RNAi agent for inhibiting expression of diacylglycerol-O-acyltransferase 2 and application thereof

Abstract

The invention relates to an RNAi agent for inhibiting DGAT2 gene expression in a cell and a pharmaceutical composition thereof. And the therapeutic use of the RNAi agent.

Description

RNAi agent for inhibiting diacylglycerol-O-acyltransferase 2 expression and its application Technical Field

[0001] The present invention relates to an RNAi agent for inhibiting the expression of diacylglycerol-O-acyltransferase 2 (DGAT2) gene in cells and a pharmaceutical composition thereof. The present invention also relates to therapeutic uses of the RNAi agent. Background Art

[0002] In mammals, metabolic energy is stored in adipose tissue as triacylglycerol (TG). Diacylglycerol-O-acyltransferase 2 (DGAT2) catalyzes the final step in TG synthesis. Previous studies have shown that DGAT2-knockout mice die within hours of birth due to TG levels falling below 90% of normal levels and a lack of a skin fat barrier, demonstrating that DGAT2 plays a key role in catalyzing TG synthesis. The protein encoded by the DGAT2 gene is highly expressed in the liver and white adipose tissue. Studies have shown that excessive TG accumulates in pancreatic islets, toxic to pancreatic β cells, and subsequently causes secondary insulin secretion impairment and insulin resistance. Elevated TG levels lead to increased lipid droplet synthesis and storage, resulting in abnormal lipid metabolism. Elevated triglyceride levels significantly increase the risk of cardiovascular disease and are closely associated with the development and progression of atherosclerosis. Increased triglycerides may also be a significant risk factor for retinal hard exudates, macular degeneration, and proliferative retinopathy, and may increase the risk of albuminuria and diabetic nephropathy. Previous studies have shown that inhibiting DGAT2 expression in obese mouse models can improve liver steatosis and blood lipid levels.

[0003] Targeting the DGAT2 gene with siRNAs can effectively and specifically inhibit the target gene and protein, reducing triglycerides, improving insulin sensitivity, and reducing hepatic steatosis, thereby providing beneficial effects in the prevention or treatment of metabolic and cardiovascular diseases, such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type 2 diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis (NAH), non-alcoholic fatty liver disease (NAFLD), homozygous or heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related diseases. Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are particularly recognized as potentially fatal diseases leading to cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Currently, no small nucleic acid drugs targeting this target are available on the market, and the development of such drugs with improved efficacy, long-lasting effects, specific targeting, and / or safety is still needed.

[0004] Summary of the Invention

[0005] One aspect of the present invention provides an RNAi agent for inhibiting the expression of the diacylglycerol-O-acyltransferase 2 (DGAT2) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NOs: 17 to 30 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0006] In some embodiments, the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.

[0007] In some embodiments, the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.

[0008] In some embodiments, the RNAi agent comprises one or two blunt ends, preferably one blunt end.

[0009] In some embodiments, the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

[0010] In some embodiments, the overhang is located at the 3' end of the sense strand, the 3' end of the antisense strand, or at both the 3' end of the sense strand and the 3' end of the antisense strand; preferably, the overhang is located at the 3' end of the antisense strand, and further preferably, the RNAi agent has a blunt end.

[0011] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NOs: 1 to 14 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0012] In some embodiments, the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 17 to 30; the sense strand has no more than 21 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 1 to 14.

[0013] In some embodiments, in the RNAi agent:

[0014] The sense strand comprises or is the sequence set forth in SEQ ID NO: 1, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0015] The sense strand comprises or is the sequence set forth in SEQ ID NO: 2, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 18, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0016] The sense strand comprises or is the sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0017] The sense strand comprises or is the sequence set forth in SEQ ID NO:4, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:20, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0018] The sense strand comprises or is the sequence set forth in SEQ ID NO:5, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:21, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0019] The sense strand comprises or is the sequence set forth in SEQ ID NO:6, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:22, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0020] The sense strand comprises or is the sequence set forth in SEQ ID NO:7, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:23, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0021] The sense strand comprises or is the sequence set forth in SEQ ID NO:8, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:24, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0022] The sense strand comprises or is the sequence set forth in SEQ ID NO:9, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:25, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0023] The sense strand comprises or is the sequence set forth in SEQ ID NO: 10, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 26, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0024] The sense strand comprises or is the sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 27, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0025] The sense strand comprises or is the sequence set forth in SEQ ID NO: 12, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 28, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0026] The sense strand comprises or is the sequence shown in SEQ ID NO: 13, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 29, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; or

[0027] The sense strand comprises or is the sequence shown in SEQ ID NO: 14, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 30, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0028] In some embodiments, the RNAi agent comprises duplex 1002, 1003, 1004, 1005, 1008, 1018, 1023, 1024, 1029, 1035, 1044, 1068, 1069, or 1072.

[0029] In some embodiments, the sense strand and / or antisense strand of the RNAi agent comprises at least one modified nucleotide independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), open circle nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl 5'-phosphate mimetic, and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising phosphorothioate internucleotide linkages, and combinations thereof; and / or preferably, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified.

[0030] In some embodiments, in the RNAi agent, in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

[0031] In some embodiments, in the RNAi agent, the antisense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: (i) between the first and second nucleotides at the 5' end of the antisense strand; (ii) between the second and third nucleotides at the 5' end of the antisense strand; (iii) between the first and second nucleotides at the 3' end of the antisense strand; and (iv) between the second and third nucleotides at the 3' end of the antisense strand.

[0032] In some embodiments, in the RNAi agent, in the 5' to 3' direction, the 7th and 9th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0033] In some embodiments, the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present between (i) the first and second nucleotides at the 5' end of the sense strand; and / or (ii) the second and third nucleotides at the 5' end of the sense strand.

[0034] In some embodiments, the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

[0035] In some embodiments, the antisense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 34, 35, 36, 39, 40, 41, 44, 45, 46, 56, 57, 58, 60, 61 or 62, or a nucleotide sequence that differs from each of them by 1, 2 or 3 nucleotides.

[0036] In some embodiments, the sense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 33, 37, 38, 42, 43, 47, 64 or 31, or a nucleotide sequence that differs from each of them by 1, 2 or 3 nucleotides.

[0037] In some embodiments, in the RNAi agent:

[0038] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 34, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0039] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0040] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0041] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0042] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0043] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0044] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0045] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0046] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:42, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0047] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:42, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0048] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:44, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0049] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:45, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0050] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0051] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:47, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:45, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0052] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:47, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0053] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:56, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0054] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:57, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0055] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:58, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0056] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:31, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:60, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0057] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0058] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 31, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 62, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0059] In some embodiments, the RNAi agent comprises duplex 1002-60, 1002-10, 1002-11, 1002-34, 1002-35, 1003-60, 1003-10, 1003-11, 1003-34, 1003-35, 1029-60, 1029-10, 1029-11, 1029-34, 1029-35, 1008-60, 1008-10, 1008-11, 1023-60, 1023-10, or 1023-11.

[0060] In some embodiments, the RNAi agent further includes a ligand targeting hepatocytes, preferably, the ligand includes a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, further preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further includes a ligand targeting non-hepatocytes, preferably, the ligand contains a lipophilic monomer, and the lipophilic monomer is preferably Y132 to Y135, Y158, Y165 to Y168, L10, L57, L321, L322, Q361 to Q367, Q361s to Q367s, Q370, Q377 to Q379, or Q383.

[0061] In some embodiments, the ligand comprises a structure as shown in formula (I):

[0062] in,

[0063] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0064] A or C is independently 0 or an integer between 1 and 14, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0065] B is 0 or an integer between 1 and 12, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0066] The wavy lines represent the location of attachment to the rest of the RNAi agent, wherein the ligand is attached to the 5' and / or 3' end of the sense and / or antisense strand; preferably, the ligand is attached to the 5' and / or 3' end of the sense strand; more preferably, the ligand is attached to the 3' end of the sense strand.

[0067] In some embodiments, the structure of the ligand is as shown in formula (II) or formula (III):

[0068] in,

[0069] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0070] Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -S-, -SS-, wherein R1 is an alkyl group containing 1-6 (e.g., 1, 2, 3, 4, 5, or 6) carbon atoms or hydrogen;

[0071] W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0072] The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand;

[0073] A, C or F is 0 or an integer between 1 and 14, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14;

[0074] B or E is 0 or an integer between 1 and 12, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0075] D is an integer between 1 and 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0076] in,

[0077] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0078] Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3 (e.g., 1, 2 or 3), q is an integer from 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9 or 10); j is 0 or 1;

[0079] W does not exist or is The wavy line on the right indicates the position where it is connected to the sense strand or antisense strand;

[0080] The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand;

[0081] A or C is 0 or an integer between 1 and 14 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14);

[0082] B is 0 or an integer between 1 and 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0083] In some embodiments, the structure of the ligand is as shown in Formula (IV) or Formula (V):

[0084] The wavy line represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand; preferably, the ligand is attached to the 3' end of the sense strand; wherein the targeting ligand is attached to the sense strand or antisense strand via a phosphate group, a phosphorothioate group, or a phosphonate group.

[0085] In some embodiments, the 3' end of the sense strand of the RNAi agent is linked to a ligand and comprises or is a nucleotide sequence shown in SEQ ID NO: 50, 51, 52, 53, 54, 55, 59 or 15, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

[0086] In some embodiments, in the RNAi agent:

[0087] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:34, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0088] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0089] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0090] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:51, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0091] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:51, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0092] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:39, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0093] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0094] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0095] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0096] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0097] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:54, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:44, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0098] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:54, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0099] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:56, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0100] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:57, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0101] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:58, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0102] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:60, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0103] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0104] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:62, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0105] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 15, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 61, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0106] In some embodiments, the RNAi agent comprises or is duplex 1002-60-001, 1002-10-001, 1002-11-001, 1002-34-001, 1002-35-001, 1003-60-001, 1003-10-001, 1003-11-001, 1003-34-001, 1003-35-001, 1029-60-001, 1029-11-001, 1008-60-001, 1008-10-001, 1008-11-001, 1023-60-001, 1023-10-001, 1023-10-L96, or 1023-11-001.

[0107] Another aspect of the present invention provides a pharmaceutical composition comprising any RNAi agent of the present invention and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

[0108] Another aspect of the present invention provides the use of any one of the RNAi agents of the present invention in the preparation of the following drugs:

[0109] (i) a drug for reducing the expression level of DGAT2 in cells;

[0110] (ii) a drug for preventing or treating a disease caused by increased DGAT2 expression level; or

[0111] (iii) Medicaments for the prevention or treatment of metabolic diseases, such as 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 or heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia.

[0112] Accordingly, the present invention also provides a method for reducing the expression level of DGAT2 in a cell, comprising administering a therapeutically effective amount of any RNAi agent of the present invention to a subject in need thereof. The present invention also provides a method for preventing or treating a disease caused by elevated DGAT2 expression levels, comprising administering a therapeutically effective amount of any RNAi agent of the present invention to a subject in need thereof. The present invention also provides a method for preventing or treating a metabolic disease, comprising administering a therapeutically effective amount of any RNAi agent of the present invention to a subject in need thereof. The metabolic disease is, for example, one or more of hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia. The present invention also provides the use of any RNAi agent of the present invention in the preparation of a medicament for preventing or treating the above-mentioned metabolic diseases. The present invention also provides any RNAi agent of the present invention for use in preventing or treating the above-mentioned metabolic diseases.

[0113] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] FIG1 shows the changes in the average triglyceride (TG) content in the liver of mice before and after administration.

[0115] FIG2 shows the inhibition efficiency of different groups on target gene mRNA expression levels. DETAILED DESCRIPTION

[0116] definition

[0117] Diacylglycerol-O-Acyltransferase 2 (DGAT2), also known as ARAT, GS1999FULL, HMFN1045, refers herein to the DGAT2 protein or its encoding gene. The human DGAT2 gene is located on chromosome 11q13.5, Gene ID: 84649. This gene encodes one of the enzymes that catalyzes the final reaction in triglyceride synthesis, in which diacylglycerol is covalently bound to a long-chain fatty acyl-CoA. The encoded protein catalyzes this reaction under low magnesium chloride concentrations. Multiple transcript variants of this gene have been identified, encoding different protein isoforms. Exemplary NCBI reference sequence numbers for human DGAT2 mRNA transcripts include NM_032564.5, NM_001253891.2, XM_011545304.3, XM_047427716.1, XM_054370204.1, and XM_054370205.1. Exemplary NCBI reference sequence numbers for human DGAT2 proteins include NP_115953.2, NP_001240820.1, XP_011543606.1, XP_047283672.1, XP_054226179.1, and XP_054226180.1.

[0118] As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that is capable of downregulating the expression of a target gene (herein, the DGAT2 gene) through an RNA interference mechanism when introduced into a cell. The term "RNAi agent of the present invention," "RNAi agent described herein," or similar expressions includes both modified RNAi agents of the present invention, regardless of sequence and target gene, and RNAi agents of the present invention having a specific sequence for interfering with the DGAT2 gene. RNA interference refers to a process in which a nucleic acid molecule induces the cutting and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, such as through an RNA-induced silencing complex (RISC) pathway. RNAi agents herein include siRNA, shRNA, and DNA / RNA hybrid molecules, sometimes also collectively referred to herein as double-stranded RNA (dsRNA), which comprise two antiparallel continuous nucleotide chains that are sufficiently complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically through hydrogen bonds (e.g., Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds, or reversed Hoogsteen hydrogen bonds) between complementary bases in the two polynucleotides. "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that hybridize to form base pairs, thereby forming a double strand between the two polynucleotide chains.

[0119] The term "antisense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the target sequence. The term "positive strand" or "sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, for example, 5, 4, 3, or 2 at the 5' and / or 3' ends of the dsRNA.

[0120] "siRNA" refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNAs are typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length.

[0121] shRNA refers to a short hairpin RNA that includes two short inverted repeats and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. The stem-loop may be 10 or fewer nucleotides. The stem-loop may be 8 or fewer unpaired nucleotides. The stem-loop may be 4 to 10 unpaired nucleotides. The stem-loop may be 4 to 8 nucleotides.

[0122] The two substantially complementary chains of a dsRNA need not but may also be covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA may also comprise one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides that extend beyond the double-stranded region at the end of the chain. When the 3' end of a chain extends beyond the 5' end of the other chain, or when the 5' end of a line extends beyond the 3' end of the other line, nucleotide overhangs are usually produced. For example, at least one chain comprises a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. For another example, at least one chain comprises a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. In other embodiments, both the 3' end and the 5' end of a chain of the dsRNA comprise an overhang of at least 1 nucleotide.

[0123] As used herein, the terms "blunt-ended" or "blunt-ended" with respect to dsRNA refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt-ended, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with both ends blunt-ended, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded throughout their entire length. As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0124] The dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose, bases or backbone components of ribonucleic acid, as described herein or modifications known in the art. Any such modification, as used in double-stranded ribonucleic acid molecules (such as siRNA, shRNA, etc.), is encompassed by the term "dsRNA" for the purposes of this disclosure. "Modified" nucleotides refer to nucleotides independently having modified sugar moieties, modified internucleotide linkages and / or modified core bases. Therefore, the term "modified nucleotides" includes substitutions, additions or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties or core bases. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art. For example, the modified nucleotides are selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), open ring nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl The present invention relates to a nucleotide modified by the present invention, wherein the nucleotide modified by the present invention comprises a base-modified nucleotide, an abasic nucleotide, an inverted abasic nucleotide, an inverted nucleotide, a morpholino nucleotide, a phosphoramidate, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a 5'-phosphate-containing nucleotide, and a nucleotide containing a 5'-phosphate mimetic; preferably selected from 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, and nucleotides containing phosphorothioate bonds between nucleotides.

[0125] The term "ligand" refers to a cell or tissue targeting agent that binds to a specified cell type (e.g., hepatocytes), such as a lectin, glycoprotein, lipid, or protein (e.g., an antibody). Exemplary targeting agents include thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (e.g., divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, and RGD peptide mimetics. In a preferred embodiment, the ligand is a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. For example, the ligand can be a derivative comprising GalNAc. In a preferred embodiment, the ligand comprises one or more N-acetylgalactosamine derivatives attached via a bivalent or trivalent branched linker.

[0126] The term "therapeutically effective amount" refers to an amount of a RNAi agent of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0127] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0128] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or delivering a RNAi agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

[0129] The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need thereof, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0130] RNAi agents for inhibiting DGAT2 gene expression

[0131] One aspect of the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and a nucleotide sequence that differs from each of these by 1, 2, or 3 nucleotides.

[0132] In the present invention, when referring to 1, 2, or 3 nucleotide differences, the 1, 2, or 3 nucleotide differences may be located on the sense strand and / or antisense strand outside the double-stranded region. In some embodiments, the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region. In some embodiments, a portion of the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region, and another portion is located on the sense strand and / or antisense strand outside the double-stranded region. With respect to the antisense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most end and the 5'-most end of the antisense strand. In some embodiments, one or two of the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand, and the other one or two are located between the 3'-most end and the 5'-most end of the antisense strand. For the sense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most or 5'-most end of the sense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most and 5'-most ends of the sense strand. In some embodiments, one or two of the 1, 2, or 3 nucleotide differences are located at the 3'-most or 5'-most end of the sense strand, and the other one or two are located between the 3'-most and 5'-most ends of the sense strand.

[0133] In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having one nucleotide difference with any one of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), wherein the one nucleotide difference is located at the extreme 3' end of the antisense strand. In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having two nucleotide differences from any one of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and the two nucleotide differences are consecutively located at the 3'-most end of the antisense strand.

[0134] In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no more than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30).

[0135] In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30).

[0136] One aspect of the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30) and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0137] In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30).

[0138] In some embodiments, the present invention provides an RNAi agent for inhibiting DGAT2 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of the nucleotide sequences of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30).

[0139] In some embodiments, the length of the double-stranded region is about 17 to about 23 base pairs. For example, a double-stranded region of suitable length is about 17 to about 22 base pairs, about 17 to about 21 base pairs, about 17 to about 20 base pairs, about 17 to about 19 base pairs, about 17 to about 18 base pairs, about 18 to about 23 base pairs, about 18 to about 22 base pairs, about 18 to about 21 base pairs, about 18 to about 20 base pairs, about 19 to 23 base pairs, about 19 to 22 base pairs, about 19 to 21 base pairs, about 20 to 23 base pairs, about 20 to 22 base pairs, or about 21 to 23 base pairs. In certain embodiments, the length of the double-stranded region is about 18 to about 21 base pairs. In other embodiments, the length of the double-stranded region is about 19 base pairs.

[0140] Therefore, in some embodiments of the present invention, an RNAi agent for inhibiting DGAT2 gene expression is provided, which comprises a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 17 to about 23 base pairs in length, and the antisense strand being no longer than 23 nucleotides and comprising at least 15 consecutive nucleotides selected from the group consisting of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30) and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0141] In some embodiments of the present invention, an RNAi agent for inhibiting DGAT2 gene expression is provided, comprising a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 18 to about 21 base pairs in length, and the antisense strand being no more than 23 nucleotides in length and comprising at least 15 consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30) and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0142] In some embodiments of the present invention, an RNAi agent for inhibiting DGAT2 gene expression is provided, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the double-stranded region is about 19 base pairs in length, and the antisense strand is no more than 23 nucleotides in length and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0143] In some embodiments, the length of the sense strand and the antisense strand in the RNAi agent of the invention is each independently about 17 to about 23 nucleotides, such as about 18 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 17 to about 22 nucleotides, about 17 to about 21 nucleotides, about 17 to about 20 nucleotides, about 17 to about 19 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 19 to about 22 nucleotides, about 19 to about 21 nucleotides, or about 20 to about 22 nucleotides. In certain embodiments, the length of the sense strand and the antisense strand is each independently about 17, about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides.

[0144] In some embodiments, the sense and antisense strands are of the same length but form a double-stranded region that is shorter than the strand, such that the RNAi agent for inhibiting DGAT2 gene expression has a two-nucleotide overhang. For example, in one embodiment, the RNAi agent for inhibiting DGAT2 gene expression comprises (i) a sense and antisense strand each of 21 nucleotides in length, (ii) a double-stranded region of 19 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi agent for inhibiting DGAT2 gene expression comprises (i) a sense and antisense strand each of 23 nucleotides in length, (ii) a double-stranded region of 21 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

[0145] In other embodiments, the sense and antisense strands are of the same length and form a double-stranded region over their entire length, such that no nucleotides protrude from either end of the double-stranded molecule. In one such embodiment, the RNAi agent for inhibiting the expression of the DGAT2 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region of 21 base pairs in length. In another such embodiment, the RNAi agent for inhibiting the expression of the DGAT2 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region of 23 base pairs in length. In another such embodiment, the RNAi agent for inhibiting the expression of the DGAT2 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region of 19 base pairs in length.

[0146] In other embodiments, the sense strand or the antisense strand is longer than the other strand, and the two strands form a double-stranded region with a length equal to the length of the shorter strand, such that the RNAi agent for inhibiting DGAT2 gene expression includes at least one nucleotide overhang. For example, in some embodiments, the sense strand is 1 to 4 nucleotides longer than the antisense strand, and the double-stranded region formed by the two strands is equal to the length of the antisense strand, such that the sense strand forms an overhang with 1 to 4 unpaired nucleotides. In other embodiments, the antisense strand is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region formed by the two strands is equal to the length of the sense strand, such that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the length of the nucleotide overhang is 1, 2, 3, or 4 nucleotides. In a specific embodiment, the overhang includes 2 nucleotides. In certain embodiments, the overhang includes a single nucleotide.

[0147] The nucleotides that protrude can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides that protrude are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides) or combinations thereof. For example, in one embodiment, the nucleotides that protrude are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides that protrude are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides or combinations thereof. In other embodiments, the protrusion comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can include a ribonucleotide or modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the protrusion comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When there is a nucleotide overhang in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (such as UU, TT, AA, GG, etc.).

[0148] The nucleotide overhang may be at the 5' or 3' end of one or both strands. For example, in one embodiment, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises nucleotide overhangs at both the 5' and 3' ends of the antisense strand. In another embodiment, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises nucleotide overhangs at both the 5' and 3' ends of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises nucleotide overhangs at both the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises nucleotide overhangs at both the 3' end of the sense strand and the 3' end of the antisense strand. In some embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of a DGAT2 gene comprises only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting DGAT2 gene expression includes only a nucleotide overhang at the 5' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting DGAT2 gene expression includes only a nucleotide overhang at the 5' end of the sense strand.

[0149] The RNAi agent used to inhibit the expression of the DGAT2 gene can include a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent used to inhibit the expression of the DGAT2 gene includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi agent used to inhibit the expression of the DGAT2 gene includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand.

[0150] Specifically, for example, in one embodiment, the RNAi agent for inhibiting the expression of the DGAT2 gene comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the length of the sense strand. In another embodiment, the RNAi agent for inhibiting the expression of the DGAT2 gene comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the length of the sense strand.

[0151] In some embodiments, an RNAi agent for inhibiting DGAT2 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being no longer than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the RNAi agent comprises an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides.

[0152] In some embodiments, a RNAi agent for inhibiting DGAT2 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being no longer than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, and the RNAi agent comprising an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0153] In some embodiments, an RNAi agent for inhibiting DGAT2 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the RNAi agent comprises an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0154] In some embodiments, an RNAi agent for inhibiting DGAT2 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no more than 23 nucleotides in length and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, the sense strand is 19 to 21 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0155] In some embodiments, an RNAi agent for inhibiting DGAT2 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0156] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is any one nucleotide sequence selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30), the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0157] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30), and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from any one of SEQ ID NOs: 1 to 14 (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14), and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0158] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30) and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand having no more than 21 nucleotides and comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 14 (SEQ ID NOs: 1 to 15). NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14) and at least 15 (e.g., 15, 16, 17, 18 or 19) consecutive nucleotides of any nucleotide sequence of the present invention and a nucleotide sequence that has 1, 2 or 3 nucleotide differences therefrom.

[0159] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 17 to 30 (SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30), and the sense strand having no more than 21 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 14 (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14).

[0160] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein:

[0161] The sense strand comprises or is the sequence set forth in SEQ ID NO: 1, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0162] The sense strand comprises or is the sequence set forth in SEQ ID NO: 2, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 18, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0163] The sense strand comprises or is the sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0164] The sense strand comprises or is the sequence set forth in SEQ ID NO:4, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:20, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0165] The sense strand comprises or is the sequence set forth in SEQ ID NO:5, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:21, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0166] The sense strand comprises or is the sequence set forth in SEQ ID NO:6, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:22, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0167] The sense strand comprises or is the sequence set forth in SEQ ID NO:7, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:23, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0168] The sense strand comprises or is the sequence set forth in SEQ ID NO:8, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:24, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0169] The sense strand comprises or is the sequence set forth in SEQ ID NO:9, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:25, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0170] The sense strand comprises or is the sequence set forth in SEQ ID NO: 10, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 26, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0171] The sense strand comprises or is the sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 27, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0172] The sense strand comprises or is the sequence set forth in SEQ ID NO: 12, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 28, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0173] The sense strand comprises or is the sequence shown in SEQ ID NO: 13, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 29, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; or

[0174] The sense strand comprises or is the sequence shown in SEQ ID NO: 14, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 30, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0175] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the DGAT2 gene, comprising a sense strand and an antisense strand that form complementary double-stranded regions, wherein the sense strand and the antisense strand pair to form any one of duplexes 1002, 1003, 1004, 1005, 1008, 1018, 1023, 1024, 1029, 1035, 1044, 1068, 1069, and 1072 as described herein.

[0176] Nucleotide-modified RNAi agents

[0177] For any of the embodiments of the RNAi agent of the present invention described in the section "RNAi agent for inhibiting DGAT2 gene expression", the sense strand and / or antisense strand of the RNAi agent may comprise at least one modified nucleotide.

[0178] In some embodiments, the sense strand and the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention each contain at least one modified nucleotide.

[0179] In some embodiments, each nucleotide in the sense strand and the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified.

[0180] In any of the above embodiments, the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), open circle nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, and combinations thereof.

[0181] In a preferred embodiment, each nucleotide of the sense strand and the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and the modification mode is selected from one of STC (Alnylam), ESC (Alnylam), Advanced ESC (Alnylam), ESC+ (Alnylam), AD1-3 (Arrowhead), AD5 (Arrowhead) and GalXC (Dicerna) (see, for example, Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020; 5(1): 101. Published 2020Jun 19. doi: 10.1038 / s41392-020-0207-x).

[0182] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0183] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 12, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0184] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 14 and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0185] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage exists in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand. In a preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0186] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0187] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0188] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0189] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 5, 7, and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0190] For example, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7, 9, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0191] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and between the second and third nucleotides at the 5' end of the sense strand.

[0192] In a preferred embodiment, the antisense strand has at least one phosphorothioate internucleotide linkage. Preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0193] Therefore, in some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand; in the 5' to 3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the sense strand and / or between the second and third nucleotides at the 5' end of the sense strand.

[0194] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting DGAT2 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between: the first nucleotide and the second nucleotide at the 5' end of the antisense strand, and between the second nucleotide and the third nucleotide at the 5' end of the antisense strand. , between the first nucleotide and the second nucleotide at the 3' end of the antisense strand, and between the second nucleotide and the third nucleotide at the 3' end of the antisense strand; in the 5' to 3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between the first nucleotide and the second nucleotide at the 5' end of the sense strand and between the second nucleotide and the third nucleotide at the 5' end of the sense strand.

[0195] In a preferred embodiment, the antisense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 34, 35, 36, 39, 40, 41, 44, 45, 46, 56, 57, 58, 60, 61 or 62, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

[0196] In a preferred embodiment, the sense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 33, 37, 38, 42, 43, 47, 64 or 31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

[0197] In a preferred embodiment, the antisense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 34, 35, 36, 39, 40, 41, 44, 45, 46, 56, 57, 58, 60, 61 or 62, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; and the sense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 33, 37, 38, 42, 43, 47, 64 or 31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

[0198] In some embodiments, in the RNAi agents provided herein:

[0199] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 34, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0200] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0201] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0202] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0203] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0204] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO: 39, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0205] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0206] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0207] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:42, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0208] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:42, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0209] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:44, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0210] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:45, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0211] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:43, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0212] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:47, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:45, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0213] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:47, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0214] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:56, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0215] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:57, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0216] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:58, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0217] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:31, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:60, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0218] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0219] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 31, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 62, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0220] In preferred embodiments, the RNAi agent provided herein comprises duplex 1002-60, 1002-10, 1002-11, 1002-34, 1002-35, 1003-60, 1003-10, 1003-11, 1003-34, 1003-35, 1029-60, 1029-10, 1029-11, 1029-34, 1029-35, 1008-60, 1008-10, 1008-11, 1023-60, 1023-10 or 1023-11.

[0221] Ligand-linked RNAi agents

[0222] For any of the embodiments of the RNAi agent of the present invention described in the sections "RNAi agents for inhibiting DGAT2 gene expression" and "Nucleotide-modified RNAi agents" above, the RNAi agent may comprise a ligand. As used herein, "ligand" refers to any compound or molecule that is capable of interacting directly or indirectly with another compound or molecule. The interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiating a signal transduction cascade, inducing receptor-mediated endocytosis), or may simply be a physical connection. The ligand may alter one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance of the RNA molecule.

[0223] The DGAT2 gene is primarily expressed in the liver and adipocytes. Therefore, in certain embodiments, it is desirable to specifically deliver the RNAi agent of the present invention to hepatocytes. Therefore, in certain embodiments, the ligand is targeted to specifically deliver the RNAi agent to hepatocytes using various methods described in more detail below. In certain embodiments, the RNAi agent is targeted to hepatocytes using a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0224] In some embodiments, RNAi agents can be specifically targeted to the liver by using ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may include an antigen binding protein (e.g., an antibody or its binding fragment (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as asialoglycoprotein receptors and LDL receptors. In a specific embodiment, the ligand includes an antibody or its binding fragment that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. Exemplary antibodies and their binding fragments that can be used as ligands for targeting the RNAi agents of the present invention to the liver are described in WO 2017 / 058944, which is incorporated herein by reference in its entirety. Other antibodies or their binding fragments that specifically bind to ASGR1, LDL receptor, or other liver-surface expressed proteins suitable for use as ligands in the RNAi agents of the present invention are purchased from commercial sources.

[0225] In some embodiments, when used for extrahepatic delivery, including but not limited to, the central nervous system (CNS) (e.g., brain, spine, or eye), muscle, lung, or fat, the RNAi agent may comprise one or more lipophilic monomers conjugated to one or more positions of the antisense strand and / or sense strand. For example, the lipophilic monomer may be connected to the antisense strand and / or sense strand via a nucleobase, a sugar moiety, or an internucleoside bond. In some embodiments, a lipophilic monomer having a phosphoramidite group is coupled to the 3' end or 5' end of the sense strand or antisense strand in the final synthesis cycle. In some embodiments, the lipophilic monomer has an octanol-water partition coefficient greater than 0, 1, 1.5, 2, 3, 4, 5, or 10. Suitable lipophilic monomers may be aliphatic, alicyclic, polyalicyclic, steroid, straight or branched aliphatic hydrocarbon. Exemplary lipophilic monomers are, for example, lipophilic monomers Y132 to Y135, Y158, Y165 to Y168, L10, L57, L321, L322, Q361 to Q367, Q361s to Q367s, Q370, Q377 to Q379, Q383, etc. described in WO 2021 / 092371.

[0226] In certain embodiments, part comprises carbohydrate." carbohydrate " refers to the compound that is formed by one or more monosaccharide units with at least 6 carbon atoms (can be straight chain, branched or cyclic), and each carbon atom is connected with oxygen, nitrogen or sulphur atom.Carbohydrate includes but is not limited to sugar (for example, monosaccharide, disaccharide, trisaccharide, tetrasaccharide and the oligosaccharide containing about 4,5,6,7,8 or 9 monosaccharide units) and polysaccharide (such as starch, glycogen, cellulose and polysaccharide glue). In some embodiments, the carbohydrate that is attached to the part is disaccharide and the trisaccharide that is selected from pentose, hexose or heptose and comprises such monosaccharide unit. In other embodiments, the carbohydrate that is attached to the part is amino sugar, for example galactosamine, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.

[0227] In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose can be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine can be selected from fructosamine, galactosamine, glucosamine or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine or N-acetylgalactosamine. In specific embodiments, ligands comprising glucose, galactose and N-acetylgalactosamine (GalNAc) are particularly effective in targeting RNA to hepatocytes because these ligands bind to ASGR expressed on the surface of hepatocytes. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi agents of the invention are described in USP 7,491,805, 8,106,022, and 8,877,917; US Patent Publication No. US20030130186; and WIPO Publication No. WO 2013 / 166155, all of which are incorporated herein by reference in their entirety.

[0228] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units that are capable of independently binding to or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The "valency" of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" refer to carbohydrate moieties having one, two, three, and four binding domains, respectively, with respect to a carbohydrate moiety. The multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetylgalactosamine moiety, a multivalent N-acetylglucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety can be divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bivalent or trivalent. In a specific embodiment, the multivalent N-acetylgalactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the RNAi agents of the present invention are described in detail below.

[0229] The ligand can be directly or indirectly connected or conjugated to the RNA molecule of the RNAi agent. For example, in some embodiments, the ligand is directly covalently connected to the sense strand or antisense strand of the RNAi agent. In other embodiments, the ligand is covalently connected to the sense strand or antisense strand of the RNAi agent through a linker. The ligand can be connected to the core base, sugar moiety or internucleotide junction of the sense strand or antisense strand of the RNAi agent of the present invention.

[0230] In some embodiments, the ligand can be attached to the 3' or 5' end of the sense or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached at the 5'-position of the 5'-terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3'-position of the 3'-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached to the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached to the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0231] In certain embodiments, the ligand is connected to the sense strand or antisense strand by a joint." joint " refers to an atom or a group of atoms by which a ligand is covalently connected to a polynucleotide component of an RNAi agent. The length of the joint can be about 1 to about 30 atoms, about 2 to about 28 atoms, about 3 to about 26 atoms, about 4 to about 24 atoms, about 6 to about 20 atoms, about 7 to about 20 atoms, about 8 to about 20 atoms, about 8 to about 18 atoms, and about 12 to about 18 atoms. In some embodiments, the joint can include a bifunctional linking portion, which generally includes an alkyl portion with two functional groups. One of the functional groups is selected to be combined with a compound of interest (such as the sense or antisense strand of an RNAi agent chain), while the other functional group is selected to be combined with substantially any selected group, such as a ligand as described herein. In certain embodiments, the joint includes an oligomer of a chain structure or a repeating unit, such as ethylene glycol or an amino acid unit. The example of the functional group typically used in the bifunctional linking portion includes but is not limited to an electrophilic reagent for reacting with a nucleophilic group and a nucleophilic reagent for reacting with an electrophilic group. In some embodiments, the bifunctional linking moiety includes an amino group, a hydroxyl group, a carboxylic acid, a thiol group, an unsaturated bond (eg, a double bond or a triple bond), and the like.

[0232] Linkers that can be used to attach the ligand to the sense or antisense strand of the RNAi agent of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, 6-aminohexanoic acid, substituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl or substituted or unsubstituted C2-C 20 Preferred substituents for such linkers include, but are not limited to, hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0233] In certain embodiments, joint is cleavable.Cleaving joint is a kind of enough stable outside cell but is cracked to release two parts that combine together by joint when entering target cell.In some embodiments, cleavable joint is in target cell or under the first reference condition (it can for example be selected as simulation or represent cell condition) than cracking in experimenter's blood fast at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times.

[0234] Cleavable linkers are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activity in cells. Examples of such cleavage agents include: redox agents selected for specific substrates or having no substrate specificity, including, for example, oxidases or reductases present in cells; esterases; endosomes or reagents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0235] Cleavable linkers can include pH-sensitive moieties. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0, and lysosomes have a more acidic pH (about 5.0). Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand in the cell, or releasing it into the desired organelle of the cell. The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cell to be targeted. For example, a liver targeting ligand can be connected to the RNA molecule by a linker that includes an ester group. Hepatocytes are rich in esterases, so the linker is more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Other types of cells rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases, such as hepatocytes and synovial cells, a linker containing a peptide bond can be used.

[0236] Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi agents of the invention are known in the art, such as those described in U.S. Patents 7,723,509, 8,017,762, 8,828,956, 8,877,917, and 9,181,551, all of which are incorporated herein by reference in their entirety.

[0237] In some embodiments, the ligand covalently attached to the sense strand or antisense strand of the RNAi agent of the present invention includes a GalNAc moiety, such as a multivalent GalNAc. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 5' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0238] In some embodiments, the ligand is L96, NAG25, and NAG37, each having the structural formula shown below, wherein the wavy line represents the position of attachment to the sense or antisense strand of the RNAi agent.

[0239] In some embodiments, the RNAi agent of the present invention comprises a ligand comprising a structure as shown in Formula I:

[0240] in,

[0241] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0242] A or C is independently 0 or an integer between 1 and 14 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14);

[0243] B is 0 or an integer between 1 and 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12);

[0244] The wavy lines represent the locations of attachment to the rest of the RNAi agent; wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand; preferably, the ligand is attached to the 5' and / or 3' end of the sense strand; more preferably, the ligand is attached to the 3' end of the sense strand.

[0245] In a preferred embodiment, the ligand is covalently attached to the 5' and / or 3' end of the sense strand via a linker. In one embodiment, the linker is a PEG linker.

[0246] In a preferred embodiment, the structure of the ligand is as shown in formula (II) or formula (III):

[0247] in,

[0248] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0249] Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -S-, -SS-, wherein R1 is an alkyl group containing 1-6 (e.g., 1, 2, 3, 4, 5, or 6) carbon atoms or hydrogen;

[0250] W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0251] The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand;

[0252] A, C or F is 0 or an integer between 1 and 14 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14);

[0253] B or E is 0 or an integer between 1 and 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12);

[0254] D is an integer between 1 and 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);

[0255] in,

[0256] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-,

[0257] Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3 (e.g., 1, 2 or 3), q is an integer from 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9 or 10), and j is 0 or 1;

[0258] W does not exist or is The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0259] The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand;

[0260] A or C is 0 or an integer between 1 and 14 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14);

[0261] B is 0 or an integer between 1 and 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0262] Preferably, exemplary trivalent and / or tetravalent GalNAc ligands that can be linked to double-stranded RNA molecules in the RNAi agents of the present invention are provided in Structural Formula IV or V below:

[0263] Wherein, the wavy line represents the position of connection to the sense strand or antisense strand; preferably, the ligand is connected to the 5' and / or 3' end of the sense strand; more preferably, the ligand is connected to the 3' end of the sense strand; wherein, the targeting ligand is connected to the sense strand or antisense strand via a phosphate group, a thiophosphate group or a phosphonate group.

[0264] In some embodiments, the RNAi agent of the present invention further complexes a metal cation, preferably a monovalent metal cation, preferably Na + , K+ 、Ag + ; divalent metal cation, preferably Ca 2+ Mg 2+ 、Cu 2+ 、Fe 2+ 、Zn 2+ 、Mn 2+ ; or a trivalent metal cation, preferably Fe 3+ 、Al 3+ In some embodiments, the metal cation is complexed with the nitrogen heterocyclic structure in any one of Formula II to Formula V.

[0265] For example, in one embodiment, the RNAi agent provided by the present invention has the structure shown below, wherein K+ is complexed to the nitrogen heterocyclic structure of the ligand, wherein represents the duplex portion of the RNAi agent for DGAT2 gene expression,

[0266] For the convenience of description, the ligand represented by formula (IV) is represented herein by the code name YHZY12001, and the ligand represented by formula (V) is represented herein by the code name YHZY12002.

[0267] In some embodiments, the ligand is linked to the 3' end of the sense strand, which comprises or is the nucleotide sequence shown in SEQ ID NO: 50, 51, 52, 53, 54, 55, 59 or 15, or a nucleotide sequence that differs from each of them by 1, 2 or 3 nucleotides.

[0268] In some embodiments, the ligand is linked to the 3' end of the sense strand, which comprises or is a nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 59 or 15, or a nucleotide sequence that differs from each of them by 1, 2, or 3 nucleotides; and the antisense strand comprises or is a nucleotide sequence set forth in SEQ ID NO: 34, 35, 36, 39, 40, 41, 44, 46, 56, 57, 58, 60, 61 or 62, or a nucleotide sequence that differs from each of them by 1, 2, or 3 nucleotides.

[0269] In some embodiments, the present invention provides RNAi wherein:

[0270] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:34, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0271] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0272] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:50, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0273] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:51, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:35, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0274] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:51, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:36, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0275] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:39, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0276] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0277] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0278] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:40, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0279] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:41, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0280] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:54, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:44, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0281] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:54, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:46, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0282] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:56, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0283] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:57, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0284] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:55, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:58, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0285] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:60, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0286] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0287] The sense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence set forth in SEQ ID NO:62, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0288] The sense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 15, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 61, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0289] In some embodiments, the RNAi agent provided herein comprises or is duplex 1002-60-001, 1002-10-001, 1002-11-001, 1002-34-001, 1002-35-001, 1003-60-001, 1003-10-001, 1003-11-001, 1003-34-001, 1003-35-001, 1029-60-001, 1029-11-001, 1008-60-001, 1008-10-001, 1008-11-001, 1023-60-001, 1023-10-001, 1023-10-L96, or 1023-11-001.

[0290] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO. 5: 5'-UGGUGAAGCUCUUCGACAA-3' and has a length of no more than 21 nucleotides, the antisense strand comprises SEQ ID NO. 21: 5'-UUGUCGAAGAGCUUCACCAGG-3' and has a length of no more than 23 nucleotides, each nucleotide of the sense strand and the antisense strand of the RNAi agent is modified; and the 3'-end of the sense strand is conjugated to a ligand targeting hepatocytes.

[0291] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.31: 5'-mU*mG*mGmUfGmAfAfGfCmUmCmUmUmCmGmAmCmAmA-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.57: 5'-mU*fU*mGmUfCmGfAmAmGmAmGmCmUfUmCfAmCmCmA*mG*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, and the 3'-end of the sense chain is conjugated to a hepatocyte-targeting ligand.

[0292] In the above particularly preferred embodiment, the ligand may be a structure shown in Formula IV:

[0293] It is linked to the sense strand via a phosphate group or a phosphorothioate group.

[0294] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.55: 5'-mU*mG*mGmUfGmAfAfGfCmUmCmUmUmCmGmAmCmAmA-YHZY12001-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.57: 5'-mU*fU*mGmUfCmGfAmAmGmAmGmCmUfUmCfAmCmCmA*mG*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, wherein YHZY12001 has a structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

[0295] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO. 7: 5'-CUACUUUCGAGACUACUUU-3' and is no longer than 21 nucleotides, the antisense strand comprises SEQ ID NO. 23: 5'-AAAGUAGUCUCGAAAGUAGCG-3' and is no longer than 23 nucleotides, each nucleotide of the sense strand and the antisense strand of the RNAi agent is modified; and the 3'-end of the sense strand is conjugated to a ligand targeting hepatocytes.

[0296] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.31: 5'-mC*mU*mAmCfUmUfUfCfGmAmGmAmCmUmAmCmUmUmU-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.61: 5'-mA*fA*mAmGfUmAfGmUmCmUmCmGmAfAmAfGmUmAmG*mC*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, and the 3'-end of the sense chain is conjugated to a hepatocyte-targeting ligand.

[0297] In the above particularly preferred embodiment, the ligand may be a structure shown in Formula IV:

[0298] It is linked to the sense strand via a phosphate group or a phosphorothioate group.

[0299] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting DGAT2 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.59: 5'-mC*mU*mAmCfUmUfUfCfGmAmGmAmCmUmAmCmUmUmU-YHZY12001-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.61: 5'-mA*fA*mAmGfUmAfGmUmCmUmCmGmAfAmAfGmUmAmG*mC*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, wherein YHZY12001 has a structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

[0300] Pharmaceutical composition

[0301] The present invention also includes pharmaceutical compositions and preparations comprising the RNAi agents described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and preparations can be used to reduce the expression of the DGAT2 gene in patients in need thereof. In the case of considering clinical applications, pharmaceutical compositions and preparations will be prepared in a form suitable for the intended application. Typically, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0302] The composition and method for preparing the pharmaceutical composition depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the condition or the dosage to be administered. In some embodiments, the pharmaceutical composition is prepared based on the expected route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral administration forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (such as a ligand containing GalNAc as described herein or containing an antibody).

[0303] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi agent described herein. An "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce the expression of the DGAT2 gene in a patient's specific tissue or cell type (e.g., liver or hepatocytes). The effective amount of the RNAi agent of the present invention can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, and can be administered daily, weekly, monthly, or at longer intervals. Accurately determining a specific effective dosage and dosing frequency may be based on several factors, including the patient's size, age, and general condition, the type of disease to be treated (e.g., myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the specific RNAi agent used, and the route of administration.

[0304] The administration of the pharmaceutical composition of the present invention can be carried out by any common route, as long as the target tissue can be obtained by the route. These routes include but are not limited to parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, oral, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or delivered by the portal vein. In some embodiments, the pharmaceutical composition is parenteral. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0305] Colloidal dispersion systems can be used as delivery vehicles for the RNAi agents of the present invention, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Commercially available fat emulsions suitable for transporting nucleic acids of the present invention include Intralipid (Baxter International Inc.), Liposyn (Abbott Pharmaceuticals), Lipsyn II (Hospira), Liposyn III (Hospire), Nutrilipid (B.Braun Medical Inc.), and other similar fat emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi agent of the present invention can be encapsulated within a liposome, or can form a complex therewith, particularly with a cationic liposome. Alternatively, the RNAi agent of the present invention can be complexed with lipids, particularly with cationic lipids. Suitable cationic lipids are, for example, diol tetramethylaminopropyl (DOTAP) and diol phosphatidylethanolamine (DOTMA).

[0306] Liposomal formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agents to the skin. In some embodiments, liposomes are used to deliver RNAi agents to epidermal cells and also enhance the penetration of RNAi agents into dermal tissue, such as the skin.

[0307] RNAi agent of the present invention can be fully encapsulated in lipid formulations, such as LNP or other nucleic acid-lipid particles. As used herein, term " LNP " refers to stable nucleic acid-lipid particles. LNP generally comprises cationic lipids, non-cationic lipids and the lipid (such as PEG-lipid conjugate) that prevents particle aggregation. LNP is very useful for systemic application because they show prolonged circulation time after intravenous (iv) injection, and accumulate in distal sites (such as the position physically separated from the administration site). LNP includes " pSPLP ", which includes the condensing agent-nucleic acid complex of the encapsulation described in WO00 / 03683. LNP particles of the present invention generally have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and substantially nontoxic. In addition, when nucleic acid is present in nucleic acid-lipid particles of the present invention, it has resistance to the degradation of nuclease in aqueous solution. Nucleic acid-lipid particles and methods for preparing the same are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication Nos. 2010 / 0324120 and WO 96 / 40964. In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to RNAi agent ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0308] The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA). Dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dihydroxypropoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dihydroxypropoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-di Linoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin -K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)didecadecan-2-ol (Tech G1) or mixtures thereof.The cationic lipid may comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0309] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. In some embodiments, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (molar percentage), have a particle size of 63.0±20 nm, and a siRNA / lipid ratio of 0.027.

[0310] The ionizable / non-cationic lipids can be anionic lipids or neutral lipids, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl DP-acylglyceroglycerophosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine ...DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC The non-cationic lipid may be present in an amount of about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % (if cholesterol is included) of the total lipids present in the particle.

[0311] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 20 mol% of the total lipid present in the particle, or about 2 mol%. In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 60 mol% of the total lipid present in the particle, or about 48 mol%.

[0312] In one embodiment, lipidoid ND98·4HCl (molecular weight 1487) (see U.S. Patent Application No. 12 / 056,230, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be mixed in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing.

[0313] Other exemplary lipid-siRNA formulations can be found in, for example, WO2009 / 127060 (SNALP), PCT / US2010 / 022614 (XTC), US2010 / 0324120 (MC3), PCT / US09 / 63933 (ALNY-100), and WO2010 / 129709 (C12-200).

[0314] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In general, these preparations are sterile and, to a certain extent, fluid and easy to inject. The preparation should remain stable under production and storage conditions and should be preserved to prevent contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0315] Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent along with any other ingredients (e.g., those listed above) and then sterilizing by filtration. Typically, dispersions are prepared by adding the various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and the desired other ingredients, e.g., as described above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0316] The compositions of the present invention can generally be formulated in neutral form or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium, potassium, ammonium, calcium or iron oxide) or organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the RNAi agent of the present invention is formulated as a sodium salt.

[0317] For example, for parenteral administration in the form of an aqueous solution, the solution is generally appropriately buffered, and the liquid diluent is first made isotonic with, for example, enough saline or glucose. Such an aqueous solution can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid, or injected at the infusion site of the suggestion. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local Food and Drug Administration. In certain embodiments, the pharmaceutical composition of the present invention comprises sterile saline solution and RNAi agent as described herein or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises RNAi agent as described herein and sterile water (e.g., water for injection, WFI) or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises RNAi agent as described herein and phosphate buffered saline (PBS) or consists of it.

[0318] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored within a drug delivery device. Devices for injecting formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, intracorporeal syringes, and injection pens. Devices for aerosolizing or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for use in treating or preventing one or more diseases or conditions described herein.

[0319] Treatment methods and uses

[0320] The present invention provides a method for reducing or inhibiting the expression of the DGAT2 gene in a cell (e.g., a hepatocyte) by contacting the cell with any of the RNAi agents described herein. The cell can be in vitro or in vivo. DGAT2 gene expression can be assessed by measuring the amount or level of DGAT2 mRNA or DGAT2 protein. Reduction of DGAT2 expression in cells or animals treated with the RNAi agents of the present invention can be determined relative to DGAT2 expression in cells or animals not treated with the RNAi agent or treated with a control RNAi agent. For example, in some embodiments, reduction of DGAT2 expression is assessed by (a) measuring the amount or level of DGAT2 mRNA in hepatocytes treated with a RNAi agent of the invention, (b) measuring the amount or level of DGAT2 mRNA in hepatocytes treated with a control RNAi agent (e.g., an RNAi agent directed against an RNA molecule not expressed in hepatocytes or an RNAi agent having a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the DGAT2 mRNA levels measured in the treated cells in (a) with the DGAT2 mRNA levels in the control cells in (b). Prior to comparison, the DGAT2 mRNA levels in the treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). DGAT2 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, and the like.

[0321] In some embodiments, the method for assessing DGAT2 expression levels is performed in vitro in cells that naturally express the DGAT2 gene (e.g., hepatocytes) or cells that have been engineered to express DGAT2. In certain embodiments, the method is performed in hepatocytes in vitro. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human or non-human primate hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the hepatocytes are HuH-7 cells. In another embodiment, the hepatocytes are primary human hepatocytes.

[0322] In other embodiments, the method for assessing DGAT2 expression levels is performed in vivo. The RNAi agent and any control RNAi agent can be administered to an animal (e.g., a transgenic animal or non-human primate expressing a DGAT2 gene), and DGAT2 mRNA or serum DGAT2 protein levels can be assessed in liver tissue harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with DGAT2 expression can be assessed in the treated animal. For example, DGAT2 protein is an expression product of DGAT2 mRNA in serum or plasma. Therefore, serum or plasma levels of DGAT2 protein can be measured in animals treated with the RNAi agents of the present invention to assess the functional efficacy of reducing DGAT2 expression.

[0323] In certain embodiments, the expression of DGAT2 in hepatocytes is reduced by at least 40%, at least 45%, or at least 50% by the RNAi agents of the invention. In some embodiments, the expression of DGAT2 in hepatocytes is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by the RNAi agents of the invention. In other embodiments, the expression of DGAT2 in hepatocytes is reduced by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by the RNAi agents of the invention. The percentage reduction in DGAT2 expression can be measured by any of the methods described herein, as well as other methods known in the art.

[0324] The present invention provides methods for reducing or inhibiting the expression of the DGAT2 gene, thereby reducing or inhibiting the production of DGAT2 protein, in a patient in need thereof, as well as methods for treating or preventing diseases or conditions associated with DGAT2 expression or activity. A "disease or condition associated with DGAT2 expression" refers to a disease or condition in which DGAT2 expression levels are altered, or a condition or condition in which elevated DGAT2 expression levels are associated with an increased risk of developing the disease or condition. Diseases or conditions associated with DGAT2 expression can also include those caused by abnormal changes in lipoprotein metabolism, such as changes that result in abnormal or elevated levels of DGAT2 protein, cholesterol, lipids, triglycerides, etc., or impaired clearance of these molecules. In certain embodiments, the RNAi agents of the present invention are particularly useful for treating or preventing metabolic diseases and reducing circulating levels of DGAT2.

[0325] Diseases and conditions associated with DGAT2 expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, metabolic diseases such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0326] In certain embodiments, the present invention provides a method for reducing DGAT2 expression in a patient in need thereof, comprising administering to the patient any of the RNAi agents described herein. Preferably, after administration of the RNAi agent, the level of DGAT2 expression in the patient's hepatocytes is reduced compared to the level of DGAT2 expression in the patient who did not receive the RNAi agent, or compared to the level of DGAT2 expression in the patient before administration of the RNAi agent. In some embodiments, after administration of the RNAi agent of the present invention, DGAT2 expression in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percentage reduction in DGAT2 expression can be measured by any of the methods described herein, as well as other methods known in the art. In certain embodiments, the percent reduction in DGAT2 expression is determined by assessing DGAT2 protein levels in the patient's serum or plasma according to the methods described herein.

[0327] In certain embodiments, the patient in need of reducing DGAT2 expression is a patient diagnosed with a metabolic disease or at risk for a metabolic disease. Therefore, the present invention includes a method for treating or preventing a metabolic disease in a patient in need thereof by administering any of the RNAi agents of the present invention. In some embodiments, the present invention includes the use of any of the RNAi agents described herein in the preparation of a medicament for treating or preventing a metabolic disease in a patient in need thereof. In other embodiments, the present invention provides a DGAT2-targeting RNAi agent for use in a method for treating or preventing a metabolic disease in a patient in need thereof. As described above, the metabolic disease is, for example, hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, or statin-resistant hypercholesterolemia. In a preferred embodiment, the disease to be treated or prevented according to the methods of the present invention is nonalcoholic fatty liver disease. In a preferred embodiment, the disease to be treated or prevented according to the methods of the present invention is nonalcoholic steatohepatitis.

[0328] In certain other embodiments, the patient in need of reduced DGAT2 expression is a patient with elevated circulating DGAT2 levels. Thus, in some embodiments, the present invention provides a method for reducing serum or plasma levels of DGAT2 protein in a patient in need thereof by administering to the patient any of the RNAi agents described herein. In some embodiments, the present invention includes the use of any of the RNAi agents described herein in the preparation of a medicament for reducing serum or plasma levels of DGAT2 protein in a patient in need thereof. In other embodiments, the present invention provides a DGAT2-targeted RNAi agent for use in a method for reducing serum or plasma levels of DGAT2 protein in a patient in need thereof.

[0329] Sequence Listing

[0330] Table 1 Unmodified DGAT2 siRNA

[0331] Table 2 Nucleotide-modified DGAT2 siRNA

[0332] Where m represents 2'-O-methyl (2'-OMe) modification, f represents 2'-fluoro (2'-F) modification, * represents phosphorothioate modification, SS represents sense strand, and AS represents antisense strand.

[0333] Table 3. Nucleotide-modified DGAT2 siRNAs linked to GalNAc ligands

[0334] Where m represents 2'-O-methyl (2'-OMe) modification, f represents 2'-fluoro (2'-F) modification, * represents phosphorothioate modification, SS represents sense strand, and AS represents antisense strand.

[0335] Example

[0336] Experimental methods and materials

[0337] Target sequence screening

[0338] siRNA was designed based on the full-length DGAT2 mRNA sequence (NM_006488.3), and all sequences were derived from the NCBI gene database.

[0339] GalNAc resin synthesis

[0340] GalNAc is a terminally reactive compound. Examples include Gal-C7, Gal-C6, Gal-C5, Gal-C4, and Gal-C3, which react with carboxylic acids, and Gal-C7-NH2, Gal-C6-NH2, Gal-C5-NH2, Gal-C4-NH2, and Gal-C3-NH2, which react with amino groups. These compounds were synthesized according to reported methods, and detailed information is shown in Table 4.

[0341] Table 4. Chemical structures, nomenclature, and synthesis methods of compounds containing terminal reactive groups (GalNAc).

[0342] Synthesis of key intermediate compound A

[0343] Synthesis of compound A-2:

[0344] Compound A-1 (3.0 g, 5.24 mmol, 1.0 equiv.) was dissolved in DMF (20 mL), and HBTU (3.9 g, 10.5 mmol, 2.0 equiv.) and DIPEA (1.73 mL, 2.0 equiv.) were added sequentially. The mixture was stirred at room temperature for 5 minutes. 1,9-Diaminononane (993 mg, 6.28 mmol) dissolved in DMF (10 mL) was then added, and the mixture was stirred at room temperature for 5 hours. After the reaction was complete, the reaction solution was quenched with water (30 mL) and extracted with ethyl acetate (20 mL). The organic phase was washed sequentially with 5% sodium bicarbonate solution (20 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product A-2 (3.65 g). The crude product was used directly in the next step. LCMS: (ESI) m / z = 714 [M+H] + .

[0345] Synthesis of compound A-3:

[0346] The crude product A-2 (3.65 g, 5.13 mmol) obtained in the previous step was dissolved in THF (10 mL), and Cbz-OSu (1.28 g, 5.13 mmol) and saturated sodium bicarbonate solution (10 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3×20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was separated and purified by preparative chromatography to obtain A-3 (1.1 g, yield: 25.6%) as a white solid. LCMS: (ESI) m / z = 847.5 [M+H] + .

[0347] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 5% B to 95% B in 20 minutes.

[0348] Synthesis of compound A:

[0349] Intermediate A-3 (1.1 g, 1.30 mmol) was dissolved in DCM (5 mL) and cooled to -10°C using a cryogenic reactor. A mixture of TFA and DCM (15 mL, v:v = 1:1) was slowly added. The reaction was stirred at -10°C for 15 hours. LCMS confirmed the reaction was complete. Cold methyl tert-butyl ether (40 mL) was added, resulting in a white turbidity. The mixture was centrifuged (3200 rpm) for 3 minutes, and the supernatant was discarded. Methyl tert-butyl ether (40 mL) was added to the solid residue, and the mixture was sonicated for 3 minutes. The reaction was centrifuged (3200 rpm) for 3 minutes, and the supernatant was discarded. The solid residue was dried to afford A (660 mg, yield: 74.9%) as a white solid. LCMS: (ESI) m / z = 679 [M+H] + .

[0350] Synthesis of Class IA molecules

[0351] 3.1 Synthesis of I-A10-5FAM

[0352] Synthesis of intermediate I-A10-A

[0353] 1-tert-Butyloxycarbonyl-ethylenediamine (71 mg, 0.442 mmol, 5.0 equiv.) and Intermediate A (60 mg, 0.0884 mmol) were dissolved in DMF (3 mL). EDCI (68 mg, 0.354 mmol), HOAt (48 mg, 0.354 mmol), and DIPEA (151 μl, 0.884 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative chromatography to obtain I-A10-A as a white solid (68 mg, yield: 68.0%). LCMS: (ESI) m / z = 1127.7 [M+Na] + ;1105.7[M+H] + ; 553.5[M+2H] / 2 + .

[0354] HPLC purification conditions: Column: Welch Topsil C18, 21.2×250mm, 5μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 20% B to 70% B in 20 minutes.

[0355] Synthesis of intermediate I-A10-B

[0356] I-A10-A (68 mg, 0.0435 mmol, 1.0 equiv.) was dissolved in DCM (3 mL), cooled to -5°C in an ice-salt bath, and TFA (3 mL) was slowly added dropwise. The reaction mixture was stirred for 2 hours. The reaction solution was concentrated to dryness to obtain the trifluoroacetate salt of intermediate I-A10-B as a colorless, transparent liquid (80 mg, yield >100%). LCMS: (ESI) m / z = 805.7 [M+H] + ; 403.5[M+2H] / 2 + .

[0357] Synthesis of intermediate I-A10-C

[0358] Intermediate I-A10-B (80 mg, 0.0435 mmol) and Gal-C4 (94 mg, 0.218 mmol) were dissolved in DMF (2 mL), and HOAt (30 mg, 0.218 mmol), EDCI (42 mg, 0.218 mmol) and DIPEA (74 μL, 0.435 mmol) were added in sequence. The reaction solution was stirred at room temperature overnight. After the reaction monitoring was substantially completed, the reaction solution was directly purified by preparative chromatography to obtain I-A10-C as a white solid (56 mg, yield: 62.7%). LCMS: (ESI) m / z = 1026.6 [M+2H] / 2 + ; 684.5[M+3H] / 3 + .

[0359] HPLC purification conditions: Column: Xbridge C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 38% B to 48% B in 20 minutes.

[0360] Synthesis of intermediate I-A10-E

[0361] I-A10-C (25 mg, 0.0122 mmol) was dissolved in methanol (3 mL), and potassium carbonate (10 mg) and Pd / C (10% w / w adsorbed on activated carbon, 5 mg) were added sequentially. The mixture was stirred under hydrogen for 2 hours, and the reaction was monitored by LC-MS. The reaction solution was filtered, and approximately 2 g of dry ice was added to the filtrate, stirred for 5 minutes, and concentrated to obtain the crude product I-A10-E, which was used directly in the next reaction. LCMS: (ESI) m / z = 770.2 [M+2H] / 2 + ; 668.5[M+2H-204] / 2 + .

[0362] Synthesis of compound I-A10-5FAM

[0363] The crude product I-A10-E (0.0122 mmol) obtained in the previous step was dissolved in water (2 mL) and acetonitrile (0.5 mL). 5-FAM-OSu (8 mg, 0.0165 mmol) and saturated sodium bicarbonate solution (0.5 mL) were added sequentially, and the reaction was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative chromatography to obtain I-A10-5FAM (10.0 mg, two-step yield: 43.3%) as an orange solid. LCMS: (ESI) m / z = 967.8 [M+H+K] / 2 + ; 949.3[M+2H] / 2 + ;645.8[M+2H+K] / 3 + ; 633.3[M+3H] / 3 + .

[0364] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 14% B to 24% B in 20 minutes.

[0365] HPLC: 91.1578% (214nm), RT=13.049min; 7.2549% (214nm), RT=13.406min.

[0366] Mobile phase: A: water (0.05% TFA), B: acetonitrile (0.05% TFA)

[0367] Gradient: 5% B equilibration for 3 min, increasing to 65% B in 20 min, increasing to 95% B in 5 min.

[0368] Flow rate: 1.0 mL / min.

[0369] Chromatographic column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0370] Column temperature: 60℃.

[0371] 3.2 Synthesis of I-A11-5FAM

[0372] Synthesis of intermediate I-A11-A

[0373] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-propylenediamine (73 mg, 0.442 mmol) in DMF (3 mL). After purification, I-A11-A (70 mg, yield: 68.0%) was obtained as a white solid. LCMS: (ESI) m / z = 1147.8 [M+H] + .

[0374] Synthesis of intermediate I-A11-B

[0375] Following the method for synthesizing intermediate I-A10-B, I-A11-B (80 mg, yield: quantitative) was obtained from 70 mg of I-A11-A (0.0436 mmol) as a colorless oily liquid. LCMS: (ESI) m / z = 847.7 [M+H] + .

[0376] Synthesis of intermediate I-A11-C

[0377] Following the method for synthesizing intermediate I-A10-C, I-A11-C (32 mg, yield: 35.1%) was obtained from the crude product I-A11-B in the previous step as a white solid. LCMS: (ESI) m / z = 1047.7 [M+2H] / 2 + .

[0378] HPLCC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 41% B to 51% B in 20 minutes.

[0379] Synthesis of intermediate I-A11-E

[0380] According to the method for synthesizing intermediate I-A10-E, I-A11-e (40 mg, quantitative) was obtained from 32 mg of I-A11-C (0.0156 mmol) as a white solid. LCMS: (ESI) m / z = 791.0 [M+2H] / 2 + .

[0381] Synthesis of compound I-A11-5FAM

[0382] According to the method for synthesizing I-A10-5FAM, I-A11-5FAM (6.3 mg, yield: 20.8%) was obtained from 40 mg of I-A11-C as an orange solid. LCMS: (ESI) m / z = 989.2 [M+H+K] / 2 + .

[0383] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 13% B to 23% B in 20 minutes.

[0384] HPLC purity: 60.0611% (214 nm), RT = 12.894 min; 37.2783% (214 nm), RT = 13.256 min.

[0385] The analytical conditions were the same as those for I-A10-5FAM.

[0386] Synthesis of I-A12-5FAM

[0387] Synthesis of intermediate I-A12-A

[0388] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-butanediamine (75 mg, 0.396 mmol) in DMF (2 mL). After purification, I-A12-A (73 mg, yield: 69.5%) was obtained as a white solid. LCMS: (ESI) m / z = 595.5 [M+2H] / 2 + .

[0389] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 20% B to 80% B in 20 minutes.

[0390] Synthesis of intermediate I-A12-B

[0391] I-A12-A (73 mg, 0.061 mmol) was dissolved in DCM (2 mL) and TFA (2 mL) was slowly added dropwise. The mixture was allowed to react at room temperature for 1 hour. The reaction solution was concentrated to dryness to obtain I-A12-B (53 mg, yield: quantitative) as a colorless oil. LCMS: (ESI) m / z = 911.7 [M+Na] + ; 445.4[M+2H] / 2 + .

[0392] Synthesis of intermediate I-A12-C

[0393] Gal-C4 (1.2 g, 2.77 mmol, 5.0 eq.) was dissolved in DMF (20 mL), and I-A12-B (900 mg, 0.553 mmol), EDCI (530 mg, 2.77 mmol), HOAt (376 mg, 2.77 mmol), and DIPEA (944 μl, 5.53 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to obtain I-A12-C (900 mg, yield: 76%). LCMS: (ESI) m / z = 1068.8 [M+2H] / 2 + ;903.8[M+2H-330] / 2;712.8[M+3H] / 3 + . 1 H NMR (400MHz, DMSO) δ8.07-7.71(m,10H,10NH),7.39-7.19(m,6H,Bn-5H+1NH),5.20(d,J=3.2Hz,3H,3Gal-OCHO),5.00(s,2H,Ph- CH2),4.96(dd,J=3.2,11.2Hz,3H,3Gal-OCHC),4.48(d,J=8.4Hz,3H,3Gal-OCHC),4.02(brs,9H),3.92-3.80(m,3H,3Gal-OCHC) ,3.70-3.63(m,3H,3Gal-OCHC),3.47-3.39(m,3H,3Gal-OCHC),3.131-2.90(m,26H),2.72-2.52(m,14H),2.16-2.03(m,6H),2.1 0(s,9H,3CH3),1.99(s,9H,3CH3),1.89(s,9H,3CH3),1.76(s,9H,3CH3),1.74-1.61(m,6H),1.47-1.30(m,16H),1.23(brs,10H).

[0394] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 10 minutes.

[0395] Synthesis of intermediate I-A12-E

[0396] According to the method for synthesizing intermediate I-A10-E, I-A12-E (33 mg, quantitative) was obtained from 30 mg of I-A12-C (0.0140 mmol) as a white solid. LCMS: (ESI) m / z = 812 [M+2H] / 2 + .

[0397] Synthesis of compound I-A12-5FAM

[0398] According to the method for synthesizing I-A10-5FAM, I-A12-5FAM (8.5 mg, yield: 21.5%) was obtained as an orange solid from 33 mg of I-A12-E (0.020 mmol). LCMS: (ESI) m / z = 991.0 [M+H+K] / 2 + ;674.0[M+2H+K] / 3 + .

[0399] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0400] HPLC purity: 9.5195% (214 nm), RT = 13.588 min; 87.8220% (214 nm), RT = 13.008 min.

[0401] The analytical conditions were the same as those for I-A10-5FAM.

[0402] 3.4 Synthesis of I-A12-Cy5

[0403] According to the method for synthesizing I-A12-5FAM, I-A12-Cy5 (8.0 mg, yield: 26.5%) was obtained from 30 mg of I-A12-E (0.020 mmol) as an orange solid. LCMS: (ESI) m / z = 1044.5 [M+2H] / 2 +,709.5[M+3H+K] / 3 + ,696.8[M+3H] / 3 + .

[0404] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0405] HPLC purity: 84.9% (214 nm), RT = 17.535 min; 12.1% (214 nm), RT = 17.945 min.

[0406] 3.5 Synthesis of Compound I-A12-Ac-5FAM

[0407] I-A12-C (40 mg, 0.0187 mmol) was dissolved in methanol (5 mL) and Pd / C (10% w / w adsorbed on activated carbon, 8 mg) was added. The mixture was stirred under hydrogen for 13 hours and the reaction was monitored by LC-MS. The reaction solution was filtered, concentrated, and purified to obtain 5 mg of I-A12-Ac (yield: 13.3%), which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 + ;680.7[M+2H+K] / 3 + .

[0408] I-A12-Ac (5 mg, 0.00250 mmol) obtained in the previous step was dissolved in water (2 mL) and acetonitrile (0.5 mL). 5-FAM-OSu (1.2 mg, 0.0165 mmol) and saturated sodium bicarbonate solution (0.5 mL) were added sequentially, and the reaction was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative chromatography to obtain I-A12-Ac-5FAM (3.5 mg, yield: 59.3%) as an orange solid. LCMS: (ESI) m / z = 800.4 [M+2H+K] / 3 + ;787.5[M+3H] / 3 + .

[0409] HPLC purification conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 35% B to 55% B in 20 minutes.

[0410] HPLC purity: 75.08% (214 nm), RT = 17.237 min; 18.82% (214 nm), RT = 17.738 min.

[0411] The analytical conditions were the same as those for I-A10-5FAM.

[0412] Synthesis of 3.6I-A13-5FAM

[0413] Synthesis of intermediate I-A13-A

[0414] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-pentanediamine (89 mg, 0.442 mmol) in DMF (2 mL) at room temperature for 2 hours. The reaction solution was directly purified to obtain I-A13-A (72 mg, yield: 66.7%) as a white solid. LCMS: (ESI) m / z = 1231.7 [M+H] + ; 616.3[M+2H] / 2 + .

[0415] HPLC purification conditions: HPLC preparative column: Xbridge C18, 19×250 mm, 10 μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 33% B to 43% B in 20 minutes.

[0416] Synthesis of intermediate I-A13-B

[0417] Following the method for synthesizing intermediate I-A12-B, I-A13-B (65 mg, yield: quantitative) was obtained from 72 mg of I-A13-A (0.058 mmol) as a colorless oily liquid. LCMS: (ESI) m / z = 953.8 [M+Na] + ;931.7[M+H] + ; 466.5[M+2H] / 2 + .

[0418] Synthesis of intermediate I-A13-C

[0419] According to the method for synthesizing intermediate I-A10-C, crude product I-A13-B from the previous step was reacted to obtain I-A13-C (20 mg, yield: 13.4%) as a white solid. LCMS: (ESI) m / z = 1089.4 [M+2H] / 2 + ;924[M+2H-330] / 2 + .

[0420] HPLC purification conditions: HPLC preparative column: Xbridge C18, 19×250 mm, 10 μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 43% B to 53% B in 10 minutes.

[0421] Synthesis of intermediate I-A13-E

[0422] According to the method for synthesizing intermediate I-A10-E, I-A13-E (20 mg, quantitative) was obtained from 20 mg of I-A13-C (0.0090 mmol) as a white solid. LCMS: (ESI) m / z = 833.0 [M+2H] / 2 + ;731.4[M+2H-204] / 2 + .

[0423] Synthesis of intermediate I-A13-5FAM

[0424] According to the method for synthesizing I-A10-5FAM, I-A13-5FAM (10.0 mg, yield: 29.4%) was obtained as an orange solid from 20 mg of I-A13-E (0.0090 mmol). LCMS: (ESI) m / z = 1031 [M+H+K] / 2 + ;1012.7[M+2H] / 2 + ;687.0[M+2H+K] / 3 + ; 675.3[M+3H] / 3 + .

[0425] HPLC purification conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0426] HPLC purity: 24.2020% (214 nm), RT = 14.195 min; 71.6996% (214 nm), RT = 13.440 min.

[0427] The analytical conditions were the same as those for I-A10-5FAM.

[0428] Synthesis of class IB molecules

[0429] 4.1 Synthesis of I-B11-5FAM

[0430] Synthesis of intermediate A-4

[0431] 5-Hydroxypentylamine (5.0 g, 23.9 mmol) was dissolved in DMF (50 mL), and N-benzyloxycarbonyl-glycine (2.96 g, 28.7 mmol), HOBt (4.84 g, 35.9 mmol), EDCI (6.9 g, 35.9 mmol), and DIPEA (14.7 mL, 81.1 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was quenched with 50 mL of water and extracted with ethyl acetate (60 mL). The organic phase was washed with 5% NaHCO₃ (40 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to give compound A-4 (6.3 g, yield: 90%) as a white solid. LCMS: (ESI) m / z = 295.1 [M+H] + .

[0432] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 95% B in 20 minutes.

[0433] Synthesis of intermediate A-5

[0434] Add the activated Molecular sieves (3.0 g), freshly prepared GalNAc-1 (2.0 g, 6.079 mmol), and intermediate A-4 (1.96 g, 6.687 mmol) were added and the atmosphere was replaced with nitrogen three times. Anhydrous 1,2-dichloroethane (20 mL) was added and the mixture was stirred at room temperature for 10 minutes. TMSOTf (110 μl, 0.608 mmol) was added and the reaction was stirred at room temperature for 3 hours. The reaction mixture was filtered to remove the molecular sieves, and the filtrate was quenched with saturated aqueous NaHCO₃ (15 mL). The organic phase was separated and the aqueous phase was extracted twice with DCM (20 mL). The combined organic phases were dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to afford intermediate A-5 (400 mg, yield: 10.5%) as a white solid. LCMS: (ESI) m / z = 624.1 [M+H] + .

[0435] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 20% B to 60% B in 20 minutes.

[0436] Synthesis of intermediate A-6

[0437] A 50 mL round-bottom flask was charged with intermediate A-5 (400 mg, 0.642 mmol) and Pd / C (10%, 40 mg). The reaction system was purged with hydrogen twice, and THF (5 mL) was added. The mixture was stirred at room temperature under hydrogen for 90 minutes. The reaction mixture was filtered and concentrated to give the crude product A-6 (320 mg) as a light black oily liquid, which was used directly in the next reaction. LCMS: (ESI) m / z = 490.1 [M+H] + .

[0438] Synthesis of intermediate I-B11-A

[0439] Compound A (50 mg, 0.074 mmol) was dissolved in DMF (2 mL), and HOBt (35 mg, 0.258 mmol), EDCI (45 mg, 0.258 mmol), DIPEA (88 μl, 0.516 mmol), and intermediate A-6 (126 mg, 0.258 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by reverse phase preparative chromatography to obtain I-B11-A (50 mg, yield: 32.3%) as a white solid. LCMS: (ESI) m / z = 1047.0 [M+2H] / 2 + .

[0440] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 40% B to 50% B in 10 minutes.

[0441] Synthesis of intermediate I-B11-C

[0442] I-B11-A (50 mg, 0.024 mmol), potassium carbonate (10 mg, 0.150 mmol), and Pd / C (10%, 12 mg) were added to a 50 mL round-bottom flask, followed by methanol (5 mL). The mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-B11-C (40 mg, crude product) as a white solid. LCMS: (ESI) m / z = 791.0 [M+2H] / 2 + .

[0443] Synthesis of compound I-B11-5FAM

[0444] The crude product I-B11-C (40 mg, 0.024 mmol) obtained in the previous step was dissolved in a mixture of water (2 mL) and acetonitrile (0.5 mL). 5FAM-OSu (14 mg, 0.030 mmol) and saturated NaHCO3 solution (0.5 mL) were added sequentially. The mixture was stirred at room temperature for 60 minutes. The reaction solution was directly separated and purified by preparative chromatography to obtain I-B11-5FAM (9.5 mg, yield: 19.8%) as an orange solid. LCMS: (ESI) m / z = 989 [M+H+K] / 2 + .

[0445] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 17% B to 27% B in 20 minutes.

[0446] HPLC purity: 9.5962% (214 nm), RT = 13.531 min; 88.8773% (214 nm), RT = 13.301 min.

[0447] The analytical conditions were the same as those for I-A10-5FAM.

[0448] 4.2 Synthesis of I-B12-5FAM

[0449] Synthesis of intermediate A-7

[0450] 5-Hydroxypentylamine (5.0 g, 22.4 mmol) was dissolved in DMF (50 mL), and N-benzyloxycarbonyl-β-alanine (2.77 g, 26.9 mmol), HOBt (4.54 g, 33.6 mmol), EDCI (6.46 g, 33.6 mmol), and DIPEA (18.3 mL, 101 mmol) were added in sequence. The mixture was stirred at room temperature for 3 hours. The reaction solution was quenched with 50 mL of water and extracted with ethyl acetate (60 mL). The organic phase was washed with 5% NaHCO3 (40 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to give compound A-7 (7.0 g, yield: 92%) as a white solid. LCMS: (ESI) m / z = 309.1 [M+H] + .

[0451] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 95% B in 20 minutes.

[0452] Synthesis of intermediate A-8

[0453] Add the activated Molecular sieves (3.0 g) and DCM (20 mL) were added to freshly prepared GalNAc-1 (2.0 g, 6.08 mmol) and intermediate A-7 (2.0 g, 6.69 mmol), and stirred at room temperature for 10 minutes. TMSOTf (110 μl, 0.608 mmol) was added, and the reaction was stirred at room temperature for 3 hours. The reaction solution was filtered to remove the molecular sieves, and the filtrate was quenched with saturated aqueous NaHCO3 (15 mL). The organic phase was separated, and the aqueous phase was extracted twice with DCM (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to obtain intermediate A-8 (2.0 g, yield: 51.7%) as a white solid. LCMS: (ESI) m / z = 638.2 [M+H] + .

[0454] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 65% B in 20 minutes.

[0455] Synthesis of intermediate A-9

[0456] A 50 mL round-bottom flask was charged with intermediate A-8 (400 mg, 0.628 mmol) and Pd / C (10%, 40 mg). The reaction system was purged with hydrogen twice, and THF (5 mL) was added. The mixture was stirred at room temperature under hydrogen for 90 minutes. The reaction mixture was filtered and concentrated to give the crude product A-9 (320 mg) as a light black oily liquid, which was used directly in the next reaction. LCMS: (ESI) m / z = 504.1 [M+H] + .

[0457] Synthesis of intermediate I-B12-A

[0458] Compound A (45 mg, 0.066 mmol) was dissolved in DMF (2 mL), and HOBt (31 mg, 0.232 mmol), EDCI (45 mg, 0.232 mmol), DIPEA (79 μl, 0.464 mmol), and intermediate A-9 (117 mg, 0.232 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by reverse phase preparative chromatography to obtain I-B12-A (72 mg, yield: 51.1%) as a white solid. LCMS: (ESI) m / z = 1068.4 [M+2H] / 2 + .

[0459] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 10 minutes.

[0460] Synthesis of intermediate I-B12-C

[0461] I-B12-A (72 mg, 0.034 mmol), potassium carbonate (28 mg, 0.202 mmol), and Pd / C (10%, 5 mg) were added to a 50 mL round-bottom flask, followed by methanol (2 mL). The mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-B11-C (60 mg, crude product) as a white solid. LCMS: (ESI) m / z = 812.1 [M+2H] / 2 + ;711.0[M+2H] / 2 + ; 541.8[M+3H] / 3 + .

[0462] Synthesis of compound I-B12-5FAM

[0463] The crude product I-B12-C (40 mg, 0.025 mmol) obtained in the previous step was dissolved in a mixture of water (1.2 mL) and acetonitrile (0.3 mL). 5FAM-OSu (14 mg, 0.029 mmol) and saturated NaHCO3 solution (0.3 mL) were added sequentially. The mixture was stirred at room temperature for 60 minutes. The reaction solution was directly separated and purified by preparative chromatography to obtain I-B11-5FAM (12.5 mg, yield: 25.3%) as an orange solid. LCMS: (ESI) m / z = 1010.3 [M+H+K] / 2 + ;991.0[M+2H] / 2 + ;673.7[M+2H+K] / 3 + ; 661.3[M+3H] / 3 + .

[0464] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 17% B to 27% B in 20 minutes.

[0465] HPLC purity: 93.1525% (214 nm), RT = 13.435 min; 4.4401% (214 nm), RT = 13.806 min.

[0466] The analytical conditions were the same as those for I-A10-5FAM.

[0467] 4.3 Synthesis of I-B12-Cy5

[0468] According to the method for synthesizing I-A12-Cy5, I-B12-Cy5 (5.0 mg, yield: 19.2%) was obtained from I-B12-E (0.0124 mmol) as a dark blue solid. LCMS: (ESI) m / z = 1045.3 [M + +H] / 2 + ;697.0[M + +2H] / 3 + .

[0469] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 31% B to 41% B in 20 minutes.

[0470] HPLC purity: 90.9250% (214 nm), RT = 17.723 min; 8.5560% (214 nm), RT = 17.878 min.

[0471] Synthesis of IC molecules

[0472] 5.1 Synthesis of Compound I-C10-5FAM

[0473] Synthesis of intermediate I-C10-A

[0474] Compound A (100 mg, 0.147 mmol) was dissolved in DMF (3 mL), and HOBt (138 mg, 1.029 mmol), EDCI (198 mg, 1.029 mmol), and GalNAc-C7-NH2 (408 mg, 0.884 mmol) were added sequentially. The reaction was stirred at room temperature for 16 hours. The reaction was quenched by adding water (5 mL), extracted with ethyl acetate (8 mL), and the organic phase was washed with 5% aqueous sodium bicarbonate solution (10 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was separated and purified by preparative chromatography to obtain I-C10-A (170 mg, yield: 57.6%) as a white solid. LCMS: (ESI) m / z = 1003.0 [M+2H] / 2 + .

[0475] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 20% B to 80% B in 20 minutes.

[0476] Synthesis of intermediate I-C10-C

[0477] I-C10-A (50 mg, 0.025 mmol), potassium carbonate (21 mg, 0.150 mmol), and Pd / C (10%, 12 mg) were added to a 50 mL round-bottom flask. Methanol (5 mL) was then added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-C10-C (50 mg, crude product) as a white solid. LCMS: (ESI) m / z = 766.5 [M+H+K] / 2 + .511.6[M+2H+K] / 3 + .

[0478] Synthesis of compound I-C10-5FAM

[0479] The crude product I-C10-C (50 mg, 0.025 mmol) obtained in the previous step was dissolved in a saturated NaHCO3 solution in CH3CN / H2O (1.8 mL, v / v = 1:4:4), followed by the addition of 5-FAM-OSu (19 mg, 0.040 mmol). The mixture was stirred at room temperature for 1 hour. The reaction solution was directly separated and purified by preparative chromatography to obtain I-C10-5FAM (23 mg, yield: 37.7%) as an orange solid. LCMS: (ESI) m / z = 946 [M+H+K] / 2 + .

[0480] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 22% B to 32% B in 20 minutes.

[0481] HPLC purity: 7.4271% (214 nm), RT = 14.574 min; 91.5431% (214 nm), RT = 15.705 min.

[0482] Synthesis of ID molecules

[0483] 6.1 Synthesis of Compound I-D11-5FAM

[0484] Synthesis of GalNAc ligand-loaded resin for solid-phase oligonucleotide synthesis:

[0485] 7.1 Synthesis of Intermediate A-13

[0486] Compound A-10 (3.2 g, 5.0 mmol, 1.0 eq.) was dissolved in DCM (50 mL), and benzyl succinate (1.25 g, 6.0 mmol, 1.2 equiv.), DCC (1.24 g, 6.0 mmol, 1.2 equiv.) and DMAP (122 mg, 1 mmol, 0.2 equiv.) were added in sequence. The reaction solution was stirred at room temperature overnight and concentrated. DCM (20 mL) was added to the residue, stirred and dissolved, and the insoluble white solid was filtered off. The filtrate was concentrated and dried to give the crude product A-11 as a light yellow oily liquid, which was directly used in the next reaction.

[0487] The crude intermediate A-11 from the previous step was dissolved in DCM (25 mL), and diethylamine (25 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. After reaction monitoring, the reaction mixture was concentrated and dried to obtain the crude product A-12 as a light yellow oil, which was used directly in the next reaction.

[0488] The crude intermediate A-12 from the previous step was dissolved in DCM (100 mL), and succinic anhydride (1.5 g, 15 mmol), triethylamine (2.1 mL, 15 mmol), and DMAP (122 mg) were added sequentially. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by reverse-phase column chromatography to obtain intermediate A-13 (2.95 g, 83% yield over 3 steps) as a white solid. LCMS: (ESI) m / z = 708.2 [MH] - .

[0489] Reverse phase purification conditions: chromatographic column: 120 g C18 column; mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 20% B to 60% B in 20 minutes.

[0490] 7.2 Synthesis of Resin I-A12-H-PS

[0491] Synthesis of intermediate I-A12-D

[0492] Intermediate I-A12-C (900 mg, 0.422 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (40 mL) and methanol (5 mL). Pd / C (10%, 200 mg) was added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford crude product I-A12-D, which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 + ; 681.1[M+3H] / 3 + .

[0493] Synthesis of intermediate I-A12-G

[0494] Intermediate A-13 (359 mg, 0.506 mmol, 1.2 eq.) was dissolved in DMF (10 mL), and I-A12-D (863 mg, 0.422 mmol), EDCI (97 mg, 0.506 mmol), HOAt (69 mg, 0.506 mmol), and DIPEA (430 μl, 2.52 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to obtain I-A12-G (420 mg, yield: 37%) as a white solid. LCMS: (ESI) m / z = 1196.1 [M+2H] / 2 + ;797.7[M+3H] / 3 + . 1HNMR(400MHz,DMSO)δ8.08-7.73(m,11H,11NH),7.37-7.15(m,14H,DMTr+Bn),6.88(d,J=7.2Hz,4H,DMTr),5.38-5.31(m,1H,proline-CH-OH),5.21(d,J= 3.2Hz,3H,GalNAc-OCHO-),5.09(s,2H,Ph-CH2-),4.96(dd,J=11.6,3.2Hz,3H,GalNAc),4.48(d,J=7.6Hz,3H,GalNAc),4.25-4.07(m,2H),4.02(brs,9H) ,3.93-3.70(m,5H),3.73(brs,6H),3.70-3.53(m,4H),3.49-3.37(m,5H),3. 23-2.87(m,28H),2.70-2.53(m,17H),2.46-2.02(m,5H),2.10(s,9H,GalNAc -OCOCH3),1.99(s,9H,GalNAc-OCOCH3),1.89(s,9H,GalNAc-OCOCH3),1.78(s,9H,GalNAc-NHCOCH3),1.74-1.61(m,7H),1.36(brs,17H),1.21(brs,11H).

[0495] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 51% B to 61% B in 20 minutes.

[0496] Synthesis of intermediate I-A12-H

[0497] I-A12-G (400 mg, 0.149 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (12 mL) and methanol (4.0 mL). Pd / C (10%, 100 mg) was added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated, and the crude product was purified by preparative chromatography to afford I-A12-H ​​(350 mg, yield: 90%) as a white solid. LCMS: (ESI) m / z = 1321.2 [M+H+K] / 2 + ,1150.7[M+2H-DMTr] / 2 + . 1H NMR(400MHz,DMSO)δ8.08-7.73(m,11H,11NH),7.34-7.15(m,9H,DMTr),6.88(d,J=8.8Hz,4H,DMTr),5.38-5.31(m,1H,proline-CH-OH),5.21(d,J =3.2Hz,3H,GalNAc-OCHO-),4.95(dd,J=11.2,3.2Hz,3H,GalNAc),4.48(d,J=8.0Hz,3H,GalNAc),4.25-4.07(m,2H),4.02(brs,9H),3.93-3.70(m ,5H),3.73(brs,6H),3.70-3.53(m,5H),3.49-3.37(m,5H),3.23-2.87(m ,28H),2.70-2.39(m,19H),2.46-2.02(m,5H),2.10(s,9H,GalNAc-OCOCH 3),1.99(s,9H,GalNAc-OCOCH3),1.89(s,9H,GalNAc-OCOCH3),1.78(s,9H,GalNAc-NHCOCH3),1.74-1.61(m,7H),1.37(brs,17H),1.22(brs,11H).

[0498] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 20 minutes.

[0499] HPLC purity: >99% (214 nm), RT=14.992 min.

[0500] Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile.

[0501] Gradient: 5% B equilibration for 1 min, increase to 95% B in 20 min, increase to 95% B in 5 min.

[0502] Flow rate: 1.0 mL / min.

[0503] HPLC analytical column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0504] Column temperature: 45℃.

[0505] Synthesis of Resin I-A12-H-PS

[0506] Macroporous aminomethyl resin (250 mg, 0.4 mmol / g, 2.17 equiv.) was placed in a 5 mL plastic syringe with a filter membrane and soaked in DCM (3 mL) for 30 minutes. The resin was then washed with DCM (3 mL × 3) and then DMF (3 mL × 3) by aspiration, shaking, and extrusion. I-A12-H ​​(125 mg, 0.0461 mmol, 1.0 equiv.), DIPEA (39 μL, 0.231 mol, 5 equiv.), and PyAOP (52 mg, 0.101 mmol, 2.2 equiv.) were dissolved in DMF (3 mL) one by one. The clear solution was pipetted into the washed resin and shaken at 25°C for 24 hours. A slightly blue color was detected by Kaiser assay. The resin was washed with DMF (3 mL × 6). Unreacted amino groups on the resin were capped with a mixture of pyridine and acetic anhydride (3 mL, 2:1) by shaking for 3 hours. The resin was then washed with DMF (3 mL x 6), and Kaiser assay revealed a colorless product. The resin was then washed with DCM (3 mL x 3) and MTBE (3 mL x 3), and vacuum dried overnight to yield an off-white resin, I-A12-H-PS (415 mg).

[0507] Test resin loading

[0508] To 18.2 mg of the resin, 8.0 mL of a 0.48 N HCl / MeCN solution was accurately added. The mixture was gently shaken for 10 minutes. 2.0 mL of the clear solution was collected and examined by LCMS. The absorption peak area was 1340.29. The loading was calculated as 164.8 μmol / g using the following formula.

[0509] Cutting resin

[0510] The residual resin after DMTr removal was washed with acetonitrile (2 mL x 3) and 2 mL of concentrated aqueous ammonia was added. The mixture was shaken at 60°C for 3 hours, filtered, and the filtrate was analyzed by HPLC to confirm that the resin was loaded with high-purity GalNAc ligand (I-A12-I). LCMS: (ESI) m / z = 931.0 [M+H+K] / 2 + ;911.7[M+2H] / 2 + ; 608.3[M+3H] / 3 + HPLC purity: 87.80% (214 nm), RT = 9.588 min; 12.20% (214 nm), RT = 10.398 min.

[0511] Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile.

[0512] Gradient: 5% B equilibrate for 3 minutes, increase to 65% B in 20 minutes, equilibrate for 2 minutes.

[0513] Flow rate: 1.0 mL / min.

[0514] HPLC analytical column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0515] Column temperature: 60℃.

[0516] 7.3 Synthesis of Resin I-B12-F-PS

[0517] Synthesis of intermediate I-B12-B

[0518] Intermediate I-B12-A (400 mg, 0.187 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (10 mL) and methanol (1 mL). Pd / C (10%, 100 mg) was added and the mixture was stirred under hydrogen for 16 hours. The reaction mixture was filtered and concentrated to afford I-B12-B as a crude product, which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 + ; 668.2[M+3H] / 3 + .

[0519] Synthesis of intermediate I-B12-E

[0520] Intermediate A-13 (153 mg, 0.22 mmol, 1.2 equiv.) was dissolved in DMF (10 mL), and I-B12-B (used directly from the previous step), EDCI (42 mg, 0.22 mmol), HOAt (30 mg, 0.22 mmol), and DIPEA (92 μl, 0.54 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to yield I-B12-E (350 mg, yield: 72%) as a white solid. LCMS: (ESI) m / z = 1196.1 [M+2H] / 2 + .

[0521] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 54% B to 64% B in 20 minutes.

[0522] Synthesis of intermediate I-B12-F

[0523] I-B12-E (200 mg, 0.074 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (6 mL) and methanol (2.0 mL). Pd / C (10%, 40 mg) was added and the mixture was stirred under hydrogen for 6 hours. The reaction mixture was filtered and concentrated. The crude product was purified by HPLC to obtain I-B12-F (150 mg, yield: 78%) as a white solid. LCMS: (ESI) m / z = 1321.2 [M+H+K] / 2 + ;1169.7[M+2H-DMTr] / 2 + .

[0524] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; gradient: from 39% B to 49% B in 20 minutes.

[0525] HPLC purity: >99% (214 nm), RT = 15.734 min

[0526] The analytical conditions were the same as those for I-A12-H.

[0527] Synthesis of Resin I-B12-F-PS

[0528] I-B12-F-PS was synthesized similarly to I-A12-H-PS resin. The loading value was 118.0 μmol / g as determined by the same method.

[0529] The residual resin after the DMTr group removal was washed with acetonitrile (2 mL x 3), and 2 mL of concentrated aqueous ammonia was added. The mixture was shaken at 60°C for 3 hours, filtered, and the filtrate was analyzed by HPLC to confirm that the resin was loaded with a high-purity peptide. LCMS: (ESI) m / z = 930.8 [M+H+K] / 2 + ,911.8[M+2H] / 2 + ; 608.3[M+3H] / 3 + .

[0530] GalNAc ligand-loaded resin-synthesized oligonucleotide conjugates

[0531] GalNAc-siRNA-SS-3' was synthesized on a DNA / RNA synthesizer using the following method:

[0532] The siRNA synthesis method is no different from the conventional phosphoramidite solid-phase synthesis method (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.), including four steps of deprotection, coupling, capping, and oxidation or sulfurization. When synthesizing the sense strand with GalNAc delivery, the above-synthesized amino resin carrier connected to GalNAc (I-A12-H-PS resin, I-B12-F-PS resin, etc.) is used instead of the conventional Universal-CPG carrier. Using the solid phase carrier as the starting cycle, nucleoside monomers are connected one by one from 3' to 5'. The nucleoside phosphoramidite monomer raw materials 2'-FRNA, 2'-OMe RNA, etc. were purchased from Shanghai Zhaowei Technology Development Co., Ltd. After the solid phase synthesis is completed, the solid phase carrier is transferred to a centrifuge tube and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours to cleave the oligonucleotide from the solid phase carrier into the solution. The supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, then redissolved in deionized water and purified using a C18 reverse-phase column with a mobile phase of 0.1 M TEAA and acetonitrile. The target oligonucleotide was collected and lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm). The resulting sense and antisense strands were annealed at a fixed molar ratio to yield complementary double-stranded siRNA, which was then adjusted to the desired concentration.

[0533] HepG2 transfection

[0534] On the first day, a predetermined amount of the diluted compound was added to an equal volume of RNAiMAX Opti-MEM® solution, mixed thoroughly, and incubated at room temperature for 15 minutes. HepG2 cells were washed with DPBS and then trypsinized to adjust the cell density to 2.2 x 10^5 / mL. Simultaneously with the cell seeding, 20 μL of the Opti-MEM RNAiMax and compound mixture was added to a cell culture plate and seeded into a 96-well plate at a density of 20,000 cells per well (100 μL / well). The final volume of culture medium per well was 120 μL. The cells were cultured in a 5% CO2, 37°C incubator for 24 hours.

[0535] RNA extraction and reverse transcription

[0536] RNA was extracted according to the instructions of the RNA extraction kit (Qiagen, 74182), and RNA was reverse transcribed into cDNA according to the instructions of the FastKing cDNA first-strand synthesis kit (TianGen, KR116-02).

[0537] QPCR detection and data analysis

[0538] Target gene cDNA was detected by qPCR. The qPCR reaction system was prepared as shown in Table 1. GAPDH was used as an internal reference gene. qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: 50°C for 2 minutes, 95°C for 2 minutes; then 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds; and finally 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0539] Table 5. RT-PCR reaction system

[0540] Data analysis: After qRT-PCR, the CT value of the internal control is recorded at the same time, which is called Ct(GAPDH), and the CT value of the sample is called Ct(sample).

[0541] ΔCt(sample)=Ct(sample)-Ct(GAPDH)

[0542] ΔCt(control)=Ct(control)-Ct(GAPDH)

[0543] ΔΔCt = ΔCt(sample) - ΔCt(control)

[0544] Relative gene expression = 2^-ΔΔCt

[0545] Inhibition rate % = (1-relative expression level of sample / average relative expression level of control group) * 100

[0546] Example 1. Study on the hepatocyte endocytic activity of poly-GalNAc ligands with macrocyclic skeleton structures

[0547] Flow cytometry was used to evaluate the uptake efficiency of FAM fluorescently labeled test products in HepG2 cells.

[0548] Human hepatocellular carcinoma cell line (HepG2) was purchased from ATCC (USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, ThermoFisher Scientific, USA) containing 10% fetal bovine serum (ExCell Bio, South America), 100 units / mL penicillin, and 100 units / mL streptomycin in a 37°C, 5% CO2 incubator.

[0549] On day 0, the HepG2 cell suspension was adjusted to an appropriate density (1.5E+05 / wells) and the cells were seeded into 48-well plates. On day 1, the diluted fluorescently labeled test article was added. The test article was diluted to different concentrations, 1.6, 8, 40, 200 and 1000 nM. The GalNAc ligand was used for GivosRNA as a positive control, and 5FAM was used as a negative control. After incubation of the test article with the cells for 4 hours or 24 hours, all liquid was removed, and the cells were washed twice with PBS and collected. After digestion and fixation of the cells, the corresponding fluorescence in HepG2 cells was detected by flow cytometry (BD FACSCantoTMII, Becton Dickinson, America). The mean fluorescence intensity (MFI) of FAM of the test compound was analyzed with the mean fluorescence intensity of the positive compound. Table 6 shows the data at the 4-hour time point, and Table 7 shows the data at the 24-hour time point.

[0550] Table 6. Ratio of MFI of GalNAc ligand endocytosis in hepatocytes to MFI of positive compounds (FAM fluorescence channel, 4 hours time point)

[0551] Table 7. Ratio of MFI of GalNAc ligand endocytosis in hepatocytes to MFI of positive compounds (FAM fluorescence channel, 24 h time point)

[0552] Example 2. Screening of unmodified siRNA sequences

[0553] In order to evaluate the in vitro inhibitory activity of the modified siRNA on DGAT2 mRNA in the HepG2 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 8.

[0554] Table 8. In vitro inhibitory activity of unmodified siRNA against DGAT2 mRNA in HepG2 cell line

[0555] The results showed that the siRNA of the present invention can effectively reduce the DGAT2 mRNA level in HepG2 cells.

[0556] In order to evaluate the in vitro inhibitory activity of the nucleotide-modified siRNA against DGAT2 mRNA in the HepG2 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 9.

[0557] Table 9 In vitro inhibitory activity of modified siRNA against DGAT2 mRNA in HepG2 cell line

[0558] The results showed that the siRNA of the present invention can effectively reduce the DGAT2 mRNA level in HepG2 cells.

[0559] In order to evaluate the in vitro inhibitory activity of GalNAc-modified siRNA on DGAT2 mRNA in HepG2 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 10.

[0560] Table 10 In vitro inhibitory activity of GalNAc-modified siRNA against DGAT2 mRNA in HepG2 cell line

[0561] The results showed that the siRNA of the present invention can effectively reduce the DGAT2 mRNA level in HepG2 cells.

[0562] To evaluate the in vivo efficacy of DGAT2 siRNA in mice, ob / ob mice were used for in vivo efficacy testing. The mice were randomly divided into 5 groups, with 6 mice in each group, namely:

[0563] G1: Chow Diets, PBS, negative control group;

[0564] G2: GAN diets, PBS, negative control group;

[0565] G3: GAN diets, 1008-10-001 (10 mg / kg), treatment group;

[0566] G4: GAN diets, 1023-10-001 (10 mg / kg), treatment group;

[0567] G5: GAN diets, 1023-10-L96 (10 mg / kg), treatment group;

[0568] On Day 0, the day of administration, the G1 group was fed Chow Diets simultaneously; the G2-G5 groups began to feed GAN diets (Gubra-Amylin NASH diet, Research Diets D09100310) at the same time for 3 weeks.

[0569] On Day 1, mice were weighed and grouped by weight. The day of dosing was designated Day 0. Single administration was performed. Administration: sc. Dosage: 10 mpk; Dosing volume: 5 μL / g × mouse weight (g).

[0570] Day 21 marked the endpoint of the in vivo study. After fasting for 5-6 hours, mice were euthanized and liver tissue was harvested for analysis of triglyceride (TG) levels. The results demonstrated that the compounds 1008-10-001, 1023-10-001, and 1023-10-L96 provided by the present invention significantly reduced the average triglyceride (TG) content in the livers of mice induced by the GAN diet (Figure 1).

[0571] To evaluate the inhibitory efficacy of DGAT2 siRNA against the target gene, RNA was extracted from mouse liver tissue at the endpoint of the in vivo experiment and reverse transcribed into double-stranded cDNA. After purification, a library was constructed, and the library, once qualified, was subjected to transcriptome sequencing. After data was downloaded, linkers and low-quality sequencing data were removed, and gene / transcript quantification was performed after alignment with the reference genome (GRCm39). The results demonstrated that the compounds 1008-10-001, 1023-10-001, and 1023-10-L96 provided by the present invention significantly knocked down the mRNA expression levels of the target genes, with an average inhibitory efficiency exceeding 90% (Figure 2).

Claims

1. An RNAi agent for inhibiting the expression of diacylglycerol-O-acyltransferase 2 (DGAT2) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NOs: 17 to 30 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

2. The RNAi agent according to claim 1, wherein the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.

3. The RNAi agent according to claim 1 or 2, wherein the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.

4. The RNAi agent according to any one of claims 1 to 3, wherein the RNAi agent comprises one or two blunt ends, preferably one blunt end.

5. The RNAi agent according to any one of claims 1 to 4, wherein the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

6. The RNAi agent according to claim 5, wherein the overhang is located at the 3' end of the sense strand, the 3' end of the antisense strand, or at both the 3' end of the sense strand and the 3' end of the antisense strand; preferably, the overhang is located at the 3' end of the antisense strand, and further preferably, the RNAi agent has a blunt end.

7. The RNAi agent according to any one of claims 1 to 6, wherein the sense strand comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NOs: 1 to 14 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

8. The RNAi agent according to any one of claims 1 to 7, wherein the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 17 to 30; the sense strand has no more than 21 nucleotides and comprises a nucleotide sequence of SEQ ID NOs: 1 to 14.

9. The RNAi agent of claim 8, wherein: (a1) the sense strand comprises or is the sequence shown in SEQ ID NO: 1, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 17, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a2) the sense strand comprises or is the sequence shown in SEQ ID NO: 2, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 18, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom. Nucleotide sequences that differ by 1, 2, or 3 nucleotides; (a3) the sense strand comprises or is the sequence shown in SEQ ID NO: 3, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 19, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a4) the sense strand comprises or is the sequence shown in SEQ ID NO:4, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:20, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a5) the sense strand comprises or is the sequence shown in SEQ ID NO:5, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:21, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a6) the sense strand comprises or is the sequence shown in SEQ ID NO:6, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:22, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a7) the sense strand comprises or is the sequence shown in SEQ ID NO:7, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:23, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a8) the sense strand comprises or is the sequence shown in SEQ ID NO:8, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:24, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a9) the sense strand comprises or is the sequence shown in SEQ ID NO:9, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:25, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a10) the sense strand comprises or is the sequence shown in SEQ ID NO: 10, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 26, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a11) the sense strand comprises or is the sequence shown in SEQ ID NO: 11 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom. which have nucleotide sequences that differ by 1, 2, or 3 nucleotides; (a12) the sense strand comprises or is the sequence shown in SEQ ID NO: 12, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (a13) the sense strand comprises or is the sequence shown in SEQ ID NO: 13, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 29, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; or (a14) the sense strand comprises or is the sequence shown in SEQ ID NO: 14, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 30, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

10. The RNAi agent of any one of claims 1 to 9, wherein the RNAi agent comprises duplex 1002, 1003, 1004, 1005, 1008, 1018, 1023, 1024, 1029, 1035, 1044, 1068, 1069 or 1072.

11. The RNAi agent according to any one of claims 1 to 10, wherein the sense strand and / or antisense strand of the RNAi agent comprises at least one modified nucleotide independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), open ring nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleosides. Acid, 2'-methoxyethyl modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing thiophosphate bond internucleotide linkages, and combinations thereof; and / or preferably, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified.

12. The RNAi agent according to claim 11, wherein from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

13. The RNAi agent according to claim 11 or 12, wherein the antisense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: (i) between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; (ii) between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; (iii) between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and (iv) between the second nucleotide and the third nucleotide from the 3' end of the antisense strand.

14. The RNAi agent according to any one of claims 11 to 13, wherein in the direction from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

15. The RNAi agent according to any one of claims 11 to 14, wherein the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage exists between (i) the first nucleotide and the second nucleotide at the 5' end of the sense strand; and / or (ii) the second nucleotide and the third nucleotide at the 5' end of the sense strand.

16. The RNAi agent of claim 11, wherein the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

17. The RNAi agent according to any one of claims 11 to 16, wherein the antisense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 34, 35, 36, 39, 40, 41, 44, 45, 46, 56, 57, 58, 60, 61 or 62, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

18. The RNAi agent according to any one of claims 11 to 17, wherein the sense strand of the RNAi agent comprises or is a sequence shown in SEQ ID NO: 33, 37, 38, 42, 43, 47, 64 or 31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

19. The RNAi agent according to any one of claims 11 to 18, wherein: (b1) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:33, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:34, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b2) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:33, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:35, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b3) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:33, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:36, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b4) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 37, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO: 35, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b5) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:37, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:36, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b6) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:38, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:39, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b7) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:38, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:40, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b8) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:38, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:41, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b9) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:42, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:40, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b10) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:42, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:41, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b11) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:43, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:44, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b12) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:43, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:45, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b13) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:43, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:46, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b14) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:47, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:45, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b15) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:47, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:46, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b16) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:64, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:56, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b17) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:64, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:57, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b18) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:64, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:58, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b19) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:60, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (b20) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:61, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; or (b21) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:31, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:62, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

20. The RNAi agent of claim 19, wherein the RNAi agent comprises duplex 1002-60, 1002-10, 1002-11, 1002-34, 1002-35, 1003-60, 1003-10, 1003-11, 1003-34, 1003-35, 1029-60, 1029-10, 1029-11, 1029-34, 1029-35, 1008-60, 1008-10, 1008-11, 1023-60, 1023-10 or 1023-11.

21. The RNAi agent according to any one of claims 1 to 20, wherein the RNAi agent further comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, and further preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further comprises a ligand targeting non-hepatocytes, preferably, the ligand comprises a lipophilic monomer, and the lipophilic monomer is preferably Y132 to Y135, Y158, Y165 to Y168, L10, L57, L321, L322, Q361 to Q367, Q361s to Q367s, Q370, Q377 to Q379, or Q383.

22. The RNAi agent of claim 21, wherein the ligand comprises a structure as shown in formula (I): in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-, A or C is independently 0 or an integer between 1 and 14; B is 0 or an integer between 1 and 12; The wavy lines represent locations where the rest of the RNAi agent is attached.

23. The RNAi agent according to claim 22, wherein the structure of the ligand is as shown in formula (II) or formula (III): in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-, Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -S-, -SS-, Wherein R1 is an alkyl group containing 1 to 6 carbon atoms or hydrogen; W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand; The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand; A, C or F is 0 or an integer between 1 and 14; B or E is 0 or an integer between 1 and 12; D is an integer between 1 and 20; in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S-, Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3, q ​​is an integer from 3 to 10, and j is 0 or 1; W does not exist or is The wavy line on the right is the position connected to the sense strand or antisense strand; The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand; A or C is 0 or an integer between 1 and 14; B is 0 or an integer between 1 and 12.

24. The RNAi agent according to claim 22 or 23, wherein the ligand structure is as shown in formula (IV) or formula (V): in, The wavy line represents the position of connection to the sense strand or antisense strand, and the ligand is connected to the 5' and / or 3' end of the sense strand; preferably, the ligand is connected to the 3' end of the sense strand; preferably, wherein the ligand is connected to the sense strand or antisense strand via a phosphate group, a thiophosphate group or a phosphonate group.

25. The RNAi agent according to any one of claims 21 to 24, wherein the sense strand comprises or is a nucleotide sequence shown in SEQ ID NO: 50, 51, 52, 53, 54, 55, 59 or 15, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

26. The RNAi agent of claim 25, wherein: (c1) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:50, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:34, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c2) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:50, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:35, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c3) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:50, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:36, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c4) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:51, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:35, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c5) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:51, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:36, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c6) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:52, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:39, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; (c7) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:52, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:40, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; (c8) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:52, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:41, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c9) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:53, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:40, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c10) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:53, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:41, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c11) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:54, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:44, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c12) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:54, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:46, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c13) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:55, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:56, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c14) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:55, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:57, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c15) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:55, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:58, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c16) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:59, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:60, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c17) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:59, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:61, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; (c18) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:59, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:62, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; or (c19) the sense strand comprises or is the nucleotide sequence shown in SEQ ID NO:15, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the nucleotide sequence shown in SEQ ID NO:61, or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

27. The RNAi agent of claim 26, wherein the RNAi agent comprises or is a duplex 1002-60-001, 1002-10-001, 1002-11-001, 1002-34-001, 1002-35-001, 1003-60-001, 1003-10-001, 1003-11-001, 1003-34-001, 1003-35-001, 1029-60-001, 1029- 11-001, 1008-60-001, 1008-10-001, 1008-11-001, 1023-60-001, 1023-10-001, 1023-10-L96 or 1023-11-001.

28. A double-stranded RNAi agent for inhibiting the expression of DGAT2 in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.5: 5'-UGGUGAAGCUCUUCGACAA-3' and the length of the sense strand does not exceed 21 nucleotides, the antisense strand comprises SEQ ID NO.21: 5'-UUGUCGAAGAGCUUCACCAGG-3' and the length does not exceed 23 nucleotides, each nucleotide of the sense strand and the antisense strand of the RNAi agent is modified; and the 3'-end of the sense strand is conjugated to a ligand targeting hepatocytes.

29. The double-stranded RNAi agent of claim 28, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.31: 5'-mU*mG*mGmUfGmAfAfGfCmUmCmUmUmCmGmAmCmAmA-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.57: 5'-mU*fU*mGmUfCmGfAmAmGmAmGmCmUfUmCfAmCmCmA*mG*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, and the 3'-end of the sense strand is conjugated to a hepatocyte-targeting ligand.

30. The double-stranded RNAi agent according to claim 29, wherein the ligand is a structure shown in Formula IV: It is linked to the sense strand via a phosphate group or a phosphorothioate group.

31. A double-stranded RNAi agent for inhibiting the expression of DGAT2 in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.55: 5'-mU*mG*mGmUfGmAfAfGfCmUmCmUmUmCmGmAmCmAmA-YHZY12001-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.57: 5'-mU*fU*mGmUfCmGfAmAmGmAmGmCmUfUmCfAmCmCmA*mG*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, wherein YHZY12001 has a structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

32. A double-stranded RNAi agent for inhibiting the expression of DGAT2 in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.7: 5'-CUACUUUCGAGACUACUUU-3' and the length of the sense strand does not exceed 21 nucleotides, the antisense strand comprises SEQ ID NO.23: 5'-AAAGUAGUCUCGAAAGUAGCG-3' and the length does not exceed 23 nucleotides, each nucleotide of the sense strand and the antisense strand of the RNAi agent is modified; and the 3'-end of the sense strand is conjugated to a ligand targeting hepatocytes.

33. The double-stranded RNAi agent of claim 32, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.31: 5'-mC*mU*mAmCfUmUfUfCfGmAmGmAmCmUmAmCmUmUmU-3' and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.61: 5'-mA*fA*mAmGfUmAfGmUmCmUmCmGmAfAmAfGmUmAmG*mC*mG-3' and the length does not exceed 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, and the 3'-end of the sense strand is conjugated to a hepatocyte-targeting ligand.

34. The double-stranded RNAi agent according to claim 33, wherein the ligand is a structure shown in Formula IV: It is linked to the sense strand via a phosphate group or a phosphorothioate group.

35. A double-stranded RNAi agent for inhibiting the expression of DGAT2 in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.59: 5'-mC*mU*mAmCfUmUfUfCfGmAmGmAmCmUmAmCmUmUmU-YHZY12001-3' and the length of the sense strand is no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO.61: 5'-mA*fA*mAmGfUmAfGmUmCmUmCmGmAfAmAfGmUmAmG*mC*mG-3' and the length is no more than 23 nucleotides, wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, wherein YHZY12001 has a structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

36. A pharmaceutical composition comprising the RNAi agent of any one of claims 1 to 35 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

37. Use of the RNAi agent according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 36 in the preparation of the following drugs: (i) a drug for reducing the expression level of DGAT2 in cells; (ii) a drug for preventing or treating a disease caused by increased expression level of DGAT2; or (iii) Drugs for the prevention or treatment of metabolic diseases, such as 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 or heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia.

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