to siRNAs that inhibit ANGTPL3 and their modifications and applications. Background Technology

[0002] Angiopoietin-like 3 (also known as ANGPTL3, ANGPL3, ANG3, or angiopoietin-like protein 3) is an angiopoietin protein encoded by the human angiopoietin-like 3 gene, which has been reported to be involved in regulating lipid metabolism. ANGPTL3 is a 460-amino acid polypeptide composed of a signal peptide, an N-terminal coiled-coil domain, and a C-terminal fibrinogen (FBN)-like domain. ANGPTL3 is known to be primarily produced in human hepatocytes and secreted into circulation after synthesis. ANGPTL3 acts as an inhibitor of lipoprotein lipase (which catalyzes the hydrolysis of triglycerides) and endothelial lipase (which hydrolyzes lipoprotein phospholipids). Inhibition of these enzymes leads to elevated plasma levels of triglycerides, high-density lipoprotein (HDL), and phospholipids. Furthermore, loss-of-function mutations in ANGPTL3 result in familial hypolipoproteinemia, characterized by low plasma levels of triglycerides and low-density lipoprotein (LDL-C).

[0003] In humans, loss of ANGPTL3 function is also associated with a reduced risk of atherosclerotic cardiovascular disease. Effective therapies targeting ANGPTL3 can have beneficial effects on the treatment (including preventative treatment) of cardiometabolic diseases such as hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases. Summary of the Invention

[0004] The technical problem this application aims to solve is how to inhibit the expression of the ANGPTL3 gene in cells in order to obtain drugs for preventing or treating abnormal ANGPTL3 gene expression or diseases caused by abnormal ANGPTL3 gene expression.

[0005] To address the aforementioned technical problems, this application provides a double-stranded RNA molecule, which may be any one of 32 types of siRNA. The siRNA includes a sense strand and an antisense strand that at least partially form a double-stranded region. The sense strand includes a nucleotide sequence whose nucleotide sequence is any odd-numbered sequence from Sequence 1 to 64, and / or the antisense strand includes a nucleotide sequence whose nucleotide sequence is any even-numbered sequence from Sequence 1 to 64.

[0006] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 15-30 bp.

[0007] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 23-27 bp.

[0008] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 21-23 bp.

[0009] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 19-21 bp.

[0010] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 17-25 bp.

[0011] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 17-23 bp.

[0012] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can be 17-19 bp.

[0013] Furthermore, the length of the double-stranded region of the double-stranded RNA molecule can specifically be 19 bp.

[0014] Furthermore, the length of the sense strand of the double-stranded RNA molecule does not exceed 30 nucleotides, and / or the length of the antisense strand does not exceed 30 nucleotides.

[0015] Furthermore, the length of the sense strand of the double-stranded RNA molecule does not exceed 21 nucleotides, and / or the length of the antisense strand does not exceed 23 nucleotides.

[0016] Furthermore, the length of the sense strand of the double-stranded RNA molecule is 19 nucleotides, and / or the length of the antisense strand is 21 nucleotides.

[0017] Furthermore, at least one of the sense and antisense strands of the double-stranded RNA molecule contains a 3' overhang with at least one nucleotide, or at least one of the strands contains a 3' overhang with at least two nucleotides.

[0018] Furthermore, the antisense strand of the double-stranded RNA molecule comprises a nucleotide sequence whose nucleotide sequence is any even numbered sequence from 1 to 64.

[0019] Furthermore, in the double-stranded RNA molecule, the nucleotide sequence of the sense strand of the 32 siRNAs can be any odd-numbered sequence from Sequence 1 to 64, the sequence of position 1 to 19 of any odd-numbered sequence from Sequence 1 to 64, or a sequence with more than 90% identity to any odd-numbered sequence; and / or, the nucleotide sequence of the antisense strand can be any even-numbered sequence from Sequence 1 to 64, or a sequence with more than 90% identity to any even-numbered sequence.

[0020] The nucleotide sequence number of the sense strand of the 32 siRNAs can be n, and the nucleotide sequence number of the antisense strand of the 32 siRNAs can be n+1, where n can be any odd number from 1 to 64.

[0021] The nucleotide sequences of the positive strand of the 32 siRNAs also include sequences that have more than 90% identity with any of the sequences shown in the odd-numbered sequences 1-64;

[0022] The nucleotide sequences of the antisense strands of the 32 siRNAs also include sequences that have more than 90% identity with any of the even-numbered sequences shown in sequences 1-64.

[0023] This application also provides double-stranded RNA molecule modifiers, which can be compounds containing modified nucleotides obtained by modifying at least one nucleotide of the double-stranded RNA molecule.

[0024] The double-stranded RNA molecule modifier may be a compound containing a modified nucleotide obtained by modifying at least one nucleotide of the above-mentioned double-stranded RNA molecule, wherein the positive strand of the double-stranded RNA molecule may be a nucleotide sequence that is any odd-numbered sequence from position 1 to 19 of sequence 1-64.

[0025] Furthermore, at least one nucleotide in the sense or antisense strand of the double-stranded RNA molecule modification may be a modified nucleotide.

[0026] Furthermore, in the double-stranded RNA molecule modification, the modified nucleotide may be a compound formed by replacing the 2' hydroxyl group of the ribosyl group of the nucleotide with other groups, or it may be a compound formed by modifying the bases on the nucleotide.

[0027] Further, in the double-stranded RNA molecule modification, the modified nucleotide is selected from at least one of the following: 2'-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing thiophosphate groups, deoxynucleotides, 2'-deoxynucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate groups, and nucleotides containing 5'-phosphate mimics.

[0028] Furthermore, in the aforementioned double-stranded RNA molecule modification, the modified nucleotides can be a1)-a3).

[0029] a1) 2'-methoxy modified nucleotides;

[0030] a2) 2'-fluorinated modified nucleotides;

[0031] a3)2'-deoxy-modified nucleotides.

[0032] Furthermore, in the aforementioned double-stranded RNA molecule modification, both the sense and antisense strands of the double-stranded RNA molecule modification contain the modified nucleotides described in a1) and a2); or,

[0033] The sense strand of the double-stranded RNA molecule modifier contains the modified nucleotides described in a1) and a2), and the antisense strand of the double-stranded RNA molecule modifier contains the modified nucleotides described in a1), a2) and a3).

[0034] Furthermore, the double-stranded RNA molecule modifier comprises at least one backbone modified with a thiophosphate group (i.e., the double-stranded RNA molecule modifier further comprises linking the modified or unmodified nucleotides via a thiophosphate group).

[0035] Furthermore, the double-stranded RNA molecule modification includes M1) or M38):

[0036] The double-stranded RNA molecule modification described in M1) includes a 2'-methoxy-modified nucleotide, wherein the 2'-methoxy-modified nucleotide is located in the antisense and sense strands of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1-6 and 10-19 of the sense strand may be 2'-methoxy-modified nucleotides; and at least the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand may be 2'-methoxy-modified nucleotides.

[0037] Furthermore, the double-stranded RNA molecule modifier further includes nucleotides modified with 2'-fluorination, wherein the nucleotides modified with 2'-fluorination are located in the antisense strand and the sense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 8, and 9 of the sense strand may be nucleotides modified with 2'-fluorination, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand may be nucleotides modified with 2'-fluorination.

[0038] Further, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by phosphate thioester groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by phosphate thioester groups; or,

[0039] The double-stranded RNA molecule modification described in M38 includes a 2'-methoxy-modified nucleotide, wherein the 2'-methoxy-modified nucleotide is located in the antisense and sense strands of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1-6 and 10-19 of the sense strand may be 2'-methoxy-modified nucleotides; and at least the nucleotides at positions 1, 3-4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand may be 2'-methoxy-modified nucleotides.

[0040] Furthermore, the double-stranded RNA molecule modifier also includes 2'-fluorinated nucleotides, which are located in the antisense and sense strands of the double-stranded RNA molecule modifier. In addition, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 8, and 9 of the sense strand can be 2'-fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand can be 2'-fluorinated nucleotides.

[0041] Furthermore, the double-stranded RNA molecule modifier includes a 2'-deoxy modified nucleotide, the 2'-deoxy modified nucleotide being located in the antisense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand may be 2'-deoxy modified nucleotides;

[0042] Furthermore, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

[0043] Furthermore, the double-stranded RNA molecule modification includes M1 or M38.

[0044] The double-stranded RNA molecule modification described in M1) includes nucleotides modified with 2'-methoxy groups, wherein the 2'-methoxy-modified nucleotides are located in the antisense and sense strands of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand may be nucleotides modified with 2'-methoxy groups; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand may be nucleotides modified with 2'-methoxy groups.

[0045] Furthermore, the double-stranded RNA molecule modifier also includes nucleotides modified with 2'-fluorination, wherein the nucleotides modified with 2'-fluorination are located in the antisense strand and the sense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand may be nucleotides modified with 2'-fluorination, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand may be nucleotides modified with 2'-fluorination.

[0046] Further, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by phosphate thioester groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by phosphate thioester groups; or,

[0047] The double-stranded RNA molecule modification described in M38 includes nucleotides modified with 2'-methoxy groups, wherein the 2'-methoxy-modified nucleotides are located in the antisense and sense strands of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand may be nucleotides modified with 2'-methoxy groups; the nucleotides at positions 1, 3-4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand may be nucleotides modified with 2'-methoxy groups.

[0048] Furthermore, the double-stranded RNA molecule modifier also includes 2'-fluorinated nucleotides, which are located in the antisense and sense strands of the double-stranded RNA molecule modifier. In addition, the nucleotides at positions 7, 8, and 9 of the sense strand can be 2'-fluorinated nucleotides, and the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand can be 2'-fluorinated nucleotides.

[0049] Furthermore, the double-stranded RNA molecule modifier includes a 2'-deoxy modified nucleotide, the 2'-deoxy modified nucleotide being located in the antisense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 5 and 7 of the antisense strand may be 2'-deoxy modified nucleotides;

[0050] Furthermore, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

[0051] Furthermore, the modified nucleotide at the 5' end of the antisense strand of the double-stranded RNA molecule modification may be a compound obtained by further modifying the 5' end of the antisense strand of the double-stranded RNA molecule modification with 5'-(E)-vinyl phosphate.

[0052] Furthermore, in the double-stranded RNA molecule modification, a ligand is attached to any nucleotide at the 3'-end or 5'-end of the positive strand or in the middle of the positive strand.

[0053] Furthermore, in the double-stranded RNA molecule modification, the ligand may be a small molecule, antibody, polypeptide, protein, or aptamer.

[0054] Furthermore, in the double-stranded RNA molecule modification, the small molecule may be GalNAc.

[0055] Furthermore, in the aforementioned double-stranded RNA molecule modification, the protein may be albumin.

[0056] Furthermore, in the aforementioned double-stranded RNA molecule modification, the ligand is linked to the double-stranded RNA molecule modification via a linker.

[0057] This application also provides the use of the described double-stranded RNA molecule or the modified double-stranded RNA molecule in any of the following:

[0058] D1) Application in the preparation of compositions that inhibit ANGPTL3 gene expression;

[0059] Application of D2 in suppressing ANGPTL3 gene expression;

[0060] Application of D3 in the treatment of diseases related to the ANGPTL3 gene target;

[0061] Application of D4 in the preparation of compositions for treating diseases related to the ANGPTL3 gene target.

[0062] This application also provides a composition for inhibiting ANGPTL3 gene expression, wherein the active ingredient of the composition may be the double-stranded RNA molecule or a modified double-stranded RNA molecule.

[0063] Furthermore, the composition also includes a pharmaceutically acceptable carrier.

[0064] The composition may be a pharmaceutical composition or a kit.

[0065] The pharmaceutical composition described above also comprises an unbuffered solution.

[0066] The unbuffered solution in the pharmaceutical composition described above may be physiological saline or water.

[0067] The pharmaceutical composition described above also includes a buffer solution.

[0068] The buffer solution in the pharmaceutical composition described above comprises acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof.

[0069] The buffer solution in the pharmaceutical composition described above may be phosphate-buffered saline (PBS).

[0070] Furthermore, the composition comprises lipid formulations, nanoformulations, or lipid-containing vesicles.

[0071] Furthermore, in the composition, the lipid formulation is an LNP formulation, the nano-formulation is polymer nanoparticles, and the lipid-containing vesicles are exosomes, preferably artificially modified exosomes.

[0072] This application also provides a cell comprising the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition described herein.

[0073] This application also provides a method for treating ANGPTL3 gene target-related diseases, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition to a subject suffering from ANGPTL3 gene target-related diseases.

[0074] This application also provides a method for treating hyperlipidemia, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modifier, and / or the composition to a subject suffering from hyperlipidemia.

[0075] This application also provides a method for treating obesity, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition to a subject suffering from obesity.

[0076] This application also provides a method for inhibiting the expression of the ANGPTL3 gene in cells, the method comprising:

[0077] (a) Contact the cells with the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition.

[0078] (b) Maintain the cells produced in step (a) for a period of time sufficient for the degradation of the ANGPTL3 gene mRNA transcript, thereby suppressing ANGPTL3 gene expression in the cells.

[0079] Furthermore, in the method, the cell is located within the subject.

[0080] Furthermore, in the method, the subject may be a human.

[0081] Furthermore, in the method described, the subject suffers from ANGPTL3-related disease.

[0082] Furthermore, in the method, the expression of the ANGPTL3 gene is suppressed by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

[0083] The term "subject" as used in this document may refer to an animal, such as a mammal, including primates (e.g., humans or non-human primates, such as monkeys or chimpanzees), and non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, or whales). In some embodiments, the subject may be a mouse.

[0084] In this application, the ANGPTL3 gene target-related diseases can be diseases caused by abnormal expression of the ANGPTL3 gene.

[0085] In this application, the disease caused by abnormal ANGPTL3 gene expression can be a disease caused by upregulation of ANGPTL3 gene expression.

[0086] In this application, the diseases caused by upregulation of the ANGPTL3 gene include, but are not limited to, hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases.

[0087] In this application, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with another group, or a nucleotide in which the bases on the nucleotide are modified. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids, but whose structure differs from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0088] In one embodiment of this application, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0089] In one embodiment of this application, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art. These nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0090] In one embodiment of this application, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), the 2'-amino (2'-NH2) modified nucleotide is shown in formula (4), and the 2'-deoxynucleotide (DNA) is shown in formula (5), where Base represents the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases.

[0091] In one embodiment of this application, the nucleotide analogue refers to a group that can replace a nucleotide in nucleic acid, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0092] In one embodiment of this application, the nucleotide analog may be an isonucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0093] In one embodiment of this application, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered ring, a six-membered ring, or a seven-membered ring. Typically, the bridge is incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0094] In one embodiment of this application, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8), where Base represents modified or unmodified nucleotide bases A, U, G, C, T, or other nucleotide bases.

[0095] In one embodiment of this application, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups.

[0096] In one embodiment of this application, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0097] In one embodiment of this application, the phosphate group having the modifying group is a thiophosphate group having the structure shown in formula (9). In one embodiment of this application, the nucleotide linked to the thiophosphate group is shown in formula (10), and the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0098] In one embodiment of this application, the modified nucleotide is a vinyl phosphate modified (VP-modified) nucleotide. In one embodiment of this application, the VP-modified and methoxy-modified nucleotide, i.e., the nucleotide modified with 5'-(E)-vinyl-2'-methoxy-modified phosphonate group (5'-(E)-VP-2'-OMe), is shown in formula (11); in one embodiment of this application, the VP-modified, methoxy-modified, and thiophosphate-modified nucleotide, i.e., the 5'-PS-modified nucleotide (i.e., the 5'-(E)-vinyl-2'-methoxy-modified thiophosphate-modified nucleotide), is shown in formula (12), where Base represents the modified or unmodified nucleotide base A, U, G, C, T, or other nucleotide bases.

[0099] In the siRNA preparation methods referred to in this application, unless otherwise specified, nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes RNA phosphoramidites are also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in this application are commercially available.

[0100] In this application, the positive strand of the double-stranded RNA molecule is conjugated to a ligand attached to the 3'-end, wherein the ligand is GalNAc.

[0101] The siRNA conjugate formed by GalNAc and double-stranded RNA molecules in this application has the following structure:

[0102] It is worth noting that the raw materials used in this application are all ordinary commercially available products, and their sources are not specifically limited.

[0103] This application also provides the following technical solutions:

[0104] 1. A double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand that at least partially form a double-stranded region, the antisense strand comprising a nucleotide sequence numbered in any even number in SEQ ID NO. 1-64.

[0105] 2. The double-stranded RNA molecule according to claim 1, wherein the positive strand comprises any odd-numbered nucleotide sequence from SEQ ID NO. 1-64.

[0106] 3. The double-stranded RNA molecule according to item 1 or 2, wherein the length of the double-stranded region is 15-30 bp.

[0107] 4. The double-stranded RNA molecule according to any one of claims 1-3, wherein the length of the sense strand is no more than 30 nucleotides, and / or the length of the antisense strand is no more than 30 nucleotides.

[0108] 5. The double-stranded RNA molecule according to any one of items 1-4, wherein the length of the sense strand is no more than 21 nucleotides, and / or the length of the antisense strand is no more than 21 nucleotides.

[0109] 6. A double-stranded RNA molecule according to any one of claims 1-5, wherein at least one of the sense strand and the antisense strand comprises a 3' overhang having at least one or at least two nucleotides.

[0110] 7. A double-stranded RNA molecule according to any one of claims 1-6, wherein the sense strand and antisense strand of said double-stranded RNA molecule comprise or are selected from any combination of:

[0111] The following chains are listed: the right chain shown in SEQ ID NO.41 and the wrong chain shown in SEQ ID NO.42; the right chain shown in SEQ ID NO.43 and the wrong chain shown in SEQ ID NO.44; the right chain shown in SEQ ID NO.59 and the wrong chain shown in SEQ ID NO.60; the right chain shown in SEQ ID NO.1 and the wrong chain shown in SEQ ID NO.2; the right chain shown in SEQ ID NO.3 and the wrong chain shown in SEQ ID NO.4; the right chain shown in SEQ ID NO.5 and the wrong chain shown in SEQ ID NO.6; the right chain shown in SEQ ID NO.7 and the wrong chain shown in SEQ ID NO.8; the right chain shown in SEQ ID NO.9 and the wrong chain shown in SEQ ID NO.10; the right chain shown in SEQ ID NO.11 and the wrong chain shown in SEQ ID NO.12; the right chain shown in SEQ ID NO.13 and the wrong chain shown in SEQ ID NO.14; the right chain shown in SEQ ID NO.15 and the wrong chain shown in SEQ ID NO.16; the right chain shown in SEQ ID NO.17 and the wrong chain shown in SEQ ID NO.18; SEQ ID NO. The following chains are listed: the justice chain shown in SEQ ID NO. 19 and the antisense chain shown in SEQ ID NO. 20; the justice chain shown in SEQ ID NO. 21 and the antisense chain shown in SEQ ID NO. 22; the justice chain shown in SEQ ID NO. 23 and the antisense chain shown in SEQ ID NO. 24; the justice chain shown in SEQ ID NO. 25 and the antisense chain shown in SEQ ID NO. 26; the justice chain shown in SEQ ID NO. 27 and the antisense chain shown in SEQ ID NO. 28; the justice chain shown in SEQ ID NO. 29 and the antisense chain shown in SEQ ID NO. 30; the justice chain shown in SEQ ID NO. 31 and the antisense chain shown in SEQ ID NO. 32; the justice chain shown in SEQ ID NO. 33 and the antisense chain shown in SEQ ID NO. 34; the justice chain shown in SEQ ID NO. 35 and the antisense chain shown in SEQ ID NO. 36; the justice chain shown in SEQ ID NO. 37 and the antisense chain shown in SEQ ID NO. 38; the justice chain shown in SEQ ID NO. 39 and the antisense chain shown in SEQ ID NO. 40; and the justice chain shown in SEQ ID NO. 45 and the antisense chain shown in SEQ ID NO. 46. The antisense chain shown in NO.46, the right-sense chain shown in SEQ ID NO.47 and the antisense chain shown in SEQ ID NO.48, the right-sense chain shown in SEQ ID NO.49 and the antisense chain shown in SEQ ID NO.50, and SEQ ID NO.The same chain of justice shown in SEQ ID NO. 51 and the same chain of justice shown in SEQ ID NO. 52; the same chain of justice shown in SEQ ID NO. 53 and the same chain of justice shown in SEQ ID NO. 54; the same chain of justice shown in SEQ ID NO. 55 and the same chain of justice shown in SEQ ID NO. 56; the same chain of justice shown in SEQ ID NO. 57 and the same chain of justice shown in SEQ ID NO. 58; the same chain of justice shown in SEQ ID NO. 61 and the same chain of justice shown in SEQ ID NO. 62; the same chain of justice shown in SEQ ID NO. 63 and the same chain of justice shown in SEQ ID NO. 64.

[0112] 8. A modified double-stranded RNA molecule comprising any one of claims 1-7, wherein at least one nucleotide is chemically modified.

[0113] 9. The modified double-stranded RNA molecule according to claim 8, wherein the chemical modification includes the substitution of the 2' hydroxyl group of the ribosyl group of the nucleotide with another group, the substitution of the 5' hydroxyl group of the ribosyl group of the nucleotide with another group, the modification of the bases on the nucleotide, and / or the modification of the thiophosphate group of the phosphodiester between the nucleotides.

[0114] 10. A modified double-stranded RNA molecule according to claim 8 or 9, wherein the chemically modified nucleotides are selected from at least one of the following:

[0115] 2'-Methoxy modified nucleotides, 2'-Fluoro modified nucleotides, vinylphosphonate modified nucleotides, nucleotides containing thiophosphate groups, 2'-Methyl modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2 '-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics.

[0116] 11. A modified double-stranded RNA molecule according to any one of claims 8-10, wherein the modified double-stranded RNA molecule has any of the following modification patterns:

[0117] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by a thiophosphate group; the nucleotide at position 1 of the antisense strand is a nucleotide modified by 2'-methoxy and 5'-(E)-vinyl phosphate, the nucleotides at positions 3-5, 7-13, 15, and 17-21 are nucleotides modified by 2'-methoxy, the nucleotides at positions 2, 6, 14, and 16 are nucleotides modified by 2'-fluorinated, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by a thiophosphate group;

[0118] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate groups; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0119] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, and 10-19 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by a thiophosphate group. The nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 8-9, 14, and 16 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by a thiophosphate group.

[0120] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by a thiophosphate group; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by a thiophosphate group.

[0121] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-2, 4, 6, 10, 12, 14, 16, and 18 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 3, 5, 7-9, 11, 13, 15, 17, and 19 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by a thiophosphate group. In the antisense strand, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, and 19-21 are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, and 18 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by a thiophosphate group.

[0122] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, and 10-19 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by a thiophosphate group. In the antisense strand, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 8-9, 10, 14, and 16 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by a thiophosphate group.

[0123] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate groups; the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0124] (8) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate groups; the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0125] (9) In the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate groups; the nucleotides at positions 1, 3-4, 6, 8-9, 11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 are 2'-fluorinated modified nucleotides, the nucleotides at positions 5 and 7 are deoxyribonucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0126] (10) Following the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3, are linked by thiophosphate groups. The nucleotide at position 1 of the antisense strand is a nucleotide modified with 2'-methoxy and 5'-(E)-vinylphosphate, and the nucleotides at positions 3-4 and 6 are 2'-methoxy modified nucleotides. The nucleotides at positions 8-9, 11, 13, 15, 17, 19, and 21 are nucleotides modified with 2'-methoxy groups; the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 are nucleotides modified with 2'-fluoride groups; the nucleotides at positions 5 and 7 are deoxyribonucleotides; and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0127] 12. A modified double-stranded RNA molecule according to any one of claims 8-11, wherein the double-stranded RNA molecule is selected from any one of the following:

[0128] (1) The chain of justice is:

[0129] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0130] The antispeech chain is:

[0131] VPAmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 71);

[0132] (2) The chain of justice is:

[0133] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67),

[0134] The antispeech chain is:

[0135] VPUmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 72);

[0136] (3) The justice chain is:

[0137] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69),

[0138] The antispeech chain is:

[0139] VPAmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 73).

[0140] 13. A modified double-stranded RNA molecule according to any one of claims 8-11, wherein the double-stranded RNA molecule is selected from any one of the following:

[0141] (1) The chain of justice is:

[0142] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0143] The antispeech chain is:

[0144] AmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 66);

[0145] (2) The chain of justice is:

[0146] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67),

[0147] The antispeech chain is:

[0148] UmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 68);

[0149] (3) The justice chain is:

[0150] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69),

[0151] The antispeech chain is:

[0152] AmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 70).

[0153] 14. A modified double-stranded RNA molecule according to any one of claims 8-13, wherein the sense strand comprises a ligand.

[0154] 15. The modified double-stranded RNA molecule according to claim 14, wherein the ligand is coupled to the 3'-terminus of the positive strand.

[0155] 16. A modified double-stranded RNA molecule according to item 14 or 15, wherein the ligand is coupled to the positive strand via a linker.

[0156] 17. A modified double-stranded RNA molecule according to any one of claims 14-16, wherein the 3'-end of the sense strand is attached with a compound of the following formula:

[0157] 18. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of items 1-7 or a modified double-stranded RNA molecule as described in any one of items 8-17, and a pharmaceutically acceptable carrier.

[0158] 19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition comprises a lipid formulation, a nanoformulation, or a lipid-containing vesicle.

[0159] 20. The use of any of the double-stranded RNA molecules described in items 1-7, or any of the modified double-stranded RNA molecules described in items 8-17, or the pharmaceutical composition described in item 18 or 19, in any of the following:

[0160] D1) Application in the preparation of drugs that inhibit ANGPTL3 gene expression;

[0161] Application of D2 in suppressing ANGPTL3 gene expression;

[0162] Application of D3 in the treatment of diseases related to the ANGPTL3 gene target;

[0163] Application of D4 in the preparation of drugs for treating diseases related to the ANGPTL3 gene target.

[0164] 21. The application according to item 20, wherein the ANGPTL3 gene target-related diseases are selected from one or more of the following:

[0165] Hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases.

[0166] 22. A method for treating ANGPTL3 gene target-related diseases, comprising administering to a subject a therapeutically effective amount of any of the double-stranded RNA molecules described in items 1-7, or any of the modified double-stranded RNA molecules described in items 8-17, or the pharmaceutical composition described in item 18 or 19.

[0167] 23. The method according to item 22, wherein the ANGPTL3 gene target-related diseases are selected from one or more of the following:

[0168] Hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases.

[0169] 24. A method for inhibiting the expression of the ANGPTL3 gene in cells, the method comprising:

[0170] The cells are brought into contact with any of the double-stranded RNA molecules described in items 1-7, or any of the modified double-stranded RNA molecules described in items 8-17, or the pharmaceutical composition described in item 18 or 19.

[0171] Maintaining the cell for a sufficient period of time to allow for the degradation of the ANGPTL3 gene mRNA transcript can suppress ANGPTL3 gene expression in the cell.

[0172] 25. The method according to item 24, wherein the cells are located inside or outside the subject.

[0173] 26. The method of claim 25, wherein the subject suffers from an ANGPTL3 gene target-related disease.

[0174] 27. The method according to item 26, wherein the ANGPTL3 gene target-related diseases are selected from one or more of the following:

[0175] Hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases.

[0176] Compared with the prior art, the beneficial technical effects achieved by this application are as follows:

[0177] This application provides a novel double-stranded RNA molecule targeting the ANGPTL3 gene. In vitro and in vivo experiments have demonstrated that the provided double-stranded RNA molecule can effectively inhibit ANGPTL3 gene expression. This indicates that the provided double-stranded RNA molecule has significant drug development potential and application value in diseases with abnormal ANGPTL3 gene expression, such as hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases. Detailed Implementation

[0178] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0179] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0180] definition

[0181] In this application, "siRNA" is defined as a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, such as a transcript of a gene encoding a protein) that is complementary to it. siRNA comprises an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0182] In this application, "antisense strand" refers to a strand of siRNA that contains regions that are fully or substantially complementary to the target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence). When the complementary region is not fully complementary to the target sequence, the mismatch typically occurs in the internal or terminal regions of the molecule. In some embodiments, the double-stranded nucleic acid molecule includes nucleotide mismatches in the antisense strand.

[0183] In this application, as understood by those skilled in the art, the term “complementary” when used to describe the first nucleoside sequence relative to the second nucleoside sequence refers to the ability of an oligonucleotide containing the first nucleoside sequence to hybridize with an oligonucleotide containing the second nucleoside sequence under certain conditions and form a double-stranded structure.

[0184] In the application, the term "sense chain" refers to a chain of siRNA that includes regions substantially complementary to the region defined herein as the antisense chain.

[0185] In this application, the term "protrusion" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of the siRNA. A nucleotide protrusion exists, for example, when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. The siRNA may contain a protrusion having at least one nucleotide; alternatively, the protrusion may contain at least two, three, four, five, or more nucleotides. The nucleotide protrusion may contain or consist of a nucleotide / nucleoside analogue (including deoxynucleotides / nucleosides). One or more protrusions may be located on the sense strand, the antisense strand, or any combination thereof. Additionally, one or more nucleotides of the protrusion may be present at the 5' end, 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0186] In this application, complementary sequences within a double-stranded RNA molecule include base pairings along the entire length of the sense and antisense strands. Such sequences may be referred to as “perfectly complementary” to each other in this application. However, when the sense strand is referred to as “fundamentally complementary” or “partially complementary” relative to the antisense strand herein, the two sequences may be perfectly complementary, or they may form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably no more than 5, while maintaining the ability to hybridize under the conditions most relevant to its final application. In determining complementarity, overhangs should not be considered mismatches. For example, a double-stranded RNA molecule containing a 17-nucleotide sense strand and a 19-nucleotide antisense strand, where the longer nucleotide contains a 17-nucleotide sequence perfectly complementary to the shorter nucleotide, can still be referred to as “perfectly complementary.”

[0187] In this application, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with another group, or a nucleotide in which the bases on the nucleotide are modified bases. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids, but whose structure differs from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0188] In this application, "ligand" refers to a chemical moiety conjugated to siRNA that can alter the distribution, targeting, or lifetime of the siRNA. In a preferred embodiment, such a ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types), compartments (e.g., cell or organ compartments, tissues, organs, or regions of the body) compared to siRNA, for example, siRNA without such a ligand.

[0189] In this application, Base represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0190] In this application, the term “suppression” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of suppression.

[0191] In this application, "inhibiting ANGPTL3 expression" refers to inhibiting the expression of any ANGPTL3 gene and its variants or mutants. Therefore, the ANGPTL3 gene can be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or a transgenic ANGPTL3 gene in the case of genetically manipulated cells, cell groups, or organisms.

[0192] In this application, "inhibition of ANGPTL3 gene expression" includes inhibition of any level of the ANGPTL3 gene, such as at least partial repression of ANGPTL3 gene expression. ANGPTL3 gene expression can be assessed based on the level or changes in the level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA level, ANGPTL3 protein level, or lipid level. This level can be assessed in individual cells or in a group of cells (including, for example, samples derived from a subject).

[0193] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with ANGPTL3 expression compared to a control level. A control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only or inert control) subjects, cells, or samples.

[0194] In this application, "treatment" means a beneficial or desired outcome, including but not limited to the relief or improvement of one or more symptoms related to gene expression. "Treatment" can also mean extended survival compared to expected survival without treatment. Treatment may include prevention of the development of comorbidities, such as reducing liver injury in individuals with liver infections.

[0195] In this application, "therapeutic effective amount" is intended to include an amount of nucleic acid (e.g., siRNA) that, when administered to a patient to treat a subject with a disease, is sufficient to achieve treatment of the disease (e.g., by alleviating, improving, or maintaining an existing disease, or one or more symptoms of the disease or its associated comorbidities).

[0196] In this application, "pharmaceutically acceptable" means a compound, material, composition, or dosage form suitable for tissue contact with human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and with a reasonable benefit / risk ratio.

[0197] In this application, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, for carrying or transporting a target compound from one organ or body part to another. Each carrier must be "acceptable," meaning it is compatible with other components in the formulation and will not cause harm to the subject receiving treatment.

[0198] Double-stranded RNA molecules

[0199] On the one hand, this application provides an unmodified double-stranded RNA molecule, specifically an siRNA, wherein the double-stranded RNA molecule includes a sense strand and an antisense strand that at least partially form a double-stranded region, and the antisense strand includes any even-numbered nucleotide sequence in SEQ ID NO. 1-64.

[0200] In some embodiments, the positive strand comprises any odd-numbered nucleotide sequence from SEQ ID NO. 1-64.

[0201] In some embodiments, the length of the double-stranded region is 15-30 bp, for example, it can be 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, or 30 bp. In some embodiments, the length of the double-stranded region is 23-27 bp. In some embodiments, the length of the double-stranded region is 21-23 bp. In some embodiments, the length of the double-stranded region is 19-21 bp. In some embodiments, the length of the double-stranded region is 17-25 bp. In some embodiments, the length of the double-stranded region is 17-23 bp. In some embodiments, the length of the double-stranded region is 17-19 bp. In some embodiments, the length of the double-stranded region is 19 bp.

[0202] In some embodiments, the length of the sense strand does not exceed 30 nucleotides, and / or the length of the antisense strand does not exceed 30 nucleotides. In some embodiments, the length of the sense strand does not exceed 21 nucleotides, and / or the length of the antisense strand does not exceed 21 nucleotides.

[0203] In some embodiments, at least one of the sense and antisense strands includes a 3' overhang having at least one or at least two nucleotides.

[0204] In some embodiments, the sense strand of the double-stranded RNA molecule has a nucleotide sequence with any odd number in SEQ ID NO. 1-64, and the antisense strand has a nucleotide sequence with any even number in SEQ ID NO. 1-64.

[0205] In some embodiments, the sense and antisense strands of the double-stranded RNA molecule comprise or are any combination selected from the following:

[0206] The following chains are listed: the right chain shown in SEQ ID NO.41 and the wrong chain shown in SEQ ID NO.42; the right chain shown in SEQ ID NO.43 and the wrong chain shown in SEQ ID NO.44; the right chain shown in SEQ ID NO.59 and the wrong chain shown in SEQ ID NO.60; the right chain shown in SEQ ID NO.1 and the wrong chain shown in SEQ ID NO.2; the right chain shown in SEQ ID NO.3 and the wrong chain shown in SEQ ID NO.4; the right chain shown in SEQ ID NO.5 and the wrong chain shown in SEQ ID NO.6; the right chain shown in SEQ ID NO.7 and the wrong chain shown in SEQ ID NO.8; the right chain shown in SEQ ID NO.9 and the wrong chain shown in SEQ ID NO.10; the right chain shown in SEQ ID NO.11 and the wrong chain shown in SEQ ID NO.12; the right chain shown in SEQ ID NO.13 and the wrong chain shown in SEQ ID NO.14; the right chain shown in SEQ ID NO.15 and the wrong chain shown in SEQ ID NO.16; the right chain shown in SEQ ID NO.17 and the wrong chain shown in SEQ ID NO.18; SEQ ID NO. The following chains are listed: the justice chain shown in SEQ ID NO. 19 and the antisense chain shown in SEQ ID NO. 20; the justice chain shown in SEQ ID NO. 21 and the antisense chain shown in SEQ ID NO. 22; the justice chain shown in SEQ ID NO. 23 and the antisense chain shown in SEQ ID NO. 24; the justice chain shown in SEQ ID NO. 25 and the antisense chain shown in SEQ ID NO. 26; the justice chain shown in SEQ ID NO. 27 and the antisense chain shown in SEQ ID NO. 28; the justice chain shown in SEQ ID NO. 29 and the antisense chain shown in SEQ ID NO. 30; the justice chain shown in SEQ ID NO. 31 and the antisense chain shown in SEQ ID NO. 32; the justice chain shown in SEQ ID NO. 33 and the antisense chain shown in SEQ ID NO. 34; the justice chain shown in SEQ ID NO. 35 and the antisense chain shown in SEQ ID NO. 36; the justice chain shown in SEQ ID NO. 37 and the antisense chain shown in SEQ ID NO. 38; the justice chain shown in SEQ ID NO. 39 and the antisense chain shown in SEQ ID NO. 40; and the justice chain shown in SEQ ID NO. 45 and the antisense chain shown in SEQ ID NO. 46. The antisense chain shown in NO.46, the right-sense chain shown in SEQ ID NO.47 and the antisense chain shown in SEQ ID NO.48, the right-sense chain shown in SEQ ID NO.49 and the antisense chain shown in SEQ ID NO.50, and SEQ ID NO.The same chain of justice shown in SEQ ID NO. 51 and the same chain of justice shown in SEQ ID NO. 52; the same chain of justice shown in SEQ ID NO. 53 and the same chain of justice shown in SEQ ID NO. 54; the same chain of justice shown in SEQ ID NO. 55 and the same chain of justice shown in SEQ ID NO. 56; the same chain of justice shown in SEQ ID NO. 57 and the same chain of justice shown in SEQ ID NO. 58; the same chain of justice shown in SEQ ID NO. 61 and the same chain of justice shown in SEQ ID NO. 62; the same chain of justice shown in SEQ ID NO. 63 and the same chain of justice shown in SEQ ID NO. 64.

[0207] On the other hand, this application also provides unmodified double-stranded RNA molecules, comprising any of the aforementioned double-stranded RNA molecules, wherein at least one nucleotide is chemically modified. That is, it is obtained by modifying at least one nucleotide of any of the aforementioned unmodified double-stranded RNA molecules, resulting in a compound containing the modified nucleotide.

[0208] The chemical modification can be the substitution of the 2' hydroxyl group of the ribosyl group of the nucleotide by other groups, the modification of the bases on the nucleotide, the modification of the thiophosphate group of the phosphodiester between nucleotides, or any combination of these three types of modifications.

[0209] In some embodiments, the modified nucleotide is a compound formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, or a compound formed by modifying the bases on the nucleotide.

[0210] In some embodiments, the modified nucleotide is selected from at least one of the following:

[0211] 2'-Methoxy modified nucleotides, 2'-Fluoro modified nucleotides, Vinylphosphonate modified nucleotides, Phosphothioester modified nucleotides, 2'-Methyl modified nucleotides, Deoxy-nucleotides, 2'-Deoxy-nucleotides, 3'-Terminal Deoxy-thymidine (dT) nucleotides, 2'-Deoxy-modified nucleotides, Locked nucleotides, Non-locked nucleotides, Configuration-restricted nucleotides, Restricted ethyl nucleotides, Baseless nucleotides, 2'-Amino-modified nucleotides, 2'-O-Allyl-modified nucleotides, 2 '-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics.

[0212] In one embodiment of this application, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0213] In one embodiment of this application, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art. These nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0214] In one embodiment of this application, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide may be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3); the 2'-amino (2'-NH2) modified nucleotide is shown in formula (4); and the 2'-deoxynucleotide (DNA) is shown in formula (5).

[0215] In one embodiment of this application, the nucleotide analogue refers to a group that can replace a nucleotide in nucleic acid, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0216] In one embodiment of this application, the nucleotide analog may be an isonucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0217] In one embodiment of this application, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered ring, a six-membered ring, or a seven-membered ring. Typically, the bridge is incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0218] In one embodiment of this application, the BNA may be an LNA, an ENA, a cET BNA, etc., wherein an LNA is shown in equation (6), an ENA is shown in equation (7), and a cET BNA is shown in equation (8):

[0219] In one embodiment of this application, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups.

[0220] In one embodiment of this application, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0221] In one embodiment of this application, the phosphate group having the modifying group is a thiophosphate group having the structure shown in formula (9). In one embodiment of this application, the nucleotide linked to the thiophosphate group is shown in formula (10), and the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0222] In one embodiment of this application, the modified nucleotide is a vinyl phosphate modified (VP-modified) nucleotide. In one embodiment of this application, the VP-modified and methoxy-modified nucleotide, i.e., the nucleotide modified with 5'-(E)-vinyl-2'-methoxy-modified phosphonate group (5'-(E)-VP-2'-OMe), is shown in formula (11); in one embodiment of this application, the VP-modified, methoxy-modified, and thiophosphate-modified nucleotide, i.e., the 5'-PS-modified nucleotide (i.e., the 5'-(E)-vinyl-2'-methoxy-modified thiophosphate-modified nucleotide), is shown in formula (12).

[0223] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-6 and 10-19 of the sense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and positions 2 and 3 linked by thiophosphate groups; the nucleotide at position 1 of the antisense strand is a nucleotide modified with both 2'-methoxy and 5'-(E)-vinylphosphate, the nucleotides at positions 3-5, 7-13, 15, and 17-21 being 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 linked by thiophosphate groups.

[0224] In some embodiments, the double-stranded RNA molecule is selected from any of the following:

[0225] (1) The chain of justice is:

[0226] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0227] The antispeech chain is:

[0228] VPAmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 71);

[0229] (2) The chain of justice is:

[0230] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67),

[0231] The antispeech chain is:

[0232] VPUmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 72);

[0233] (3) The justice chain is:

[0234] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69),

[0235] The antispeech chain is:

[0236] VPAmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 73).

[0237] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-6 and 10-19 of the sense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 linked by thiophosphate groups; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 linked by thiophosphate groups.

[0238] In some embodiments, the double-stranded RNA molecule is selected from any of the following:

[0239] (1) The chain of justice is:

[0240] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0241] The antispeech chain is:

[0242] AmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 66);

[0243] (2) The chain of justice is:

[0244] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67),

[0245] The antispeech chain is:

[0246] UmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 68);

[0247] (3) The justice chain is:

[0248] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69),

[0249] The antispeech chain is:

[0250] AmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 70).

[0251] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-4, 6, and 10-19 of the sense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 being 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 being linked by thiophosphate groups; the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 8-9, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 being linked by thiophosphate groups.

[0252] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-4, 6, and 10-19 of the sense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 being linked by thiophosphate groups; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 being linked by thiophosphate groups.

[0253] In some embodiments, the modified double-stranded RNA molecule, with the nucleotides at positions 1-2, 4, 6, 10, 12, 14, 16, and 18 of the sense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 3, 5, 7-9, 11, 13, 15, 17, and 19 being 2'-fluorinated modified nucleotides, with the nucleotides at positions 1 and 2, and 2 and 3 linked by thiophosphate groups; and the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, and 19-21 of the antisense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, and 18 being 2'-fluorinated modified nucleotides, with the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 linked by thiophosphate groups.

[0254] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-4, 6, and 10-19 of the sense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 being linked by thiophosphate groups; the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 8-9, 10, 14, and 16 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 being linked by thiophosphate groups.

[0255] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-6 and 10-19 of the sense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 linked by thiophosphate groups; the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 14, and 16 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 linked by thiophosphate groups.

[0256] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-4, 6, and 10-19 of the sense strand being 2'-methoxy modified nucleotides, and the nucleotides at positions 5 and 7-9 being 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 being linked by thiophosphate groups; the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 being linked by thiophosphate groups.

[0257] In some embodiments, the modified double-stranded RNA molecule is arranged with the 5' end to the 3' end as follows: nucleotides 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, nucleotides 7-9 are 2'-fluoro modified nucleotides, and nucleotides 1 and 2, and 2 and 3 are linked by thiophosphate groups; nucleotides 1, 3-4, 6, 8-9, 11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-methoxy modified nucleotides, nucleotides 2, 10, 12, 14, 16, 18, and 20 are 2'-fluoro modified nucleotides, nucleotides 5 and 7 are deoxyribonucleotides, and nucleotides 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0258] In some embodiments, the modified double-stranded RNA molecule, with the nucleotides at positions 1-6 and 10-19 of the sense strand being 2'-methoxy modified, and the nucleotides at positions 7-9 being 2'-fluoro modified, are linked by thiophosphate groups at positions 1 and 2, and positions 2 and 3; the nucleotide at position 1 of the antisense strand is a nucleoside nucleotide modified with both 2'-methoxy and 5'-(E)-vinylphosphate. The nucleotides at positions 3-4, 6, 8-9, 11, 13, 15, 17, 19, and 21 are nucleotides modified with 2'-methoxy groups; the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 are nucleotides modified with 2'-fluoride groups; the nucleotides at positions 5 and 7 are deoxyribonucleotides; and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0259] The double-stranded RNA molecules used in this application can all be prepared using methods known in the art. For example, the phosphoramide solid-phase synthesis method used in siRNA synthesis, which is well-known to those skilled in the art. The nucleoside monomers used in this application are all commercially available.

[0260] The modified double-stranded RNA molecule of this application can be further coupled with a ligand. The ligand can be coupled to any nucleotide at the 3'-end, 5'-end, or middle of the positive strand. In some embodiments, the ligand is coupled to the 3'-end of the positive strand.

[0261] Those skilled in the art will understand that the coupling of the ligand to the sense strand can be direct coupling or indirect coupling via a linker. In some embodiments, the ligand is coupled to the 3'-terminus of the sense strand via a linker.

[0262] In some embodiments, the ligand is GalNAc, i.e., a compound represented by the following formula:

[0263] In some embodiments, the siRNA conjugate formed by the GalNAc and siRNA molecules of this application has the following structure:

[0264] The abbreviations for the nucleotide monomers used in this application are shown in the table below. It should be noted that when these monomers are present in oligonucleotides, they are interconnected via 5'-3'-phosphodiester bonds.

[0265] Pharmaceutical Composition

[0266] This application also provides a pharmaceutical composition comprising the above-described unmodified double-stranded RNA molecule or modified double-stranded RNA molecule, and a pharmaceutically acceptable carrier.

[0267] The pharmaceutically acceptable carrier may be an excipient, stabilizer, suspending agent, or diluent, as is well known to those skilled in the art.

[0268] The pharmaceutical composition of this application can be administered at a dose sufficient to inhibit gene expression. Typically, a suitable dose of the double-stranded RNA molecule of this application is from about 0.001 to about 200.0 mg per kilogram of body weight per day, usually from about 1 to 50 mg per kilogram of body weight per day. Typically, a suitable dose of the double-stranded RNA molecule of this application is from about 0.1 mg / kg to about 5 mg / kg, for example, about 1 mg / kg and about 3 mg / kg.

[0269] Repeated dosing regimens may include the administration of therapeutic doses of nucleic acid periodically (e.g., every other day or once a year). In some embodiments, the frequency of administration of nucleic acid (e.g., siRNA) is approximately once a month to approximately once a quarter (i.e., approximately once every three months).

[0270] In some embodiments, the double-stranded RNA molecule of this application is administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg, approximately once weekly, once monthly, once every two months, or once quarterly (i.e., once every three months). In some embodiments, the double-stranded RNA molecule of this application is administered to the subject once weekly. In some embodiments, the double-stranded RNA molecule of this application is administered to the subject once monthly. In some embodiments, the double-stranded RNA molecule of this application is administered once quarterly (i.e., once every three months).

[0271] After the initial treatment regimen, the frequency of treatment can be reduced. For example, after applying once a week or every two weeks for three months, it can be repeated once a month for six months or a year; or longer.

[0272] The pharmaceutical composition of this application can be administered in a variety of ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration can be local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via a nebulizer), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, via an implanted device; or intracranial, for example, via intraparenchymal, intrathecal, or intraventricular administration. In some preferred embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the double-stranded RNA molecule of this application is administered subcutaneously to the subject.

[0273] Treatment methods and therapeutic uses

[0274] This application also provides the use of the above-mentioned unmodified double-stranded RNA molecules or modified double-stranded RNA molecules in any of the following:

[0275] D1) Application in the preparation of drugs that inhibit ANGPTL3 gene expression;

[0276] Application of D2 in suppressing ANGPTL3 gene expression;

[0277] Application of D3 in the treatment of diseases related to the ANGPTL3 gene target;

[0278] Application of D4 in the preparation of drugs for treating diseases related to the ANGPTL3 gene target.

[0279] Inhibition of ANGPTL3 gene expression can refer to inhibition of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

[0280] Diseases related to the ANGPTL3 gene target can encompass diseases known in the field caused by abnormal ANGPTL3 gene expression.

[0281] In some embodiments, the disease caused by abnormal ANGPTL3 gene expression is a disease caused by upregulation of ANGPTL3 gene expression.

[0282] In some embodiments, the diseases resulting from the upregulation of the ANGPTL3 gene are selected from one or more of the following groups:

[0283] Hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolism disorders, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases.

[0284] This application also provides a method for inhibiting the expression of the ANGPTL3 gene in cells, the method comprising:

[0285] The cells are brought into contact with the above-mentioned unmodified double-stranded RNA molecule or modified double-stranded RNA molecule, or the above-mentioned pharmaceutical composition;

[0286] Maintaining the cell for a sufficient period of time to allow for the degradation of the ANGPTL3 gene mRNA transcript can suppress ANGPTL3 gene expression in the cell.

[0287] In some embodiments, the cells are located inside the subject. In some embodiments, the cells are located outside the subject. The subject can be a mammal, including primates (e.g., humans or non-human primates, such as monkeys or chimpanzees) and non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, or whales). In some embodiments, the subject is a human.

[0288] In some embodiments, the subject suffers from an ANGPTL3 gene target-related disease. The ANGPTL3 gene target-related disease is as described above.

[0289] Example

[0290] Related experimental materials

[0291] siRNA source: synthesized by Suzhou Gemma Gene Co., Ltd.

[0292] Source of laboratory animals:

[0293] C57BL / 6J mice were purchased from Jicui Yaokang and were 6-8 weeks old. The mice were acclimatized in the SPF barrier for one week.

[0294] The human ANGPTL3 transgenic mouse (also called C57BL / 6J-hANGPTL3 humanized mouse in the example) was purchased from Nanmo Biotechnology (NM-HU-210036).

[0295] The rearing conditions are as follows: the temperature of the rearing environment should be controlled at 20-22℃, the humidity should be maintained within the range of 40-60%, a 12-hour light / 12-hour dark cycle should be adopted, and food and water should be available freely.

[0296] Animal administration method: The following experimental animals were all administered a single dose via subcutaneous injection in the back of the neck, with 100 μL injected per animal.

[0297] The ARO-ANG3-1 sequence is derived from AD05488 in US10995335B2, but the difference between it and AD05488 is that it does not use NAG37 as the delivery head, but instead uses GalNAc as the delivery target.

[0298] The GRD342230.9 sequence originates from AD-65695 in Alnylam patent WO2016168286A1.

[0299] Example 1: Inhibition of ANGPTL3 siRNA Synthesis

[0300] In this application, ANGPTL3 mRNA refers to mRNA with GeneBank registration numbers NM_014495.4, XM_005543185.3, or NM_013913.4.

[0301] The siRNA of this application was synthesized by Suzhou Gemma Gene Co., Ltd. using a solid-phase synthesis method known in the art. Unmodified siRNAs as shown in Table 1 below were synthesized using the aforementioned solid-phase synthesis method.

[0302] Table 1 Unmodified siRNAs that inhibit ANGPTL3

[0303] Modified siRNA

[0304] The siRNAs shown in Table 1 were modified according to the modification patterns M1, M1VP, M2, M3, M4, M6, M7, M38, and M38VP, and the modified siRNAs were obtained using the solid-phase synthesis method described above.

[0305] M1 Modification Mode:

[0306] Chain of Justice 5'-3'

[0307] ms-ms-mmmmfffmmmmmmmmmm

[0308] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0309] antisense chain 5'-3'

[0310] ms-fs-mmmfmmmmmmmmfmfmm-ms-ms-m

[0311] That is, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 6, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0312] Following the M1 modification pattern, the unmodified siRNAs in Table 1 were modified to obtain M1-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M1. For example, the unmodified siRNA named ANG3-1041, after being modified with M1, becomes ANG3-1041M1. Examples of M1-modified siRNAs are as follows: For instance, the siRNA numbered ANG3-HC30 after M1 modification is ANG3-HC30M1, and its corresponding positive strand is:

[0313] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0314] The antispeech chain is:

[0315] AmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 66).

[0316] The siRNA modified according to the M1 pattern of ANG3-HC31 is numbered ANG3-HC31M1, and its corresponding positive strand is:

[0317] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67),

[0318] The antispeech chain is:

[0319] UmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 68).

[0320] The siRNA modified according to the M1 pattern of ANG3-HC83 is numbered ANG3-HC83M1, and its corresponding positive strand is:

[0321] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69),

[0322] The antispeech chain is:

[0323] AmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 70).

[0324] M1VP Modification Mode:

[0325] Chain of Justice 5'-3'

[0326] ms-ms-mmmmfffmmmmmmmmmm

[0327] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0328] antisense chain 5'-3'

[0329] VPms-fs-mmmfmmmmmmmmfmfmm-ms-ms-m

[0330] That is, the nucleotide at position 1 of the antisense strand is a nucleotide modified with 2'-methoxy and 5'-(E)-vinyl phosphate, i.e., the modified nucleotide is The nucleotides at positions 3-5, 7-13, 15, and 17-21 are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 6, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0331] Following the M1VP modification pattern, the unmodified siRNAs in Table 1 were modified to obtain M1VP-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M1VP. For example, the unmodified siRNA named ANG3-1041, after being modified with M1VP, becomes ANG3-1041M1VP. Exemplary M1VP-modified siRNAs are as follows:

[0332] For example, the siRNA modified according to the M1VP pattern of ANG3-HC30 is numbered ANG3-HC30M1VP, and its corresponding sense strand is:

[0333] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65),

[0334] The ansense chain is: VPAmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO.71).

[0335] The siRNA modified according to the M1VP pattern of ANG3-HC31 is numbered ANG3-HC31M1VP, and its corresponding sense strand is: GmsAmsAmCmUmAmCfAfUfAmUmAmAmCmUmAmCmAm (SEQ ID NO.67), and its antisense strand is: VPUmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO.72).

[0336] The siRNA modified according to the M1VP pattern of ANG3-HC83 is numbered ANG3-HC83M1VP, and its corresponding sense strand is: CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO.69), and its antisense strand is: VPAmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO.73).

[0337] M2 Modification Mode:

[0338] Chain of Justice 5'-3'

[0339] ms-ms-mmfmfffmmmmmmmmmm

[0340] That is, the nucleotides at positions 1-4, 6, and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0341] antisense chain 5'-3'

[0342] ms-fs-mmmfmffmmmmfmfmm-ms-ms-m

[0343] That is, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 6, 8-9, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0344] The unmodified siRNAs in Table 1 were modified to obtain M2-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M2. For example, the unmodified siRNA named ANG3-HC30 was modified with M2 to obtain the modified siRNA named ANG3-HC30M2.

[0345] M3 Editing Mode:

[0346] Chain of Justice 5'-3'

[0347] ms-ms-mmfmfffmmmmmmmmmm

[0348] That is, the nucleotides at positions 1-4, 6, and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0349] antisense chain 5'-3'

[0350] ms-fs-mmmfmmmmmmmmfmfmm-ms-ms-m

[0351] That is, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 6, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0352] The unmodified siRNAs in Table 1 were modified to obtain M3-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M3. For example, the unmodified siRNA named ANG3-HC30 was modified with M3 to obtain the modified siRNA named ANG3-HC30M3.

[0353] M4 Editing Mode:

[0354] Chain of Justice 5'-3'

[0355] ms-ms-fmfmfffmfmfmfmfmf

[0356] That is, the nucleotides at positions 1-2, 4, 6, 10, 12, 14, 16, and 18 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 3, 5, 7-9, 11, 13, 15, 17, and 19 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0357] antisense chain 5'-3'

[0358] ms-fs-mfmfmfmfmmmfmfmf-ms-ms-m

[0359] That is, the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, and 19-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, and 18 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0360] The unmodified siRNAs in Table 1 were modified to obtain M4-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M4. For example, the unmodified siRNA named ANG3-HC30 was modified with M4 to obtain the modified siRNA named ANG3-HC30M4.

[0361] M5 Editing Mode:

[0362] Chain of Justice 5'-3'

[0363] ms-ms-mmfmfffmmmmmmmmmm

[0364] That is, the nucleotides at positions 1-4, 6, and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0365] antisense chain 5'-3'

[0366] ms-fs-mmmfmffmmmmfmfmm-ms-ms-m

[0367] That is, the nucleotides at positions 1, 3-5, 7, 10-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 6, 8-9, 10, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0368] The unmodified siRNAs in Table 1 were modified to obtain M5-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M5. For example, the unmodified siRNA named ANG3-HC30 is modified with M5 to obtain the modified siRNA named ANG3-HC30M5.

[0369] M6 Modification Mode:

[0370] Chain of Justice 5'-3'

[0371] ms-ms-mmmmfffmmmmmmmmmm

[0372] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0373] antisense chain 5'-3'

[0374] ms-fs-mmmmmmmmmmmmfmfmm-ms-ms-m

[0375] That is, the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0376] The unmodified siRNAs in Table 1 were modified to obtain M6-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M6. For example, the unmodified siRNA named ANG3-HC30 was modified with M6 to obtain the modified siRNA named ANG3-HC30M6.

[0377] M7 Modification Mode:

[0378] Chain of Justice 5'-3'

[0379] ms-ms-mmfmfffmmmmmmmmmm

[0380] That is, the nucleotides at positions 1-4, 6, and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 5 and 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0381] antisense chain 5'-3'

[0382] ms-fs-mmmmmmmmmmmmfmfmm-ms-ms-m

[0383] That is, the nucleotides at positions 1, 3-13, 15, and 17-21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 14, and 16 are nucleotides modified with 2'-fluoride groups, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0384] The unmodified siRNAs in Table 1 were modified to obtain M7-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M7. For example, the unmodified siRNA named ANG3-HC30 was modified with M7 to obtain the modified siRNA named ANG3-HC30M7.

[0385] M38 Modification Mode:

[0386] Chain of Justice 5'-3'

[0387] ms-ms-mmmmfffmmmmmmmmmm

[0388] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0389] antisense chain 5'-3'

[0390] ms-fs-mm-(d)-m-(d)-mmfmfmfmfmf-ms-fs-m

[0391] That is, the nucleotides at positions 1, 3-4, 6, 8-9, 11, 13, 15, 17, 19, and 21 of the antisense strand are nucleotides modified with 2'-methoxy groups, the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 are nucleotides modified with 2'-fluoride groups, the nucleotides at positions 5 and 7 are deoxyribonucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0392] The unmodified siRNAs in Table 1 were modified to obtain M38-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M38. For example, the unmodified siRNA named ANG3-HC30 was modified with M38 to obtain the modified siRNA named ANG3-HC30M38.

[0393] M38VP Modification Mode:

[0394] Chain of Justice 5'-3'

[0395] ms-ms-mmmmfffmmmmmmmmmm

[0396] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphate groups.

[0397] antisense chain 5'-3'

[0398] VPms-fs-mm-(d)-m-(d)-mmfmfmfmfmf-ms-fs-m

[0399] That is, the nucleotide at position 1 of the antisense strand is a nucleotide modified with 2'-methoxy and 5'-(E)-vinyl phosphate, i.e., the modified nucleotide is The nucleotides at positions 3-4, 6, 8-9, 11, 13, 15, 17, 19, and 21 are nucleotides modified with 2'-methoxy groups; the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 are nucleotides modified with 2'-fluoride groups; the nucleotides at positions 5 and 7 are deoxyribonucleotides; and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups.

[0400] The unmodified siRNAs in Table 1 were modified to obtain M38VP-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M38VP. For example, the unmodified siRNA named ANG3-HC30 was modified with M38 to obtain the modified siRNA named ANG3-HC30M38VP.

[0401] Furthermore, based on the modification patterns of M1, M1VP, M2, M3, M4, M5, M6, M7, M38, or M38VP, GalNAc is coupled to the 3' end of the positive strand to obtain siRNAs modified with GalNAc, namely M1, M1VP, M2, M3, M4, M5, M6, M7, M38, or M38VP. The names of the siRNAs coupled with GalNAc are the naked sequence name + M1G, M1GVP, M2G, M3G, M4G, M5G, M6G, M7G, M38G, and M38GVP. For example, a siRNA with the naked sequence number ANG3-HC30, modified with M1 and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC30M1G; a siRNA with the naked sequence number ANG3-HC30, modified with M1VP and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC30M1GVP. Similarly, a siRNA with the naked sequence number ANG3-HC31, modified with M1 and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC31M1G; and a siRNA with the naked sequence number ANG3-HC31, modified with M1VP and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC31M1GVP. The siRNA with naked sequence number ANG3-HC83, modified with M1 and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC83M1G. The siRNA with naked sequence number ANG3-HC83, modified with M1VP and coupled with GalNAc at the 3' end of the positive strand, is designated ANG3-HC83M1GVP.

[0402] The structure of the siRNA coupled with GalNAc is shown in the following formula:

[0403] For example, ANG3-HC30 modified according to the M1VP pattern, and further coupled with Galnac, results in siRNA numbered ANG3-HC30M1GVP, with the corresponding positive strand being:

[0404] GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm-GalNAc (SEQ ID NO. 65),

[0405] The ansense chain is: VPAmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO.71).

[0406] The siRNA of ANG3-HC31 modified according to the M1VP pattern and further coupled with Galnac is numbered ANG3-HC31M1GVP, and its corresponding positive strand is:

[0407] GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm-GalNAc (SEQ ID NO. 67),

[0408] The ansense chain is: VPUmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm(SEQ ID NO.72).

[0409] The siRNA modified according to the M1VP pattern and further conjugated with Galnac is numbered ANG3-HC83M1GVP, and its corresponding positive strand is:

[0410] CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm-GalNAc (SEQ ID NO. 69),

[0411] The ansense chain is: VPAmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO.73).

[0412] In the above modification patterns M1, M1VP, M2, M3, M4, M5, M6, M7, M38, or M38VP, m indicates that the nucleotide is 2'-methoxy modified, f indicates that the nucleotide is 2'-F modified, s indicates that the two nucleotides are linked by a thiophosphate group, and VP indicates that the 5' end of the nucleotide is (E)-vinyl phosphate modified.

[0413] Experimental Example 2: In vivo screening in animals

[0414] The modified siRNA synthesized in Example 1 was screened in animals.

[0415] Three hANGPTL3+ / + (female, over 6 weeks old) mice in each group received a single subcutaneous administration of 3 mg / kg of GalNAc-conjugated modified siRNA or a saline control, with an administration volume of 100 μL per mouse. On day 14 post-administration, the mice were sacrificed, liver samples were collected, liver mRNA was extracted, and analyzed using RT-qPCR.

[0416] The detection steps for RT-qPCR are as follows:

[0417] Step 1: RNA extraction

[0418] 1) Take 10-20 mg of mouse liver tissue, place it in RNA protection solution, incubate overnight at 4°C, discard the RNA protection solution, add 1 mL of Trizol Lysis Buffer (purchased from Life Technology, catalog number 410701), and grind the tissue at low temperature to lyse it. After thorough grinding, transfer it to an RNase-free 1.5 ml centrifuge tube; shake vigorously for about 10-15 seconds to fully lyse the tissue cells, and let it stand at room temperature for 3-5 minutes.

[0419] 2) Carefully open the tube cap, add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925); shake vigorously for 15-20 seconds, let stand at room temperature for 2-3 minutes; centrifuge at 12000×g for 20 minutes at 4℃.

[0420] 3) After centrifugation, carefully remove the centrifuge tubes to the centrifuge tube rack, transfer the supernatant to a new 2.0 mL centrifuge tube, add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., product number 20210802) to the supernatant, and mix by inverting.

[0421] 4) Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture from step 3), let stand for 2 min; centrifuge at 10000×g for 1 min at 4℃, discard the filtrate; repeat the above steps with the remaining mixture.

[0422] 5) Add 700 μL of 80% ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate;

[0423] 6) Add 700 μL of 80% ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate;

[0424] 7) Centrifuge the purification column at 4℃, 10000×g for 2 min (empty).

[0425] 8) After centrifugation, carefully remove the purification column with the collection tube (if there is liquid in the collection tube, be careful not to splash the liquid onto the purification column), discard the collection tube, place the purification column into a new 1.5 mL centrifuge tube, add 100 μL LEPC water to the purification column, and let it stand at room temperature for 2 min; centrifuge at 4℃, 10000×g for 1 min.

[0426] 9) RNA can be temporarily stored at 4°C and used for PCR experiments within one day. (For long-term storage, it should be stored at -80°C).

[0427] Step 2: RNA reverse transcription

[0428] The experimental procedure was performed using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01) following the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe total RNA from cells. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, then at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA.

[0429] Step 3: Preparation of qPCR reaction system

[0430] For each reverse transcription reaction system, take 4 μL of the above-mentioned cDNA-containing solution as a template. Using the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), prepare a 20 μL qPCR reaction system on an ice box according to Table 2-1. Primer 1 and Primer 2 are the PCR primer sequences for amplifying the target gene ANGPTL3 and the internal reference gene GAPDH, respectively (as shown in Table 2-2). Place each qPCR reaction system in an ABIStepOnePlus Real-Time... Amplification was performed using a three-step method on a PCR instrument. The RT-qPCR reaction program for hANGPTL3 is shown in Table 2-3, and the RT-qPCR reaction program for mANGPTL3 is shown in Table 2-4, yielding product W containing amplified target gene ANGPTL3 and internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 10s, 60℃ for 1min, and 95℃ for 15s. The melting curves of target gene ANGPTL3 and internal reference gene GAPDH in product W were collected by a real-time quantitative PCR instrument, and the Ct values ​​of target gene ANGPTL3 and internal reference gene GAPDH were obtained.

[0431] Table 2-1. RNA amplification reaction system

[0432] Table 2-2 Primer Information

[0433] Table 2-3. RT-qPCR reaction procedure (hANGPTL3)

[0434] Table 2-4. RT-qPCR reaction procedure (mANGPTL3)

[0435] The relative quantification of the target gene ANGPTL3 in each test group was performed using the Ct(ΔΔCt) method, as follows:

[0436] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0437] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0438] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0439] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0440] Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value.

[0441] In the above formula, the control group refers to the saline group.

[0442] Using the control group as a baseline, the expression level of ANGPTL3 mRNA in the test group was normalized, and the expression level of ANGPTL3 mRNA in the control group was defined as 100%.

[0443] The relative expression level of ANGPTL3 mRNA in the test group = 2^(-ΔΔCT)(test group) × 100%

[0444] The ANGPTL3 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data are expressed as a percentage relative to the saline control group. The results are shown in Table 2-5.

[0445] Table 2-5 Results of in vivo screening tests in animals

[0446] Experimental Example 3: Screening for Target Activity with Modified Sequences

[0447] This embodiment provides an assay for detecting the on-target activity of modified siRNA in inhibiting ANGPTL3. A plasmid vector, psiCHECK2, was constructed for the assay. psiCHECK2 is a plasmid vector that monitors changes in the expression of a target gene fused with a reporter gene. This vector uses Renal luciferase as the primary reporter gene. The target fragment is cloned into a multiple cloning site downstream of the translation stop codon of Renal luciferase. The synthesized siRNA triggers an RNAi process targeting the target gene, leading to the cleavage and subsequent degradation of the fusion mRNA. By detecting changes in Renal luciferase activity, a targeting relationship between the siRNA and the target gene fragment can be determined. The experimental procedure is as follows:

[0448] Step 1: Construct the detection plasmid

[0449] A detection plasmid was constructed using the psiCHECKTM-2 (PromegaTM) plasmid. The detection plasmid contains the insertion sequence shown below. The insertion sequence contains a sequence that is inversely complementary to the antisense strand of the siRNA to be tested. A single copy of the insertion sequence was cloned into the Xho I / Not I site of the psiCHECKTM-2 plasmid to obtain the detection plasmid.

[0450] The inserted sequence information is shown in Table 3-1.

[0451] Table 3-1 Information on detected plasmid insertion sequences

[0452] Step 2: Cell Culture and Transfection

[0453] In a 96-well plate, 5 μl of siRNA was added to each well, along with 12.5 μl of Opti-MEM containing 20 ng of the detection plasmid, and 32.5 μl of Opti-MEM plus 0.3 μl of Lipofectamine 2000 (Invitrogen, catalog number 11668-019) was added to each well. The mixture was incubated at room temperature for 15 minutes. Then, 50 μl of DMEM complete medium (Transgen Biotech, catalog number FI101-01) containing 1 × 10⁴ 293T cells was added to each well and the mixture was incubated at 37°C for 24 hours for subsequent dual-luciferase assay.

[0454] Step 3: Dual-luciferase assay

[0455] Dilute the 5× lysis buffer from the Dual Luciferase Assay Kit (Promega, catalog number E2940) to 1× lysis buffer with water. Discard the supernatant from the cells cultured in step two. Dilute and wash each well twice with PBS buffer (Hyclone, catalog number SH30256.01). Add 50 μL / well of 1× lysis buffer to each cell plate and lyse at room temperature for 20 min to obtain lysed cell plates. Transfer 30 μL / well of lysis buffer from the lysed cell plates to opaque 96-well assay plates. Prepare two substrates according to the instructions using the Dual Luciferase Assay Kit and add 30 μL / well of substrate 1 and substrate 2 to new 96-well plates, respectively. Perform assays using a multi-mode microplate reader after each substrate addition to obtain numerical results for Firefly luciferase and Renilla luciferase.

[0456] The luminescence ratio of each well in the ELISA plate was calculated as Renilla / Firefly. The luminescence ratio of each test group or control group was the average of the luminescence ratios of the three culture wells. Using the control group's luminescence ratio as a baseline, the luminescence ratios of each test group were normalized to obtain the ratio R of luminescence ratio (test) / luminescence ratio (control), which represents the expression level of the Renilla reporter gene, i.e., its relative residual activity. The inhibition rate of siRNA was (1-R)×100%.

[0457] The modified siRNA and corresponding detection plasmid information are shown in Table 3-2.

[0458] Table 3-2

[0459] The target activity results are shown in Table 3-3.

[0460] Table 3-3

[0461] The above sequences were transfected into the Hep3B cell line as described in Example 4. The final siRNA transfection concentration was 0.1 nM, and three biological replicates were set up for each siRNA. NC, MOCK, and BLANK were set up as controls. The MOCK group received only the interfering reagent without any sequence; the BLANK group contained only cells. The inhibitory effect of each siRNA on ANGPTL3 mRNA expression is shown in Tables 3-4.

[0462] Table 3-4 Results of siRNA sequences inhibiting ANGPTL3 mRNA expression in Hep3B cells

[0463] Experiment Example 4: In vitro cell screening

[0464] The modified siRNA synthesized in Example 1 was subjected to in vitro cell activity testing.

[0465] Hep3B cells cultured in 10cm dishes were routinely trypsinized 48 hours after passage. The cells were resuspended in complete culture medium and diluted to 3 x 10⁻⁶. 5 siRNA was seeded at 50 μL / well in 96-well plates and transfected using Lipofectamine RNAiMAX. Each siRNA was configured with three biological replicates. NC, MOCK, and BLANK were set up as controls. The MOCK group received only the interference reagent (Lipofectamine RNAiMAX) without any additional sequence; the BLANK group contained only cells. The final transfection concentrations for each sequence were 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.012 nM, 0.004 nM, 0.001 nM, 0.00045 nM, and 0.00015 nM.

[0466] NC's Chain of Justice (5'-3'):

[0467] CfsAmsCfUmUfAmCfGmCfUmGfAmGfUmAfCmUfUmCfGmAf (SEQ ID NO. 80);

[0468] NC's antisense chain (5'-3'):

[0469] UmsCfsGmAfAmGfUmAfCmUfCmAfGmCfGmUfAmAfGmUfGmsAfsUm (SEQ ID NO. 81).

[0470] 48 hours after transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the magnetic bead method total RNA extraction kit (Germage-E31008-96) according to the manufacturer's instructions.

[0471] Perform qPCR to detect ANGPTL3 gene expression levels according to the following steps.

[0472] (1) RNA template preparation

[0473] Genomic DNA removal: Add samples in the order shown in the table below, and gently mix with a pipette. Reaction program: 42°C, 2 min.

[0474] Table 4-1. Genome Removal Reaction System

[0475] (2) Reverse transcription

[0476] Prepare the first-strand cDNA synthesis reaction solution (total 20 μL) as shown in the table below, and gently mix with a pipette. Incubate at 50℃ for 15 min; then at 85℃ for 2 min. After reverse transcription, dilute the cDNA 5-fold for PCR detection and store at -20℃ for later use (or store at 4℃ for short-term storage).

[0477] Table 4-2. cDNA Synthesis Reaction System

[0478] (3) RT-qPCR reaction system

[0479] Table 4-3. RT-qPCR probe method reaction system

[0480] exist Quantitative real-time PCR was performed. To calculate relative fold changes, the ΔΔCt method was used to analyze the data, and the analysis was standardized for the MOCK group.

[0481] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0482] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0483] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0484] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0485] Using the control group as a baseline, the expression level of ANGPTL3 mRNA in the test group was normalized, and the expression level of ANGPTL3 mRNA in the control group was defined as 100%.

[0486] The relative expression level of ANGPTL3 mRNA in the test group was 2^(-ΔΔCt) (test group).

[0487] For the same test group siRNA, the average relative expression level of ANGPTL3 mRNA at each concentration is the arithmetic mean of the relative expression levels of the three culture wells at that concentration.

[0488] The inhibition rate of siRNA on ANGPTL3 mRNA expression was calculated using the following equation: Inhibition rate = (1 - relative expression level of ANGPTL3 mRNA in the test group) × 100%.

[0489] The results are shown in Table 4-4.

[0490] Table 4-4 Relative expression levels of ANGPTL3 mRNA (IC50)

[0491] Experimental Example 5: In vivo screening in animals

[0492] The modified siRNA synthesized in Example 1 was screened in animals.

[0493] Female C57BL / 6J-hANGPTL3 humanized mice (8-10 weeks old) were randomly divided into groups of three according to body weight. Serum Angptl3 protein levels were collected one day before drug administration as a baseline. Animals then received a single subcutaneous injection of either 1 mg / kg or 3 mg / kg of the modified siRNA drug, with saline as a control. Blood was collected from C57BL / 6J-hANGPTL3 humanized mice on days 13, 20, 27, 34, 41, 48, 55, and 62 post-administration. Human ANGPTL3 protein levels were measured using a human ANGPTL3-specific ELISA kit according to the manufacturer's instructions (IBL America #27412). ANGPTL3 protein level normalization was performed by dividing the ANGPTL3 protein level of each animal at a given time point by its pre-treatment expression level (in this case, day-1) to determine the "normalized to pre-treatment" expression ratio. Expression at specific time points was then normalized to the saline control group by dividing the ratio of "normalized to pre-treatment" for each individual animal by the average "normalized to pre-treatment" ratio of all mice in the saline control group. This resulted in expression at each time point being normalized to the control group.

[0494] The results are shown in Table 5.

Claims

1. A double stranded RNA molecule, wherein the double stranded RNA molecule comprises a sense strand and an antisense strand forming at least in part a double stranded region, the antisense strand comprising the nucleotide sequence of any even numbered SEQ ID NO. 1-64, the sense strand comprising the nucleotide sequence of any odd numbered SEQ ID NO. 1-64.

2. The double stranded RNA molecule according to any one of claim 1, wherein the length of the sense strand is not more than 30 nucleotides, and / or the length of the antisense strand is not more than 30 nucleotides, preferably, the length of the sense strand is not more than 21 nucleotides, and / or the length of the antisense strand is not more than 23 nucleotides.

3. The double stranded RNA molecule according to claim 1 or 2, wherein the sense strand and the antisense strand of the double stranded RNA molecule comprise or are any combination selected from the group consisting of: the sense strand set forth in SEQ ID NO. 41 and the antisense strand set forth in SEQ ID NO. 42, the sense strand set forth in SEQ ID NO. 43 and the antisense strand set forth in SEQ ID NO. 44, the sense strand set forth in SEQ ID NO. 59 and the antisense strand set forth in SEQ ID NO. 60, the sense strand set forth in SEQ ID NO. 1 and the antisense strand set forth in SEQ ID NO. 2, the sense strand set forth in SEQ ID NO. 3 and the antisense strand set forth in SEQ ID NO. 4, the sense strand set forth in SEQ ID NO. 5 and the antisense strand set forth in SEQ ID NO. 6, the sense strand set forth in SEQ ID NO. 7 and the antisense strand set forth in SEQ ID NO. 8, the sense strand set forth in SEQ ID NO. 9 and the antisense strand set forth in SEQ ID NO. 10, the sense strand set forth in SEQ ID NO. 11 and the antisense strand set forth in SEQ ID NO. 12, the sense strand set forth in SEQ ID NO. 13 and the antisense strand set forth in SEQ ID NO. 14, the sense strand set forth in SEQ ID NO. 15 and the antisense strand set forth in SEQ ID NO. 16, the sense strand set forth in SEQ ID NO. 17 and the antisense strand set forth in SEQ ID NO. 18, the sense strand set forth in SEQ ID NO. 19 and the antisense strand set forth in SEQ ID NO. 20, the sense strand set forth in SEQ ID NO. 21 and the antisense strand set forth in SEQ ID NO. 22, the sense strand set forth in SEQ ID NO. 23 and the antisense strand set forth in SEQ ID NO. 24, the sense strand set forth in SEQ ID NO. 25 and the antisense strand set forth in SEQ ID NO. 26, the sense strand set forth in SEQ ID NO. 27 and the antisense strand set forth in SEQ ID NO. 28, the sense strand set forth in SEQ ID NO. 29 and the antisense strand set forth in SEQ ID NO. 30, the sense strand set forth in SEQ ID NO. 31 and the antisense strand set forth in SEQ ID NO. 32, the sense strand set forth in SEQ ID NO. 33 and the antisense strand set forth in SEQ ID NO. 34, the sense strand set forth in SEQ ID NO. 35 and the antisense strand set forth in SEQ ID NO. 36, the sense strand set forth in SEQ ID NO. 37 and the antisense strand set forth in SEQ ID NO. 38, the sense strand set forth in SEQ ID NO. 39 and the antisense strand set forth in SEQ ID NO. 40, the sense strand set forth in SEQ ID NO. 45 and the antisense strand set forth in SEQ ID NO. 46, the sense strand set forth in SEQ ID NO. 47 and the antisense strand set forth in SEQ ID NO. 48, the sense strand set forth in SEQ ID NO. 49 and the antisense strand set forth in SEQ ID NO. 50, the sense strand set forth in SEQ ID NO.51, and a reverse strand as set forth in SEQ ID NO. 52, a forward strand as set forth in SEQ ID NO. 53, and a reverse strand as set forth in SEQ ID NO. 54, a forward strand as set forth in SEQ ID NO. 55, and a reverse strand as set forth in SEQ ID NO. 56, a forward strand as set forth in SEQ ID NO. 57, and a reverse strand as set forth in SEQ ID NO. 58, a forward strand as set forth in SEQ ID NO. 61, and a reverse strand as set forth in SEQ ID NO. 62, a forward strand as set forth in SEQ ID NO. 63, and a reverse strand as set forth in SEQ ID NO.

64.

4. A modified double stranded RNA molecule comprising the double stranded RNA molecule according to any one of claims 1-3, and wherein at least one nucleotide is chemically modified, preferably, the chemical modification comprises substitution of the ribosyl 2' position hydroxyl group of a nucleotide with another group, and / or modification of the base on a nucleotide, and / or substitution of the ribosyl 5' position hydroxyl group of a nucleotide with another group, and / or modification of the phosphodiester to phosphorothioate diester group between nucleotides, further preferably, the chemically modified nucleotide is selected from at least one of the group consisting of: 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 5'-(E)-vinyl phosphonate modified nucleotide, nucleotide comprising a phosphorothioate group, 2'-methyl modified nucleotide, deoxy-nucleotide, 2'-deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, non-locked nucleotide, conformationally restricted nucleotide, restricted ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-0-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-0-alkyl-modified nucleotide, morpholino nucleotide, nucleotide comprising a non-natural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, glycerol nucleotide (GNA), unlinked nucleotide (UNA), nucleotide comprising a methylphosphonate group, nucleotide comprising a 5'-phosphate, and nucleotide comprising a 5'-phosphate mimic.

5. The modified double stranded RNA molecule according to claim 4, wherein the modified double stranded RNA molecule has any one of the following modification patterns: (1) in the direction from 5' end to 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotide at position 1 of the antisense strand is a 2'-methoxy modified and 5'-(E)-vinyl phosphate modified nucleotide, the nucleotides at positions 3-5, 7-13, 15, 17-21 are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (2) in the direction from 5' end to 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-5, 7-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (3) in the direction from 5' end to 3' end, the nucleotides at positions 1-4, 6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5, 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-5, 7, 10-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 8-9, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (4) in the direction from 5' end to 3' end, the nucleotides at positions 1-4, 6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5, 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-5, 7-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (5) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-2, 4, 6, 10, 12, 14, 16, 18 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 3, 5, 7-9, 11, 13, 15, 17, 19 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 4, 6, 8, 10, 14, 16, 18 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (6) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5, 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-5, 7, 10-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 8-9, 10, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (7) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (8) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-4, 6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5, 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 14, 16 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (9) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotides at positions 1, 3-4, 6, 8-9, 11, 13, 15, 17, 19, 21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 10, 12, 14, 16, 18, 20 are 2'-fluoro modified nucleotides, the nucleotides at positions 5 and 7 are deoxyribonucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage; (10) in the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are connected by phosphorothioate linkage; the nucleotide at position 1 of the antisense strand is a 2'-methoxy modified and 5'-(E)-vinyl phosphate modified nucleotide, the nucleotides at positions 3-4, 6, 8-9, 11, 13, 15, 17, 19, 21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 10, 12, 14, 16, 18, 20 are 2'-fluoro modified nucleotides, the nucleotides at positions 5 and 7 are deoxyribonucleotides, the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are connected by phosphorothioate linkage.

6. The modified double-stranded RNA molecule of claim 4 or 5, wherein the double-stranded RNA molecule is selected from any one of the following: (1) the sense strand is: GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65), the antisense strand is: VPAmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 71); (2) the sense strand is: GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67), the antisense strand is: VPUmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 72); (3) the sense strand is: CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69), the antisense strand is: VPAmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 73); (4) the sense strand is: GmsAmsAmGmAmAmCfUfAfCmAmUmAmUmAmAmAmCmUm (SEQ ID NO. 65), the antisense strand is: AmsGfsUmUmUmAfUmAmUmGmUmAmGmUfUmCfUmUmCmsUmsCm (SEQ ID NO. 66); (5) the sense strand is: GmsAmsAmCmUmAmCfAfUfAmUmAmAmAmCmUmAmCmAm (SEQ ID NO. 67), the antisense strand is: UmsGfsUmAmGmUfUmUmAmUmAmUmGmUfAmGfUmUmCmsUmsUm (SEQ ID NO. 68); (6) the sense strand is: CmsAmsAmAmAmUmGfGfAfAmGmGmUmUmAmUmAmCmUm (SEQ ID NO. 69), the antisense strand is: AmsGfsUmAmUmAfAmCmCmUmUmCmCmAfUmUfUmUmGmsAmsGm (SEQ ID NO. 70).

7. The double-stranded RNA molecule according to any one of claims 1-3, or the modified double-stranded RNA molecule according to any one of claims 4-6, wherein the 3'-terminus of the sense strand is coupled with a compound of the following formula:

8. A pharmaceutical composition comprising the double-stranded RNA molecule of any one of claims 1-3 or the modified double-stranded RNA molecule of any one of claims 4-7, and a pharmaceutically acceptable carrier.

9. Use of the double-stranded RNA molecule of any one of claims 1-3 or the modified double-stranded RNA molecule of any one of claims 4-7 or the pharmaceutical composition of claim 8 in any of: D1) the manufacture of a medicament for inhibiting expression of an ANGPTL3 gene; D2) inhibiting expression of an ANGPTL3 gene; D3) treating a disease associated with an ANGPTL3 gene target; D4) the manufacture of a medicament for treating a disease associated with an ANGPTL3 gene target.

10. A method of treating a disease associated with an ANGPTL3 gene target, comprising administering to a subject a therapeutically effective amount of the double-stranded RNA molecule of any one of claims 1-3 or the modified double-stranded RNA molecule of any one of claims 4-7 or the pharmaceutical composition of claim 8.

11. A method of inhibiting expression of an ANGPTL3 gene in a cell, the method comprising: contacting the cell with the double-stranded RNA molecule of any one of claims 1-3 or the modified double-stranded RNA molecule of any one of claims 4-7 or the pharmaceutical composition of claim 8; maintaining the cell for a period of time sufficient to achieve degradation of mRNA transcript of the ANGPTL3 gene to inhibit expression of the ANGPTL3 gene in the cell, preferably the cell is in a subject or in vitro, more preferably the subject is afflicted with a disease associated with an ANGPTL3 gene target.

12. Use according to claim 9, or method according to claim 10 or 11, wherein the ANGPTL3 gene target related disease is selected from one or more of the group consisting of: hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic related diseases.

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