Rnai composition and use thereof

Abstract

The present invention relates to an RNAi composition for inhibiting the expression of apolipoprotein C-III, and the use thereof. The RNAi composition can be used for treating and / or preventing APOC3-associated diseases or conditions such as cardiovascular diseases, hypertriglyceridemia and pancreatitis.

Description

RNAi Compositions and Their Applications Technical Field

[0001] This disclosure relates to the field of oligonucleotide pharmaceuticals, and more specifically to RNAi compositions that inhibit apolipoprotein C-III expression and their applications, said RNAi compositions being used to treat and / or prevent APOC3-related diseases or conditions such as cardiovascular disease, hypertriglyceridemia, and pancreatitis. Background Technology

[0002] Apolipoprotein C3 (APOC3) is a member of the apolipoprotein C family. The human APOC3 gene is located on chromosome 11 and is primarily expressed in the liver, with lower expression levels in the small intestine. The pre-APOC3 protein consists of 99 amino acids; after a 20-amino acid signal peptide is cleaved in the endoplasmic reticulum, it becomes the mature APOC3 protein, consisting of 79 amino acids. APOC3 protein is distributed in triglyceride-rich lipoproteins (TRLs), including chylomicrons (CM), very low-density lipoproteins (VLDL), and high-density lipoproteins (HDL). Studies have reported that APOC3 protein can promote the secretion of VLDL in the liver; inhibit the activity of lipoprotein lipase and hepatic lipase; and delay the catabolism and clearance of TRLs (triglyceride-rich lipoproteins) by interfering with the binding of liver receptors LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1) to apoB and apoE, and is closely related to serum triglyceride levels. See, for example, Jan Borén, The Roles of ApoC-III on the Metabolism of Triglyceride-Rich Lipoproteins in Humans, Front Endocrinol (Lausanne). 2020; 11:474, doi:10.3389 / fendo.2020.00474; and Karin E Bornfeldt, Apolipoprotein C3: Form begets function, 2023, 65(1):100475, doi:10.1016 / j.jlr.2023.100475.

[0003] Studies have reported that serum triglyceride (TG) levels are a continuous risk factor. Serum TG levels between 1.7 and 5.6 mmol / L (150-500 mg / dL) primarily indicate the risk of cardiovascular disease, while serum TG levels between 5.6 and 10.0 mmol / L (500-885 mg / dL) primarily indicate the risk of cardiovascular disease and pancreatitis. Serum TG levels ≥10.0 mmol / L (885 mg / dL) primarily indicate the risk of familial chylomicronemia syndrome and multivariate chylomicronemia syndrome (represented by milky white plasma) and pancreatitis.

[0004] As serum triglyceride levels rise, the incidence and severity of pancreatitis also increase. The mortality rate of pancreatitis is approximately 1%; however, in hospitalized patients with pancreatitis and organ failure or pancreatic necrosis, the mortality rate can be as high as 30%-40%. Studies from 1961 to 2016 have shown a global upward trend in the incidence of acute pancreatitis, with an average annual growth rate of 3.07%.

[0005] Cardiovascular disease (CVD) has a persistently high incidence and mortality rate globally. The total number of CVD cases was 271 million in 1990 and 523 million in 2019. The number of CVD deaths was 12.1 million in 1990 and 18.6 million in 2019 (Reference: Global Burden of Cardiovascular Diseases and Risk Factors, 1990-2019: Update From the GBD 2019 Study, DOI: 10.1016 / j.jacc.2020.11.010), making it a major burden on healthcare systems and the economy. Studies have shown that APOC3 loss-of-function gene mutations can reduce serum triglyceride levels and cardiovascular disease risk. Homozygous carriers of the APOC3 loss-of-function gene mutation IVS2+1G-A (rs138326449) had a 37% relative decrease in serum triglyceride levels and a significantly reduced risk of coronary artery disease (p = 0.007) (Reference: APOC3 genetic variation, serum triglycerides, and risk of coronary artery disease in Asian Indians, Europeans, and other ethnic groups).

[0006] Elevated triglyceride levels are associated with a variety of diseases, including cardiovascular disease, pancreatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes (insulin resistance), hypertension, and skin lesions (xanthoma). Therefore, inhibiting APOC3 gene expression is of great significance in the treatment and / or prevention of dyslipidemia, pancreatitis, cardiovascular disease, and other metabolic-related conditions and diseases.

[0007] Invention Overview

[0008] This disclosure provides a novel RNAi activator that inhibits APOC3 gene expression through RNA interference. The RNAi activator according to the invention is suitable for the prevention and treatment of APOC3-related diseases or conditions such as hypertriglyceridemia and cardiovascular disease.

[0009] Therefore, in a first aspect, the present invention provides an RNAi activator for inhibiting APOC3 expression, wherein the RNAi activator comprises an antisense strand and a sense strand complementary to at least a portion of the sequence in the antisense strand to form a double-stranded region.

[0010] In some embodiments, the antisense strand of the RNAi activator according to the invention comprises a sequence motif complementary to at least 15, 16, or 17, for example, 18-23 (e.g., at least 18, at least 19, at least 20, at least 21, at least 22, or all) consecutive nucleotides in an APOC3 mRNA fragment. Preferably, the complementarity refers to at least 70%, at least 80%, at least 85%, at least 90%, or 100% complementarity, wherein the APOC3 mRNA fragment is a fragment corresponding to any of the target gene sequences in SEQ ID NO: 241-360; or the APOC3 mRNA fragment is a fragment corresponding to nucleotide positions 418-464 of SEQ ID NO: 361. As those skilled in the art will appreciate, the correspondence between the mRNA fragment and the specified sequence can be determined by sequence alignment.

[0011] In some further embodiments, the antisense strand of the RNAi activator according to the invention comprises a nucleotide sequence that is anticomplementary to at least 15, 16, 17, 18, 19, 20, 21, 22 or all of the target gene sequence regions selected from SEQ ID NOs:241-360; and optionally, the sense strand of the RNAi activator comprises a nucleotide sequence that is identical to or differs from at least 15 (preferably at least 16, 17, 18, 19, 20 or 21) consecutive nucleotides in the target gene sequence region by no more than 3, 2 or 1 nucleotides.

[0012] In some embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence complementary to at least 15 (preferably at least 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleotides in a human APOC3 mRNA fragment, or a nucleotide sequence differing from said nucleotide sequence by no more than 3, 2, or 1 nucleotide; and optionally, the sense strand of the RNAi activator comprises a nucleotide sequence identical to or differing from at least 15 (preferably at least 16, 17, 18, 19, 20, or 21) consecutive nucleotides in the corresponding mRNA fragment, wherein said APOC3 mRNA fragment is the fragment corresponding to nucleotide positions 418-464 of SEQ ID NO:361.

[0013] In some embodiments, the RNAi activator according to the invention comprises an antisense strand comprising: (i) 18-23 (e.g., 18, 19, 20, 21, 22, or 23) consecutive nucleotides selected from the antisense strand nucleotide sequence of any of the following compounds: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BPR... -995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511; (ii) 18-23 (e.g., 18, 19, 20, 21, 22, or 23) consecutive nucleotides selected from the antisense strand nucleotide sequence of any of the compounds listed in Table A; or (iii) a nucleotide sequence differing from the 18-23 consecutive nucleotides of (i) or (ii) by no more than 3, 2, or 1 nucleotides, wherein the antisense strand is 18-30 nucleotides in length, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Preferably, the antisense strand is 18-23 nucleotides in length.

[0014] In some embodiments, the RNAi activator according to the invention comprises a positive strand comprising: (i) 17-21 (e.g., 17, 18, 19, 20, or 21) consecutive nucleotides selected from the positive strand nucleotide sequence of any of the following compounds: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BP R-995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511; (ii) 17-21 (e.g., 17, 18, 19, 20, or 21) consecutive nucleotides selected from the positive strand nucleotide sequence of any of the compounds listed in Table A; or (iii) a nucleotide sequence differing from the 17-21 consecutive nucleotides of (i) or (ii) by no more than 3, 2, or 1 nucleotides, wherein the positive strand is 17-28 nucleotides in length, for example, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the positive strand is 17-21 nucleotides in length.

[0015] In some preferred embodiments, the antisense and sense strands of the RNAi activator according to the present invention respectively comprise the antisense and sense strand nucleotide sequences selected from any of the following compounds, or nucleotide sequences differing from them by no more than 3, 2, or 1 nucleotides: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995047, BPR-995048, BPR-995049, BPR-995040 ... 95104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BPR-995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511. In some other preferred embodiments, the antisense and sense strands of the RNAi activator according to the invention comprise, respectively, the antisense and sense strand nucleotide sequences selected from any of the compounds listed in Table A, or nucleotide sequences differing from them by no more than 3, 2, or 1 nucleotides. In some other preferred embodiments, the antisense strand and sense strand of the RNAi activator according to the invention respectively comprise an antisense strand nucleotide sequence and a sense strand nucleotide sequence selected from any of the following compounds, or nucleotide sequences that differ from them by no more than 3, 2, or 1 nucleotide: BPR-995040, BPR-995210, BPR-995055, BPR-995174, BPR-995505, BPR-995506, BPR-995507, BPR-995508, BPR-995509, BPR-995510, BPR-995511.

[0016] In some embodiments, this disclosure provides an RNAi activator comprising an antisense strand and a sense strand forming a double-stranded region, wherein the antisense strand comprises: (i) 18-23 (e.g., 18, 19, 20, 21, 22, or 23) consecutive nucleotides selected from the antisense strand nucleotide sequence of any of the compounds listed in Table 1; or (ii) a nucleotide sequence differing from the 18-23 consecutive nucleotides of (i) by no more than 3, 2, or 1 nucleotides, wherein the antisense strand is 18-30 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, preferably, the antisense strand is 18-23 nucleotides in length. In some further embodiments, the sense strand of the RNAi activator comprises: (i) 17-21 (e.g., 17, 18, 19, 20, and 21) consecutive nucleotides selected from the sense strand nucleotide sequence of any of the compounds listed in Table 1; or (ii) a nucleotide sequence differing from the 17-21 consecutive nucleotides of (i) by no more than 3, 2, or 1 nucleotides, wherein the length of the sense strand is 17-28 (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28) nucleotides, preferably, the length of the sense strand is 17-21 nucleotides. In some embodiments, the antisense strand has the same 3' terminal 5, 4, 3, or 2 residues as the antisense strand nucleotide sequence of the compound. In some preferred embodiments, the antisense and sense strands of the RNAi activator according to the present invention respectively comprise the antisense nucleotide sequence and sense nucleotide sequence selected from any of the compounds listed in Table 1, or nucleotide sequences that differ from them by no more than 3, 2 or 1 nucleotides.

[0017] In some embodiments, the RNAi activator according to the invention comprises an antisense strand and a sense strand forming a double-stranded region, wherein the sense strand comprises a region complementary to at least 15, 16, 17, or 18, or preferably at least 19, 20, or 21 consecutive nucleotides of the antisense strand. In some embodiments, the double-stranded region formed by the complementarity of the sense strand and the antisense strand is at least 15, 16, 17, or 18 nucleotides in length, preferably at least 19, 20, or 21 nucleotides in length.

[0018] In some embodiments, the RNAi activator according to the invention comprises a double strand with a protruding end consisting of an antisense strand and a sense strand, preferably wherein the protruding end is a 3' protrusion consisting of the last 1, 2, 3 or 4 nucleotides of the antisense strand.

[0019] In some preferred embodiments, the antisense strand is 23 nucleotides long, the sense strand is 21 nucleotides long, and the double-stranded region is 21 nucleotide pairs long.

[0020] In some embodiments, the antisense strand comprises the sequence of the antisense strand of any compound selected from Table 1 or Table A; and the sense strand comprises the sequence of the sense strand of the corresponding compound, or a sequence differing from it by 1, 2 or 3 nucleotides.

[0021] In some embodiments, preferably, the antisense strand comprises an antisense strand sequence selected from any of the compounds in Table 1; and / or the sense strand comprises a sense strand sequence of the corresponding compound.

[0022] In some embodiments, more preferably, the antisense strand comprises an antisense strand sequence selected from any of the compounds in Table A; and / or the sense strand comprises a sense strand sequence of the corresponding compound.

[0023] In a second aspect, the present invention provides an RNAi activator, which is a modified form of the RNAi activator according to the first aspect of the present invention. In some embodiments, the modification comprises at least one phosphate backbone modification and / or at least one nucleotide modification. In some embodiments, substantially all or all nucleotides on the antisense and / or sense strands of the RNAi activator are modified nucleotides.

[0024] In some embodiments, the RNAi activator comprises at least one 2'-modified nucleotide, wherein the 2'-modification is selected from: 2′-deoxy, 2'-fluoro, 2'-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), and 2′-ON-methylacetamido (2′-O-NMA); particularly 2'-modified nucleotides selected from 2′-deoxy, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl (2′-O-MOE). In some embodiments, the RNAi activator further comprises at least one modified nucleotide selected from the following: 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-O-methoxyethyl modified nucleotide, 2'-deoxyribonucleotide, nonlocked nucleotide (UNA), locked nucleotide (LNA), threononucleotide (TNA), and baseless nucleotide.

[0025] In some embodiments, the RNAi activator comprises a 5'-terminal phosphate modification, preferably wherein the modification is a 5'-(E)-vinylphosphonate modification at the 5' end of the antisense strand, and / or a threonucleotide modification at the 5' end of the sense strand.

[0026] In some embodiments, the RNAi activator comprises a 4'-modified threonucleotide located at the 5' terminal of the positive strand, particularly having formula (A). The 4'-modified threonucleotide, wherein Base represents a natural or modified nucleoside base, the natural nucleoside base being A, T, C, G, or U, and wherein R represents hydrogen or an alkoxy group having 1-30 carbon atoms (e.g., 25, 23, 21, 19, 17, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 carbon atom, or any range of any two of the aforementioned values, e.g., 5-25, 5-20, 8-20, or 10-15 carbon atoms), preferably, R represents a straight-chain alkoxy group with 12 carbon atoms, and the 4'-modified threonucleotide has the structure of the following formula (A1):

[0027] In some embodiments, the positive strand of the RNAi activator comprises or consists of 21 consecutive nucleotides. In some embodiments, the 21 consecutive nucleotides, counting from the 5' end, contain 2'-fluorinated nucleotides at one or more positions selected from the following (e.g., two, three, or four positions): 7, 9, 10, 11, 13, and 17, for example, at positions 7 and 9-11 or at positions 9, 11, and 13. In some embodiments, the 21 consecutive nucleotides, counting from the 5' end, contain 2'-deoxyribonucleotides at one or more positions selected from the following (e.g., one or two positions): 2, 5, and 7, for example, at positions 5, 7, and / or position 2. In some embodiments, the 21 consecutive nucleotides contain 2'-fluorinated modifications at the positions defined above and 2'-deoxy modifications at the positions defined above. In any of the foregoing embodiments involving the 21 nucleotides, preferably, the consecutive 21 nucleotides of the positive strand contain nucleotides modified with 2'-O-methyl at the remaining positions or at all positions except the first position. In some embodiments, the consecutive 21 nucleotides of the positive strand are threonucleotides at the first position, particularly threonucleotides of formula A with a 4' modification. Preferably, the consecutive 21 nucleotides of the positive strand contain nucleotides modified with 2'-O-methyl at the remaining 18, 17, or 16 positions.

[0028] In some embodiments, the antisense strand of the RNAi activator comprises or consists of 23 consecutive nucleotides. In some embodiments, the 23 consecutive nucleotides, counting from the 5' end, contain 2'-fluorinated nucleotides at one or more positions selected from the following (e.g., two, three, or four positions): 2, 6, 12, 14, and 16. For example, 2'-fluorinated nucleotides are contained at positions 2, 6, 14, and 16, or 2'-fluorinated nucleotides are contained at positions 2, 14, and 16, or 2'-fluorinated nucleotides are contained at positions 12, 14, and 16, or 2'-fluorinated nucleotides are contained at positions 2, 14, and 16. In some embodiments, the consecutive 23 nucleotides, counting from the 5' end, contain deoxyribonucleotides at one or more positions selected from the following (e.g., two, three, or four positions): 2, 5, 7, 9, and 12, for example, at positions 5 and 7 and optionally at positions 2, 9, or 12. In some embodiments, the consecutive 23 nucleotides, counting from the 5' end, contain 2'-O-methoxyethyl modified nucleotides at one or more positions selected from the following (e.g., one or two positions): 12 and 15, for example, 2'-O-methoxyethyl modified nucleotides at position 12 and / or 15. In some embodiments, the consecutive 23 nucleotides contain 2'-fluoro modifications at positions defined above and 2'-deoxy modifications at positions defined above. In some embodiments, the consecutive 23 nucleotides contain 2'-fluoro modified nucleotides at positions defined above and 2'-O-methoxyethyl modified nucleotides at positions defined above. In some embodiments, the consecutive 23 nucleotides comprise nucleotides modified with a 2'-fluorine nucleotide at the positions defined above, deoxyribonucleotides at the positions defined above, and nucleotides modified with a 2'-O-methoxyethyl nucleotide at the positions defined above. In any of the foregoing embodiments involving the 23 nucleotides, preferably, the consecutive 23 nucleotides of the antisense strand comprise nucleotides modified with a 2'-O-methyl nucleotide at the remaining 20, 19, 18, 17, or 16 positions.

[0029] In some embodiments, the antisense strand of the RNAi contains a 23 bp sequence motif, and the sequence motif has a modification pattern selected from the following:

[0030] AS Modification Mode-1:

[0031] NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNmNm

[0032] AS Modification Mode-2:

[0033] NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmN(moe)NmNm

[0034] AS modification mode - 3:

[0035] NmNfNmNmNmNfNmNmNmNmNmNmNmNfN(moe)NfNmNmNmNmNmNmNm

[0036] AS modification mode - 4:

[0037] NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmdN

[0038] AS modification mode - 5:

[0039] NmNfNmNmdNNmdNNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm

[0040] AS modification mode - 6:

[0041] NmNfNmNmdNN(moe)dNNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmNm

[0042] AS modification mode - 7:

[0043] NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmN(moe)NmNmNmNm

[0044] AS modification mode - 8:

[0045] NmNfNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmdN

[0046] AS modification mode - 9:

[0047] NmNfNmNmdNNmdNNmNmNmNmNmNmNfN(moe)NfNmNmNmNmNmNmNm

[0048] AS modification mode - 10:

[0049] NmNfNmNmdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNmNmNmNmNmNm

[0050] AS modification mode - 11:

[0051] NmdNNmNmdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNmNmNmNmNmNm

[0052] AS modification mode - 12:

[0053] NmNfNmNmdNNmdNNmNmNmNmdNNmNfN(moe)NfNmNmNmNmNmNmNm

[0054] AS modification mode - 13:

[0055] NmNfNmNmdNNmdNNmdNNmNmNmNmNfN(moe)NfNmNmNmNmNmNmNm

[0056] AS modification mode - 14:

[0057] NmNfNmNmdNNmdNNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmNm

[0058] AS modification mode - 15:

[0059] NmNfNmNmdNNmdNNmNmNmNmN(moe)NmNfN(moe)NmNmdNNmNmNmNmNm

[0060] AS modification mode - 16:

[0061] NmNfNmNmdNNmdNNmNmNmNfNfNmNfN(moe)NfNmNmNmNmNmNmNm

[0062] AS modification mode - 17:

[0063] NmNfNmNmdNNmdNNfNmNmNmNfNmNfN(moe)NfNmNmNmNmNmNmNm

[0064] AS modification mode - 18:

[0065] NmNfNmNfdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNmNmNmNmNmNm

[0066] AS modification mode - 19:

[0067] NmNfNmNmdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNfNmNmNmNmNm

[0068] AS modification mode - 20:

[0069] NmNfNmNmdNNmdNNmNmNmNmNfNmNfN(moe)NfNmNmNmNfNmNmNm

[0070] AS Modification Mode-21:

[0071] NmNfNmNfdNNmdNNfNmNmNmNfNmNfN(moe)NfNmNfNmNfNmNmNm

[0072] AS Modification Mode-22:

[0073] NmNfNmNmdNNmdNNmNmNmNmNfNmNfN(moe)dNNmNmNmNmNmNmNm

[0074] AS Modification Mode-23:

[0075] NmNfNmNmNmNfNmNmNmNmNfN(moe)NmNfNmNfNmNmNmNmNmNmNm

[0076] Wherein, N represents a nucleotide, Nm represents a 2'-O-methyl modified nucleotide, Nf represents a 2'-fluoro modified nucleotide, dN represents a deoxyribonucleotide, and N(moe) represents a 2'-O-methoxyethyl modified nucleotide. In some embodiments, AS modification patterns -5 and 10-13 are preferred.

[0077] In some embodiments, the sense strand of the RNAi contains a 21 bp sequence motif, and the sequence motif has a modification pattern selected from the following:

[0078] SS Modification Mode -1

[0079] NmNmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0080] SS Modification Mode - 2

[0081] NmNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmNmNmNm

[0082] SS Modification Mode - 3

[0083] NmNmNmNmNmNmdNNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0084] SS Modification Mode - 4

[0085] NmNmNmNmNmNmNmNmNfNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0086] SS Modification Mode -5

[0087] NmNmNmNmNmNmNmNmNfNmNfNmNfNmNmNfNmNmNmNmNm

[0088] SS Modification Mode - 6

[0089] NmNmNmNmNmNmNmNmNfdNNfNmNfNmNmNmNmNmNmNmNmNm

[0090] SS Modification Mode - 7

[0091] NmNmNmNmNmNmNmNmNmNmNfNmNfNmNmNfNmNmNmNmNm

[0092] SS Modification Mode - 8

[0093] NmNmNmNmNmNmNfNmNfdNNfNmNmNmNmNmNmNmNmNmNmNm

[0094] Wherein, N represents a nucleotide, Nm represents a 2'-O-methyl modified nucleotide, Nf represents a 2'-fluoro modified nucleotide, and dN represents a deoxyribonucleotide. In some embodiments, SS modification patterns -1 and -2 are preferred. In some embodiments, the first nucleotide of the above-described SS modification pattern is a 4'-modified threonucleotide of formula (A) as defined above, which is also considered in this disclosure, and wherein preferably, the R group of formula (A) represents a straight-chain alkyl group of 12 carbon atoms.

[0095] In some embodiments, the sense strand of the RNAi contains a 21 bp sequence motif, and the sequence motif has a modification pattern selected from the following:

[0096] SS Modification Mode -1'

[0097] TN12NmNmNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0098] SS Modification Mode -2'

[0099] TN12NmNmNmNmNmNmNmNfNmNfNmNfNmNmNmNmNmNmNmNm

[0100] SS Modification Mode -3'

[0101] TN12NmNmNmNmNmdNNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0102] SS Modification Mode -4'

[0103] TN12NmNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm

[0104] SS Modification Mode -5'

[0105] TN12NmNmNmNmNmNmNmNfNmNfNmNfNmNmNfNmNmNmNmNm

[0106] SS Modification Mode -6'

[0107] TN12NmNmNmNmNmNmNmNfdNNfNmNfNmNmNmNmNmNmNmNm

[0108] SS Modification Mode -7'

[0109] TN12NmNmNmNmNmNmNmNmNmNfNmNfNmNmNfNmNmNmNmNm

[0110] SS Modification Mode -8'

[0111] TN12NmNmNmNmNmNfNmNfdNNfNmNmNmNmNmNmNmNmNmNmNm

[0112] Wherein, N represents a nucleotide, Nm represents a 2'-O-methyl modified nucleotide, Nf represents a 2'-fluoro modified nucleotide, dN represents a deoxyribonucleotide, and TN12 represents a 4'-modified threonucleotide of formula (A1). In some embodiments, SS modification modes -1' and -2' are preferred.

[0113] In some embodiments, the RNAi has an antisense strand of 23 consecutive nucleotides and a sense strand of 21 consecutive nucleotides, wherein the antisense strand has a modification pattern selected from AS modification patterns 5 and 10-13; and the sense strand has a modification pattern selected from SS modification patterns 1 and 2. In other embodiments, the RNAi has an antisense strand of 23 consecutive nucleotides and a sense strand of 21 consecutive nucleotides, wherein the antisense strand has a modification pattern selected from AS modification patterns 5 and 10-13; and the sense strand has a modification pattern selected from SS modification patterns 1' and 2'. Preferably, the antisense strand and sense strand each comprise the antisense strand sequence and sense strand sequence of any of the compounds listed in Table 1 or preferably Table A.

[0114] In some embodiments, the antisense strand of the RNAi activator comprises: (i) 18-23 (e.g., 18, 19, 20, 21, 22, or 23) modified consecutive nucleotides selected from the modified antisense strand nucleotide sequences of any of the compounds listed in Tables 2, 3, B, or C; or (ii) a modified nucleotide sequence that differs from the 18-23 modified consecutive nucleotides of (i) by no more than 3, 2, or 1 nucleotides.

[0115] In some embodiments, the positive strand of the RNAi activator comprises: (i) 17-21 (e.g., 17, 18, 19, 20, or 21) modified consecutive nucleotides selected from the modified positive strand nucleotide sequences of any of the compounds listed in Tables 2, 3, B, or C; or (ii) a modified nucleotide sequence that differs from the 17-21 modified consecutive nucleotides of (i) by no more than 3, 2, or 1 nucleotides.

[0116] In some embodiments, the RNAi activator comprises a modified sense strand and a modified antisense strand of any of the compounds shown in Table 2 or preferably Table B. In some embodiments, the RNAi activator comprises a modified sense strand and a modified antisense strand of any of the compounds shown in Table 3 or preferably Table C.

[0117] In some embodiments, the RNAi activator further comprises at least one (e.g., one, two, three, or four) phosphate thioester or methyl phosphate backbone modification. In some preferred embodiments, the phosphate thioester modification comprises one or two phosphate thioester links at the 5' and / or 3' ends of the antisense and / or sense strands of the RNAi activator. In some embodiments, the antisense strand comprises two phosphate thioester links at both the 5' and 3' ends. In some embodiments, the sense strand comprises two phosphate thioester links at the 5' end, or the sense strand comprises two phosphate thioester links at both the 5' and 3' ends. In some embodiments, the sense strand is free of phosphate thioester links.

[0118] More preferably, the thiophosphate modification comprises or consists of the following: in some embodiments, the 5' end and 3' end of the sense chain each contain one thiosulfate, and the 5' end and 3' end of the antisense chain each contain two thiosulfates; in some embodiments, the 5' end of the sense chain contains two thiosulfates, the 3' end of the sense chain has no thiophosphate, and the 5' end and 3' end of the antisense chain each contain two thiosulfates; in some embodiments, the sense chain has no thiophosphate, and the 5' end and 3' end of the antisense chain each contain two thiosulfates.

[0119] In a third aspect, the present invention provides an RNAi activator comprising a non-nucleoside conjugate portion conjugated to a double-stranded oligonucleotide of an RNAi activator according to the first or second aspect of the present invention. In this document, such an RNAi activator is also referred to as an RNAi conjugate.

[0120] In some embodiments, the conjugation occurs at the 5' and / or 3' end of the sense strand of the RNAi and optionally at the 3' end of the antisense strand. Optionally, the conjugation is performed via thiophosphate group linkage or phosphate group linkage.

[0121] In some preferred embodiments, the RNAi conjugate comprises an asialic acid glycoprotein receptor (ASGPR) ligand, particularly a GalNAc ligand, such as a monovalent, divalent, trivalent, or tetravalent ligand. In this document, the double-stranded RNAi conjugate of the present invention comprising such ligands is also referred to simply as a GalNAc-siRNA conjugate or GalNAc-siRNA.

[0122] In some particularly preferred embodiments, the GalNAc ligand has the P36 structure according to the invention. As a preferred example, the GalNAc-siRNA conjugate has two GalNAc ligands located at the 5' and 3' ends of the sense strand, respectively; and optionally one GalNAc ligand located at the 3' end of the antisense strand.

[0123] In some other particularly preferred embodiments, the GalNAc ligand has the L96 structure according to the invention. As a preferred example, the GalNAc-siRNA conjugate has the GalNAc ligand located at the 3' end of the positive strand. In this embodiment, optionally, the GalNAc-siRNA conjugate has a 4'-modified threonucleotide of formula A at the 5' end of the positive strand.

[0124] In some more preferred embodiments, this disclosure provides RNAi conjugates having the structures of formulas Ia-If and II of the present invention, particularly RNAi conjugates of the compounds in Tables 2 and 3 formed by formulas Ia-If and II, wherein said compounds optionally contain at least one (e.g., 1, 2, 3, or 4) thiophosphate modification, particularly thiophosphate modification as defined above. In a most preferred aspect, the present invention provides GalNAc-siRNA conjugates of compounds selected from Tables B and C formed by formulas Ia-If and II, wherein said compounds optionally contain at least one (e.g., 1, 2, 3, or 4) thiophosphate modification, particularly thiophosphate modification as defined above. For details regarding the structures of formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), or (II) of the present invention, see “Detailed Description of the Invention”.

[0125] In a fourth aspect, the present invention provides compositions comprising, for example, pharmaceutical compositions comprising, an RNAi activator or RNAi conjugate according to the invention, and their uses.

[0126] In one embodiment, the use is for use as a medicine, or for use in the preparation of a medicine. In some embodiments, the medicine is used to reduce APOC3 levels and / or expression in a subject, or for the prevention or treatment of diseases or conditions mediated by APOC3.

[0127] In some embodiments, the drug comprises GalNAc-siRNA conjugates according to the invention, particularly GalNAc-siRNA conjugates formed by the compounds of Tables B and C according to the invention via formulas Ia-If and II, wherein the compounds optionally contain at least one (e.g., 1, 2, 3 or 4) thiophosphate modification, particularly thiophosphate modification as defined above.

[0128] In a fifth aspect, the present invention also provides a method for reducing (e.g., in vitro, ex vivo, or in vivo) APOC3 levels and / or expression in cells or subjects, or for preventing or treating diseases or conditions mediated by APOC3, comprising administering to said cells or subjects in need an effective amount of the RNAi activator or RNAi conjugate according to the invention, particularly the GalNAc-siRNA conjugate according to the invention, and more particularly the GalNAc-siRNA conjugates formed by the compounds of Tables B and C according to the invention via formulas Ia-If and II, wherein said compounds optionally contain at least one (e.g., 1, 2, 3, or 4) thiophosphate modification, particularly thiophosphate modification as defined above.

[0129] In some implementations, the diseases or conditions mediated by APOC3 are selected from: cardiovascular diseases, dyslipidemia, lipid metabolism disorders, familial partial lipodystrophy, chylomicronemia syndrome, hypertriglyceridemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, nephropathy, obesity, type 2 diabetes (insulin resistance), hypertension, atherosclerosis, and pancreatitis. Preferably, the diseases or conditions are selected from cardiovascular diseases, chylomicronemia syndrome, hypertriglyceridemia, and pancreatitis.

[0130] In some implementations, the subject is a mammal, particularly a human individual.

[0131] In some embodiments, the method includes administering the RNAi active agent or RNAi conjugate according to the invention subcutaneously or intravenously.

[0132] Examples of representative RNAi sequence motifs according to the present invention are provided in Table A below. In Table A, "SS" represents the sense strand and "AS" represents the antisense strand. In some cases, in the first to fifth aspects of the present invention described above, RNAi activators or RNAi conjugates according to the present invention comprising the RNAi sequence motifs provided in Table A below are more preferred.

[0133] Table A: Sequence motifs of representative siRNA duplexes

[0134] Examples of some representative RNAi activators according to the present invention are provided in Table B below, wherein “SS” represents the sense strand and “AS” represents the antisense strand. In some cases, RNAi activators or RNAi conjugates according to the present invention comprising the modified sense and antisense strands provided in Table B below are more preferred in the first to fifth aspects of the present invention described above.

[0135] Examples of some representative RNAi activators according to the present invention are provided in Table C below, wherein “SS” represents the sense strand and “AS” represents the antisense strand. In some cases, RNAi activators or RNAi conjugates according to the present invention that contain modified sense and antisense strands as provided in Table C below are more preferred in the first to fifth aspects of the present invention described above.

[0136] Table B: Modified siRNA duplexes

[0137] Table C: Modified siRNA duplexes Note: Based on the compounds shown in Table B, the 5' end of the SS chain is modified with TN12 at position 1.

[0138] In some embodiments according to the third to fifth aspects of this disclosure, the RNAi conjugate according to the invention formed in the following manner is preferred:

[0139] The compounds in Table B are conjugates formed by formula (Ia);

[0140] The compounds in Tables B and C are conjugates formed by formula (Ib);

[0141] The compounds in Table B are conjugates formed by formula (Ic);

[0142] The compounds in Table B are conjugates formed by formula (Id);

[0143] The compounds in Table B are conjugates formed by formula (Ie);

[0144] The compounds in Table B are conjugates formed by formula (If); and

[0145] The compounds in Tables B and C are conjugates formed by formula (II).

[0146] The structures of formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), or (II) are detailed in the "Detailed Description of the Invention". In some embodiments, RNAi conjugates are formed by modifying the siRNAs shown in Tables B and C with phosphate thioesters using the above-described conjugate formation methods; information on these RNAi conjugates is shown in Table 5, which constitutes a particularly preferred aspect of this disclosure.

[0147] In embodiments of this disclosure relating to RNAi conjugates of formula (Ia), formula (Ib), formula (Ic), formula (Id), formula (Ie), formula (If), or formula (II), in some cases, preferably, the RNAi conjugate has the following phosphate thioester modification pattern:

[0148] Equation (Ia):

[0149] Chain of Justice (SS 5′-3′): P36-s-P36-s-N1-o-N2-o-N3-o-N4-…-oN j-3 -oN j-2 -sN j-1 -sN j

[0150] Antisense chain (AS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0151] Formula (Ib):

[0152] Chain of Justice (SS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -oN j -s-P36-s-P36

[0153] Antisense chain (AS 5'-3'): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0154] Formula (Ic):

[0155] Chain of Justice (SS 5′-3′): P36-s-N1-s-N2-o-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -sN j -s-P36

[0156] Antisense chain (AS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0157] Formula (Id):

[0158] Chain of Justice (SS 5′-3′): P36-s-P36-s-N1-o-N2-o-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -sN j -s-P36

[0159] Antisense chain (AS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0160] Formula (Ie):

[0161] Chain of Justice (SS 5′-3′): P36-s-N1-s-N2-o-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -oN j -s-P36-s-P36

[0162] Antisense chain (AS 5'-3'): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0163] Formula (If):

[0164] Chain of Justice (SS 5′-3′): P36-s-N1-s-N2-o-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -sNj -s-P36

[0165] Antisense chain (AS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -oN k-1 -sN k -s-P36

[0166] Equation (II):

[0167] Chain of Justice (SS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN j-3 -oN j-2 -oN j-1 -oN j -o-L96

[0168] Antisense chain (AS 5′-3′): N1-s-N2-s-N3-o-N4-…-oN k-3 -oN k-2 -sN k-1 -sN k

[0169] in:

[0170] P36 represents (P) ligands, especially P36 ligands.

[0171] L96 represents the L96 ligand.

[0172] N i This represents the i-th nucleotide residue on the oligonucleotide chain (for the SS chain, i = 1, 2, ..., j, where j is the length of the sense oligonucleotide chain; for the AS chain, i = 1, 2, ..., k, where k is the length of the antisense oligonucleotide chain).

[0173] -s- indicates a thiophosphate bond.

[0174] -o- indicates a phosphodiester bond.

[0175] "..." indicates the remainder of the oligonucleotide, in which the nucleotides are linked together by phosphodiester bonds.

[0176] Brief description of the attached diagram

[0177] Figure 1 shows the 3'UTR region sequence of human APOC3 mRNA sequence ID NM_000040.3, and the inhibition rate of different compounds with the same modification scheme against APOC3 at a concentration of 0.1 nM in the HepG2 cell line activity assessment assay.

[0178] Figure 2 shows the inhibition rate of human APOC3 protein in serum at different time points after a single subcutaneous injection of 1 mg / kg RNAi conjugate into humanized mice, relative to the pre-administration level.

[0179] Figure 3 shows the inhibition rate of human APOC3 protein in serum at different time points after a single subcutaneous injection of 1 mg / kg RNAi conjugate into humanized mice, relative to the pre-administration level.

[0180] Figure 4 shows the inhibition rate of human APOC3 protein in serum on day 35 after administration, relative to pre-administration levels, when a single subcutaneous injection of 1 mg / kg RNAi conjugate was administered to humanized mice.

[0181] Figure 5 shows the inhibition rate of human APOC3 protein in serum at different time points when a single subcutaneous injection of 0.5 mg / kg RNAi conjugate into humanized mice was administered, relative to the saline group.

[0182] Figure 6 shows the inhibition rate of serum TG on day 35 after administration of a single subcutaneous injection of 0.5 mg / kg RNAi conjugate into humanized mice, relative to pre-administration levels. The results of two independent experiments are shown on either side of the dashed line in the figure.

[0183] Invention Details

[0184] definition

[0185] In this article, unless the context clearly indicates otherwise, the singular forms “a,” “one,” and “the” cover both singular and plural referents.

[0186] In this document, the terms "about" or "approximately," when referring to a measurable value (e.g., a parameter, quantity, duration, etc.), are intended to encompass both the specified value and variations relative to that specified value, such as variations of ±10%, ±5%, ±1%, or ±0.1% or less relative to the specified value, provided that such variations apply to the disclosed technical solution. It should be understood that the specific value referred to by the terms "about" or "approximately" is itself specifically and preferably disclosed.

[0187] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the term comprises the mentioned elements, integers, or steps. For example, when referring to an oligonucleotide that “comprising” a specific sequence, it is also intended to cover oligonucleotides that comprise that specific sequence.

[0188] In this paper, when referring to the sequence or sequence motif of RNAi or oligonucleotides, it is characterized by the order of nucleoside bases in that sequence or sequence motif. Since the pairing between nucleoside bases A and U corresponds to the pairing between nucleoside bases A and T, in this paper, for modified RNAi molecules or oligonucleotides containing T replacing U, the substitution position is indicated by a U base when referring to their sequence motif.

[0189] In this article, when referring to RNAi or oligonucleotides, unless otherwise specified, it should be understood that it refers not only to the sequence of nucleoside bases in the nucleotide chain that makes up the molecule (i.e., the sequence) but also to any chemical modifications present thereon (if such modifications are present). Such modifications can include phosphate backbone modifications, nucleoside modifications, and sugar modifications to the nucleotide chain, as well as conjugations of the nucleotide chain to non-nucleoside compounds. Therefore, when referring to a modified sequence of RNAi or oligonucleotides, it refers not only to the sequence of nucleoside bases in that sequence but also to the chemical modifications on the nucleotide chain having that nucleoside base sequence.

[0190] In this disclosure, as those skilled in the art will understand, “RNAi conjugate” can be considered as a specific RNAi activator having a conjugate portion modification. Therefore, unless the context clearly contradicts this, the description of “RNAi activator” in this disclosure also applies to “RNAi conjugate” and “conjugate containing RNAi activator” in this disclosure.

[0191] In this document, APOC3 mRNA refers to the mRNA of human apolipoprotein C-III. For example, human APOC3 mRNA with the sequence shown under Genbank accession number NM_000040.3. Accordingly, unless otherwise specified, the term "target gene" as used herein refers to a gene capable of transcribing said APOC3 mRNA, and the term "target mRNA" refers to said APOC3 mRNA.

[0192] In this document, RNAi activators for targeting APOC3 specifically refer to RNA molecules containing double-stranded oligonucleotides that can bind to the APOC3 gene mRNA sequence and reduce APOC3 gene expression through an RNAi mechanism, wherein the double-stranded oligonucleotides are referred to as antisense and sense strands, respectively, based on their complementarity with the target APOC3 gene sequence. In this document, such RNAi activators with oligonucleotide double strands are also referred to as siRNA or double-stranded RNAi. Therefore, unless explicitly excluded by the context, reference to the RNAi molecules of the present invention having oligonucleotide double strands is equivalent to reference to the siRNA or double-stranded RNAi of the present invention, and vice versa.

[0193] In this paper, the term "antisense" in relation to nucleic acid molecules refers to a nucleotide sequence containing a region complementary to the target nucleic acid. Therefore, for an RNAi molecule containing an antisense strand targeting APOC3, it will contain a nucleotide sequence complementary to the "sense" nucleic acid encoding the APOC3 protein (e.g., a nucleotide sequence complementary to the coding strand in the double-stranded DNA of the APOC3 gene, or a nucleotide sequence complementary to the APOC3 mRNA).

[0194] In this document, "target sequence" or "target gene sequence" refers to a continuous nucleotide motif in the mRNA molecule formed during transcription of a target gene (e.g., the APOC3 gene). The target gene sequence associated with the RNAi of this invention should be at least long enough to be used as a substrate for RNAi-guided nucleic acid cleavage, thereby causing a cleavage at or near the sequence location in the mRNA molecule formed by the transcription of the target gene. For example, the length of the target sequence can be, for example, 15-36 nucleotides ("nt"), or any sub-length therebetween. As a non-limiting example, the length of the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 18 nt, 19-30 nt, 19-26 nt, 19-23nt, 19-22nt, 19-21nt, 19-20nt, 19nt, 20-30nt, 20-26nt, 20-25nt, 20-24nt, 20-23nt, 20-22nt, 20-21nt, 20nt, 21-30nt, 21-26nt, 21-25nt, 21-24nt, 21-23nt, or 21-22nt, 21nt, 22nt, or 23nt. In some embodiments of the invention, the target sequence is preferably at least 18 nucleotides long, more preferably at least 19 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 30 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 25 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 23 nucleotides long. In some embodiments of the invention, the target sequence is about 21 to about 23 nucleotides long. In the antisense strand of RNAi, the sequence region complementary to consecutive nucleotides in the target gene sequence is also referred to herein as a "sequence motif." Based on this core sequence motif, and applying the Watson and Crick base pairing rules, those skilled in the art who read this disclosure can readily design RNAi activator molecules that specifically target APOC3. The antisense strand of such RNAi activators can be, for example, about 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. Depending on the designed form of the RNAi activator, the antisense strand sequence outside the "sequence motif" may, in some cases, be designed to be completely or substantially complementary to the extension of the target sequence on APOC3 mRNA, or not according to the Watson and Crick base pairing rules. All RNAi activators designed in this way are an aspect of this invention.

[0195] In this document, unless otherwise specified, the terms "complementarity" or "complementarity" refer to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions and form a double-stranded structure. Those skilled in the art can determine the optimal complementarity of the two sequences and the conditions used to determine this complementarity based on the final application of the hybridized oligonucleotide or polynucleotide. Therefore, in this document, when describing the base pairing between the sense and antisense strands of RNAi, or the base pairing between the antisense strand and the target sequence of RNAi, the terms "complementarity" or "complementarity" should be understood to cover not only 100% complementarity (i.e., perfect complementarity) but also cases of less than 100% complementarity, i.e., the presence of base mismatches in the complementary double-stranded nucleic acid region that do not substantially affect the RNAi's intended function. As those skilled in the art will appreciate, in double-stranded nucleic acid molecules, when a base on one strand forms a Watson-Crick base pair with a corresponding base on the other strand in a complementary manner, the bases at that position on both strands are considered to be "complementarily paired" or "matched." For example, the purine base adenine (A) is complementary to the pyrimidine base thymine (T) or uracil (U); the purine base guanine (C) is complementary to the pyrimidine base cytosine (G). Correspondingly, a "mismatch" refers to a situation in double-stranded nucleic acids where corresponding bases on one strand are not complementary to each other. However, it should be understood that nucleotides modified in the base portion of RNA nucleosides should also be considered complementary if Watson-Crick base pairing is permitted. Therefore, in this paper, the term "complementary" nucleoside bases encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 371.4.1). In this paper, the terms "complementary" and "reverse complementary" are used interchangeably.

[0196] In this document, for the purposes of this invention, the expression “complementary” or “complementarity” associated with double-stranded RNAi activators (such as siRNA described herein) is preferably not less than 70%, meaning that at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, or the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 21-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity % of the continuous nucleotide sequence in the double-stranded region. Accordingly, in this document, the expression associated with RNAi activators, “complementary (antisense) sequence” to the target sequence, or “complementary (sense) sequence” to a portion of the antisense sequence, can be “completely complementary” or “substantially complementary.” “Completely complementary” means that the two sequences have 100% complementarity. When the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may contain one or more, but typically no more than 30%, 20%, or 10%, mismatched base pairs in the hybridized duplex, and still retain the ability to hybridize under conditions most relevant to its final application (e.g., repressing gene expression via a RISC pathway). It should be understood here that when the two oligonucleotides of an RNAi are designed to form one or more single-stranded overhangs during hybridization, such overhangs will not be considered mismatches when determining complementarity. For example, for the purposes described herein, an RNAi comprising a 21-nucleotide-long sense oligonucleotide chain and a 23-nucleotide-long antisense oligonucleotide chain may still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter sense oligonucleotide.

[0197] In this document, the term "protruding end" is used to describe an unpaired nucleotide located at the 3' or 5' end of the double-stranded region of a double-stranded oligonucleotide. In some embodiments according to the invention, the protruding end is 1 to 4 nt long and is preferably located at the 3' end of the antisense strand of the RNAi.

[0198] In this document, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the former. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0199] In this article, the nucleosides and nucleotides that make up the nucleotide chain in nucleic acid molecules (such as RNAi or siRNA molecules) may be referred to as “units” or “monomers”.

[0200] In this document, "nucleotide" refers to the structural unit of oligonucleotides and polynucleotides, and for the purposes of this invention, includes naturally occurring nucleotides and modified nucleotides. In nature, RNA nucleotides comprise a sugar moiety (ribose), a nucleobase moiety, and a phosphate ester group. In this document, a modified nucleotide refers to a nucleotide that, corresponding to a natural RNA nucleotide, has modifications in its sugar moiety and / or nucleobase moiety.

[0201] In this document, the term "modified nucleoside" or "nucleoside modification" refers to a modified nucleoside formed by introducing one or more sugar moieties and / or one or more (nuclear)base moieties, compared to the corresponding RNA nucleoside. Therefore, the term "modified nucleoside" may also be used interchangeably with the term "modified nucleotide." In some preferred embodiments of the invention, the modified nucleoside comprises a modified sugar moieties.

[0202] In this paper, the term "nucleobase" refers to the base portion of nucleosides and nucleotides. In natural nucleic acids, nucleobases are the purine portion (e.g., adenine and guanine) and the pyrimidine portion (e.g., uracil, thymine, and cytosine) of nucleosides. In this paper, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this case, "nucleobase" refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Descriptions of such variants can be found, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 371.4.1. In some cases, the nucleobase moiety can be modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolysine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolysine, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. The nucleobase moiety can be indicated by a letter code (e.g., A, T, G, C, or U) for each corresponding nucleobase. For example, in the oligonucleotides of the RNAi molecules of table AC exemplified in this disclosure, the nucleobase moiety is selected from A, T, G, C, and U.

[0203] For naturally occurring oligonucleotides, internucleotide bonds comprise phosphate groups that form phosphodiester bonds between adjacent nucleosides. Hereinafter, the term "modified internucleotide bond" is defined as a bond that covalently links two nucleosides together, other than a phosphodiester (PO) bond. The oligonucleotide chain of the RNAi according to the invention may contain one or more internucleotide bonds derived from natural phosphodiester modifications, also referred to herein as "phosphodiester backbone modifications." Modified internucleotide bonds contemplated according to the invention include, but are not limited to: thiophosphate bonds, dithiophosphate bonds, methylphosphate bonds, selenophosphate bonds, phosphoramidite bonds, etc. In some embodiments, modified internucleotide bonds can increase the stability of the oligonucleotide, such as nuclease resistance, compared to phosphodiester bonds. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)) in a manner well known in the art. In some embodiments, preferably, the modified internucleotide bond in the oligonucleotide used for the RNAi of the invention is a thiophosphate bond. In some embodiments, the oligonucleotides of the sense and / or antisense strands may have one or two phosphate-thioester nucleoside internucleotide bonds at the 5' and / or 3' ends. In some embodiments, the nucleoside linking the oligonucleotide of the RNAi of the present invention to a non-nucleotide functional group such as a conjugate moiety may be a phosphodiester bond, or in some cases, a phosphate-thioester bond.

[0204] In this document, unless otherwise stated, the term "conjugation" refers to the covalent connection between two or more chemical moieties, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by covalently connecting these chemical moieties. Accordingly, in this document, "RNAi conjugated compound" (e.g., "siRNA conjugated compound") refers to a compound formed by covalently linking one or more chemical moieties having a specific function to an oligonucleotide chain of RNAi (such as siRNA). In this document, the specific chemical moiety covalently linked to the oligonucleotide chain of RNAi (such as siRNA), or the specific compound that can conjugate the specific chemical moiety to RNAi via a reaction, is also referred to as the "conjugated moiety." In some embodiments of the invention, the conjugated moiety is a non-nucleoside or non-nucleotide chemical moiety, but this does not preclude the possibility that the conjugated moiety is linked to an oligonucleotide via a nucleoside or nucleotide or its analogue or derivative.

[0205] In this document, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "alkyl" in "optionally substituted" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0206] In this document, "alkyl" refers to a straight-chain or branched chain having a specified number of carbon atoms, which can be from 1 to 30 carbon atoms, for example, 1 to 20 carbon atoms, 12 to 16 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight-chain forms having that number of carbons, and optionally, substitutions may be made.

[0207] In this document, "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group having a specified number of carbon atoms and containing at least one double bond. Specifically, alkenyl groups have 2 to 20, 2 to 18, for example 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and are optionally substituted. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0208] In this document, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety, which may be substituted or unsubstituted. Preferably, the aryl group is (C6-C6) 10 Aryl group. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as C1-C8 or C1-C4 alkyl groups. Preferably, the aryl group is an optionally substituted phenyl group.

[0209] In this document, "GalNAc ligand" refers to an asialic acid glycoprotein receptor (ASGPR) ligand containing a structural moiety of N-acetylgalactosamine (GalNAc) or a derivative thereof. This term encompasses monovalent, divalent, trivalent, tetravalent, and multivalent GalNAc ligands providing one, two, three, four, or more structural moieties of GalNAc or GalNAc derivatives.

[0210] In this document, the term "isolated" as a modifier for a compound means that the compound is artificially prepared or that the compound is completely or at least partially isolated from the natural environment in which the compound is naturally present. It should be understood that, unless otherwise stated, the compounds used in this invention (whether chemical or biochemical) are isolated. In some cases, the isolated compound is "purified" or "partially purified" from its production or preparation environment, and thus does not contain at least some of the components present in the environment in which the compound is produced or prepared. In some embodiments, the isolated compound has a purity of at least 90%. "Purified" or "partially purified" compounds can be combined with one or more other compounds to form compositions that achieve a specific purpose. Therefore, "purified" or "partially purified" does not exclude combinations of such compositions, such as the combination of an RNAi conjugate with a second active agent for the treatment of an APOC3-related disease or condition.

[0211] In this document, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, primates (e.g., human and non-human primates), laboratory animals (e.g., rodents, such as mice and rats), farm animals (e.g., cattle, pigs, sheep, and horses), grazing animals, and pets (such as dogs and cats).

[0212] In this document, the term "treatment" of a symptom and / or disease in mammals means (i) eliminating any symptoms of the disease or symptom; (ii) suppressing the symptom or disease, i.e., preventing the occurrence or progression of symptoms; and / or (iii) alleviating the symptom or disease, i.e., causing symptom resolution. In some aspects, the RNAi active agent or RNAi conjugate according to the invention may be therapeutically administered to inhibit, reduce, alleviate, stop, or reverse the progression of APOC3-related diseases or symptom or their symptoms in a subject, or to stabilize the development or progression of said disease or symptom or its symptoms.

[0213] In this document, the term "prevention" refers to reducing or decreasing the likelihood of a subject developing a disease or disease symptoms. Therefore, in some aspects, the RNAi active agent or RNAi composition of the present invention may be administered prophylactically to prevent the occurrence or recurrence of APOC3-related diseases or conditions or their symptoms in a subject. In some aspects, the subject does not yet have, but is at risk of having, said disease or condition, or is susceptible to developing said disease or condition.

[0214] In this article, "effective amount" means a predetermined amount of active agent that can elicit a desired biological or medical response in an organization, system, animal, or human, and / or an amount that prevents, inhibits, delays, or reverses the progression of a disease state or any other adverse symptom, or otherwise improves a disease state or symptom to achieve the desired therapeutic effect.

[0215] In this document, "therapeutic effective dose" and "preventive effective dose" refer to the amount that effectively achieves the desired therapeutic or preventive outcome at the required dose and for the required duration. Therapeutic and preventive effective doses can vary depending on various factors such as the disease to be treated or prevented, the individual's age, sex, and weight. Therapeutic and preventive effective doses are amounts in which any toxic or harmful effects are less than the beneficial therapeutic / preventive effects. Compared to subjects who have not received the drug, "therapeutic effective doses" and "preventive effective doses" preferably reduce measurable parameters (e.g., serum APOC3 levels or serum triglyceride levels) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%. The ability of the RNAi active agent or RNAi conjugate of the present invention to reduce said measurable parameters can be evaluated in in vitro or animal model systems that predict therapeutic efficacy in humans. Typically, prophylactic administration is performed in subjects before the onset of disease symptoms, or before or at an earlier stage of the disease. Detailed Implementation

[0216] This invention provides RNAi activators (especially siRNAs with oligonucleotide double strands) that silence the APOC3 gene using an RNAi mechanism, their conjugates, compositions, and uses. In cell-based and animal studies, the inventors have demonstrated that the siRNA molecules of this invention specifically and efficiently mediate RNAi, resulting in a significant inhibition of APOC3 gene expression. Therefore, methods and compositions comprising these siRNAs can be used to treat diseases or conditions that would benefit from downregulation of APOC3 expression (such as hyperlipidemia and cardiovascular disease). Using these siRNAs allows for targeted degradation of APOC3 mRNA involved in the regulation of triglyceride levels.

[0217] The present invention will now be described in detail. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these aspects is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, RNAi activators, conjugates, compositions, methods, pharmaceuticals, and uses according to any aspect of the present invention may include any such combination of features.

[0218] RNAi activator

[0219] In a first aspect, the present invention provides an RNAi activator targeting APOC3. The “RNAi” or “RNAi activator” according to the present invention is an activator molecule containing RNA (or a derivative thereof), wherein the activator is capable of mediating the targeted cleavage of messenger RNA (mRNA) (i.e., APOC3 mRNA) via the RNA-induced silencing complex (RISC) pathway.

[0220] Intrinsic RNAi (RNA interference) mechanisms in organisms typically involve a series of processes, including: Dicer processing long dsRNA into short 19-21 base pairs (bp) siRNA; siRNA binding to Ago protein to form an RNA-induced silencing complex (RISC); the AGO protein cleaving the sense strand of the siRNA and releasing it; subsequently, the mature RISC bound to the antisense strand cleaves the mRNA that is anticomplementary to the antisense strand through a sequence complementation mechanism. Based on this RNA interference mechanism, various artificial RNAi molecules with different structures have been developed. These structures can enter the RNAi pathway at different stages to achieve sequence-specific cleavage of target gene transcripts. See, for example, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211 (this literature is hereby incorporated herein by reference in its entirety for the purposes of this invention). Artificial RNAi molecules with such structures include, for example, siRNA molecules having a double-stranded region and one or two overhangs, long siRNA molecules that can serve as substrates for the Dicer enzyme, short hairpin RNA (shRNA) that can be processed by Dicer to produce siRNA structures, and long single-stranded siRNA molecules containing only an antisense strand. It is understood that these molecular forms all fall within the scope of the RNAi activators of the present invention. However, in some aspects, preferably, the RNAi activator according to the present invention refers to an siRNA molecule comprising an oligonucleotide double strand, i.e., a sense strand (sense oligonucleotide) and an antisense strand (antisense oligonucleotide), wherein the sense strand is complementary to at least a portion of the sequence of the antisense strand (e.g., at least 15, 16, 17, 18, or 19 consecutive nucleotides) to form an oligonucleotide double-stranded region. For example, siRNA molecules having a 19-21 nt double-stranded region and a 2-nt 3' overhang, or siRNA molecules containing a 20-22 nt antisense strand and a 15-16 nt short sense strand, thus having an atypical long overhang, are all within the scope of the present invention. Furthermore, molecules in which the sense and antisense strands of the disclosed RNAi molecule are covalently linked together by a single nucleotide strand or other linkage (e.g., the shRNA molecule described below) are also considered in this invention.

[0221] In this paper, the RNAi-related term "antisense strand" refers to an oligonucleotide chain in an RNAi activator containing a region complementary to a consecutive nucleotide of the target sequence. The RNAi-related term "sense strand" refers to an oligonucleotide chain in an RNAi activator containing a region complementary to at least a portion of the antisense strand to form a double-stranded region. In this paper, the antisense strand is sometimes also referred to as the "guide" strand, and the sense strand as the "passenger" strand.

[0222] For the purpose of inhibiting target mRNA expression, as those skilled in the art know, the oligonucleotide used as the sense strand does not participate in direct complementary binding to the target gene, and does not need to have perfectly complementary base pairing with the antisense strand oligonucleotide in the duplex region. Therefore, in some aspects, the sense strand (passenger strand) according to the invention may include at least one or more of the following properties: substantially complementary to the consecutive nucleotides of the antisense strand in the duplex region with the antisense strand, for example, at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary; having one or more additional nucleotides forming a protrusion or loop relative to the consecutive nucleotides of the antisense strand in the duplex region; and having one or more nucleotide gaps or vacancies relative to the consecutive nucleotides of the antisense strand in the duplex region. Similarly, for the purpose of inhibiting target mRNA expression, as those skilled in the art will understand, the antisense strand, serving as the guide RNAi for specific binding to the target mRNA, may also contain sequences that are not 100% complementary to the consecutive nucleotide regions of the target mRNA; for example, the complementarity may be at least 80%, at least 90%, or 95% complementarity; however, in some cases, 100% complementarity is preferred. According to the purposes of the invention, in some aspects, when considering the sequence motif of the antisense strand to the consecutive nucleotide complementarity of the target gene sequence, the presence of insertions and deletions is preferably not permitted. With regard to the sense and antisense strands of the invention, in some aspects, mismatches may be located inside or at the end of the double-stranded regions of the sense and antisense strands, for example, mismatches of 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0223] In some embodiments, the present invention provides an RNAi activator whose antisense strand comprises a sequence motif complementary to at least 18, at least 19, at least 20, or all consecutive nucleotides in a target gene sequence. In some embodiments, complementarity refers to at least 80% complementarity, i.e., nucleotide mismatches at no more than 20% of the positions in the consecutive nucleotide regions of the target gene sequence. In other embodiments, complementarity refers to at least 85%, 90%, or 95% complementarity. In still other embodiments, complementarity refers to 100% complementarity. In some embodiments, the target gene sequence is a sequence selected from any of SEQ ID NO:241-360, or a corresponding human APOC3 mRNA fragment. The correspondence between the mRNA fragment and the specified sequence can be determined by sequence alignment. In some embodiments, the present invention also considers the case where the sequence motif of the antisense strand is complementary to at least 18, at least 19, at least 20, or all consecutive nucleotides in a region comprising the target gene sequence and two nucleotides upstream and downstream of it. In some implementations, the antisense strand is, for example, about 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides long.

[0224] In some embodiments, the present invention also provides a positive strand complementary to at least a portion of the sequence in the antisense strand according to the invention, and a double-stranded RNAi activator comprising such an antisense strand and a positive strand. In some embodiments, the at least portion of the sequence in the antisense strand has, for example, at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides. In some embodiments, the complementarity refers to at least 70% complementarity, i.e., nucleotide mismatches at no more than 20% of the positions in the consecutive nucleotide regions. In other embodiments, the complementarity refers to at least 80%, 85%, 90%, 95%, or 100% complementarity. In some embodiments, the length of the positive strand is, for example, about 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides.

[0225] In this document, when double-stranded RNAi and siRNA are involved, the double-stranded region formed by the hybridization of the sense and antisense strands can be of any length that allows for the specific degradation of APOC3 target mRNA via the RISC pathway. In some embodiments, this length is 15 to 36 base pairs (“bp”), or can be any length within this range, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bp, and any subranges therein, including but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18- 26bp, 18-23bp, 18-22bp, 18-21bp, 18-20bp, 19-30bp, 19-26bp, 19-23bp, 19-22bp, 19-21bp, 19-20bp, 19bp, 20-30bp, 20-26bp, 20-25bp, 20-24bp, 20-23bp, 20-22bp, 20-21bp, 20bp, 21-30bp, 21-26bp, 21-25bp, 21-24bp, 21-23bp, 21-22bp, 21bp, 22bp, or 23bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 19 to about 30bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 19 to about 27bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 19 to about 25 bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 19 to about 23 bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 19 to about 21 bp. In some embodiments, the double-stranded RNAi and siRNA according to the present invention have a double-stranded region of about 21 bp.

[0226] In some embodiments, in the RNAi molecule according to the invention, the oligonucleotide double strands (i.e., the sense strand and the antisense strand) forming the double-stranded region need not, but may be covalently linked. Hereinafter, when the two strands are covalently linked by a hairpin loop, the structure is generally referred to as "shRNA" in this document and in the art. When the two strands are covalently linked by a means other than a hairpin loop, the linking structure is referred to as a "connector." For siRNA molecules having an shRNA structure, the nucleotide single strand constituting the "hairpin loop" is located between the 3' end of one strand forming the double-stranded structure and the 5' end of the corresponding other strand, and may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may contain 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.

[0227] In some embodiments, the double-stranded RNAi or siRNA according to the present invention may have one or two overhangs. When the 3' end of one strand of the double-stranded oligonucleotide constituting the double-stranded RNAi or siRNA extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand, unpaired nucleotides form overhangs. The length of the overhang can be at least one nucleotide; optionally, the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The overhang can contain nucleotides or modified nucleotides, or be composed of nucleotides or modified nucleotides. The overhang can be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang can be located at the 5' end and / or the 3' end of the antisense strand or the sense strand. In some preferred embodiments, the RNAi according to the present invention comprises a double-stranded body with overhangs consisting of an antisense strand and a sense strand, and preferably, the overhang is a single 3' overhang consisting of 1, 2, 3, or 4 nucleotides at the terminal end of the 3' end of the antisense strand. More preferably, the overhang is a single 3' overhang consisting of the terminal 2 nucleotides of the antisense strand. In some embodiments, the overhang on the antisense strand may be fully complementary or substantially complementary to the extension of the target gene sequence on APOC3 mRNA. In other embodiments, the overhang on the antisense strand may be complementary, partially complementary, or non-complementary to the extension of the target gene sequence on APOC3 mRNA. In some embodiments, the overhang on the antisense strand has a sequence selected from 5'GU3', 5'GG3', 5'AG3', 5'UG3', 5'GA3', 5'GC3', and 5'UU3', especially 5'GG3' or 5'AG3'.

[0228] The RNAi activator according to the invention can also have zero protrusions. In the case of zero protrusions, the RNAi activator has blunt ends, also called a "blunt RNAi activator," and such molecules lack 3' or 5' single-stranded nucleotide protrusions.

[0229] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a sequence motif differing by 0, 1, 2, or 3 nucleotides from the antisense and sense strand sequences of any of the compounds in Table 1 or Table A. In some embodiments, the differencing nucleotide is a nucleotide substitution.

[0230] In some preferred embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise an antisense strand sequence and a sense strand sequence selected from any of the compounds in Table 1, respectively. In some further preferred embodiments, the RNAi activator according to the present invention comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise an antisense strand sequence and a sense strand sequence selected from any of the compounds in Table A, respectively.

[0231] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a nucleotide sequence differing from the antisense strand sequence and the sense strand sequence of any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the antisense strand and the sense strand each comprise the antisense strand sequence and the sense strand sequence of any of the following compounds: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, B... PR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BPR-995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511.

[0232] In addition to the RNAi activators described above, the present invention also considers other RNAi activators that target the target gene sequences or target gene sequence regions shown in Table 1 (e.g., complementary to at least nucleotides 4 through 22 of the target gene sequence, or complementary to all nucleotides of the target gene sequence). Such RNAi activator molecules can be designed according to the Watson and Crick base pairing rules. For example, the RNAi activator can be an oligonucleotide containing a portion of the target gene sequence region antisense. Such antisense oligonucleotides can be about 5, 10, 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. However, in some embodiments, preferably, the RNAi activator is a double-stranded RNAi or siRNA having the characteristics described above.

[0233] In some embodiments, the RNAi of this disclosure having the aforementioned antisense and sense strands possesses one or more of the following properties: binding to the coding region of APOC3; not binding or hardly binding to mRNA or transcripts of other genes (no or almost no "off-target effects"); not inducing or hardly inducing immunogenicity; capable of binding to APOC3 mRNA sequence segments conserved across multiple animal species (including humans, mice, rats, cynomolgus monkeys, etc.), thereby facilitating the testing of RNAi activity using experimental animals; and / or lacking specific sequences known or suspected of reducing RNAi activity. In one embodiment, the APOC3-specific RNAi activator according to the invention is a siRNA possessing any one or more of these properties.

[0234] Modification of RNAi activators

[0235] Those skilled in the art will recognize that the RNAi molecule according to the invention can be unmodified (i.e., containing naturally occurring RNA nucleosides), but can also be (and preferably is) modified, as long as it retains the desired functional activity (i.e., capable of forming the desired double-stranded structure and allowing or mediating specific degradation of the target RNA via the RISC pathway). Such RNA modification can occur at the base moiety, sugar moiety, and / or phosphate linker of the nucleotide. Therefore, in a second aspect, this disclosure provides modified forms of the RNAi activator according to the first aspect of the invention.

[0236] As a non-limiting example, modified RNAi activators can be constructed using methods known in the art, employing chemical synthesis and enzymatic ligation reactions. For instance, modified RNAi activators can be chemically synthesized using naturally occurring nucleotides or nucleotides with various modifications (designed to reduce off-target effects and / or increase the biological stability of the molecule, or to increase the physical stability of the double strand formed between antisense and sense nucleic acids).

[0237] As another non-limiting example, the oligonucleotide chain of the RNAi molecule may include at least one modified nucleoside. Alternatively, the oligonucleotide of the RNAi molecule may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more modified nucleosides, or all nucleosides of the oligonucleotide may be modified nucleosides. For each of the multiple modified nucleosides in the RNAi molecule, the modification need not be identical.

[0238] In some respects, this disclosure covers modified forms of any RNAi activator specifically described herein (e.g., Table 1). In some embodiments, variants of said modifications contain the same sequence (i.e., the nucleoside base sequence) but contain different modifications in the phosphate backbone, ribose, and bases. Examples of modifications that may be mentioned include, for example: 2'-O-methyl nucleotide modifications, 2'-fluoronucleotide modifications, 2'-deoxyribonucleotide modifications, locked nucleotide (LNA) modifications, unlocked nucleotide (UNA) modifications, threonucleotide (TNA) modifications, conformation-restricted nucleotide modifications, 2'-O-methoxyethyl nucleotide modifications, debased nucleotide modifications, 2'-amino nucleotide modifications, 2'-O-allyl-nucleotide modifications, 2'-C-alkyl-nucleotide modifications, 2'-O-alkyl nucleotides, morpholinonucleotides, aminophosphamide nucleotide modifications, nucleotide modifications with non-natural bases, tetrahydropyranonucleotide modifications, 1,5-dehydrated hexadiol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotide modifications containing thiophosphate groups, nucleotide modifications containing methylphosphate groups, nucleotide modifications containing 2'-phosphate groups, nucleotide modifications containing 5'-phosphate groups, thermostable nucleotide modifications, and 2-O-(N-methylacetamide) nucleotide modifications; and combinations thereof.

[0239] In some embodiments, the RNAi activator according to the invention may have one or more modifications inside the nucleic acid molecule or at one or both ends thereof. Examples of nucleoside base modifications, ribose moiety modifications, and phosphate backbone modifications that can be used for RNAi according to the invention are described below. Furthermore, a summary list of some oligonucleotide modifications known in the art can be found in PCT Publication WO 2003070918. However, it is understood that the modifications that can be used for RNAi according to the invention are not limited thereto.

[0240] Nucleoside base modification

[0241] “G,” “C,” “A,” “T,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such a substitution. Examples of such nucleoside base modifications that can be used to generate RNAi activators include the substitution of nucleotides containing uracil, guanine, or adenine with nucleotides containing, for example, inosine; and the replacement of adenine and cytosine in oligonucleotides with guanine and uracil, respectively, to form a GU Wobble base pair with the target mRNA. In addition, other examples of modified nucleoside bases that can be used to generate RNAi activators include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7 -Methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl queosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. These modified nucleoside bases are all within the scope of this invention.

[0242] Sugar modification

[0243] Compared to the ribose moiety present in natural RNA, the oligonucleotides of the RNAi of the present invention can contain one or more nucleosides with modified sugar moieties (i.e., glycosidic modifications). Numerous glycosidic modified nucleosides have been developed, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance, and off-target effects. Glycosidic modification can include modifications by replacing the naturally present 2'-OH group on the ribose ring of the RNA nucleoside with other groups. Furthermore, substituents can be introduced, for example, at the 2', 3', 4', or 5' positions of the sugar ring. The following are some specific examples of 2'-modified nucleotides:

[0244] In some preferred embodiments, the RNAi activator of the present invention comprises at least one 2'-modified nucleotide (i.e., a 2' sugar-modified nucleoside). Such modified nucleosides include nucleosides having a substituent at the 2' position other than –OH (2'-substituted nucleosides). A variety of 2'-substituted nucleosides have been developed for use in RNAi molecules, and many 2'-substituted nucleosides have been found to possess beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can provide oligonucleotides with enhanced binding affinity and / or increased nuclease resistance. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA nucleosides (MOE), 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. Other examples can be found, for instance, in Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. In some embodiments, the RNAi activator according to the invention comprises at least one 2'-modified nucleotide, said 2'-modification being selected from 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-allyl, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, the RNAi activator according to the invention comprises at least one 2'-modified nucleoside selected from the following: 2'-O-alkyl-RNA nucleoside, 2'-O-methyl-RNA nucleoside, 2'-alkoxy-RNA nucleoside, 2'-O-methoxyethyl-RNA nucleoside (MOE), 2'-amino-DNA nucleoside, 2'-fluoro-RNA nucleoside, and 2'-F-ANA nucleoside. In some embodiments, the RNAi activator according to the invention comprises one or more deoxyribonucleosides.

[0245] Nucleotide analogues

[0246] In some embodiments, the RNAi activator of the present invention comprises at least one nucleotide analog. Available nucleotide analogs include, for example, nucleotide analogs formed by replacing the ribocycle structure with one of the following: a hexose ring (HNA), a threonose ring (TNA), a locked nucleic acid structure (LNA, a bicyclic ring with a double-base bridge between the C2 and C4 carbons on the ribocycle), or a non-locked nucleic acid structure (UNA, a ribocycle lacking a bond between the C2 and C3 carbons). Other examples of available nucleotide analogs include, for example, bicyclic hexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798); and peptide nucleic acids (PNA) or morpholino nucleic acids.

[0247] Here are some specific examples of nucleotide analogs:

[0248] In some embodiments, the RNAi activator according to the invention comprises at least one nucleotide analog selected from the following: LNA nucleotide, UNA nucleotide and TNA nucleotide.

[0249] In some preferred embodiments of the invention, the RNAi activator according to the invention comprises a 4'-modified threonucleotide as shown in formula (A). In some embodiments, the threonucleotide is located at the 5' terminal of the positive strand of the RNAi activator. For a description of 4'-modified threonucleotides that can be used in the present invention, see the applicant's co-pending PCT application (PCT / CN2024 / 121099). This application is hereby incorporated herein by reference in its entirety for the purposes of this invention.

[0250] Phosphate ester backbone modification

[0251] Various phosphate backbone modifications for use in RNAi molecules are known in the art. Such modifications include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates and chiral phosphonates), phosphonites, and aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates). Furthermore, modifications to inter-nucleotide links or backbones that do not contain phosphorus atoms are also within the scope of this invention. A description of phosphate backbone modifications can be found in WO 2023 / 076451, which is incorporated herein by reference for the purposes of this invention. In some embodiments, a thiophosphate backbone modification is introduced into the nucleoside of the oligonucleotide of the RNAi molecule of the present invention. This modification can enhance the nuclease stability of said oligonucleotide.

[0252] In some embodiments, the RNAi activator according to the invention may optionally also comprise a chemical modification at the 5' and / or 3' ends, i.e., a non-nucleotide or nucleoside chemical moiety linked to the end of the oligonucleotide chain (sense and / or antisense strand) of RNAi. Examples of chemical moieties linked to the 3' end of the oligonucleotide chain can be found, for example, in WO 2005 / 021749 and WO 2007 / 128477. Examples of chemical moieties linked to the 5' end of the oligonucleotide chain may include, but are not limited to, a phosphate ester modification at the 5' end, preferably wherein said modification is selected from: 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analogues, and 5'-thiophosphate (5'-PS). In some preferred embodiments, the RNAi activator according to the invention comprises a 5'-(E)-vinylphosphonate modification at the 5' end of the antisense strand.

[0253] Exemplary modified RNAi activators

[0254] In some embodiments, the present invention provides modified RNAi activators, particularly modified forms of RNAi activators comprising the sense and antisense strand sequences of compounds listed in Table 1 or Table A. In some embodiments, the modification comprises at least one phosphate backbone modification and / or at least one nucleotide modification. In some embodiments, the modification comprises at least one 2'-modified nucleotide, wherein the 2'-modification is selected from 2'-deoxy, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl (2'-O-MOE). In some embodiments, the modification comprises at least one modified nucleotide selected from: 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-O-methoxyethyl modified nucleotide, deoxynucleoside nucleotide, UNA, LNA, and TNA (threonucleotide). In some embodiments, the TNA modification comprises a 4'-modified threonucleotide of formula (A), preferably located at the 5' terminal of the sense strand of the RNAi activator.

[0255] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99500101, BPR-99551205, BPR-99516101, BPR-99506301, BPR-99515301, BPR-99 518801, BPR-99504601, BPR-99505901, BPR-99513801, BPR-99513601, BPR-99508401, BPR-99501901, BPR-99501301, BPR-99521001, BPR-99500901, BPR-99504901, BPR-99505601, BPR-99503801, BPR-99513501, BPR-99500301, BPR-99508701, BPR-99509701. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.5 nM achieves a relative inhibition rate of over 60% against APOC3 expression in HepG2 cell-based assays. Preferably, the determination is performed according to the method described in Example 2.

[0256] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99500101, BPR-99551205, BPR-99516101, BPR-99506301, BPR-99515301, BPR-99518801, BPR-99504601, BPR-99505901, BPR-99513801, BPR-99513601, BPR-99508401, BPR-99501901, BPR-99501301, BPR-99521001, BPR-99500901, BPR-99504901, BPR-99505601, BPR-99503801, BPR-99513501. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.5 nM in HepG2 cell-based assays exhibits a relative inhibition rate of over 70% against APOC3 expression. Preferably, the assay is performed according to the method described in Example 2.

[0257] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99500101, BPR-99551205, BPR-99516101, BPR-99506301, BPR-99515301, BPR-99518801, BPR-99504601, BPR-99505901, BPR-99513801. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 80% against APOC3 expression at a concentration of 0.5 nM in HepG2 cell-based assays. Preferably, the determination is performed according to the method described in Example 2.

[0258] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99500101, BPR-99551205, BPR-99516101, BPR-99506301, and BPR-99515301. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 90% against APOC3 expression at a concentration of 0.5 nM in a HepG2 cell-based assay. Preferably, the assay is performed according to Example 2.

[0259] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401, BPR-99551143, BPR-99505 557, BPR-99505551, BPR-99551152, BPR-99551206, BPR-99500101, BPR-99551156, BPR-99551205, BPR-99551153, B PR-99551201, BPR-99551141, BPR-99517451, BPR-99517450, BPR-99517420, BPR-99521051, BPR-99521050, BPR-99 521060, BPR-99516174, BPR-99516101, BPR-99516150, BPR-99515301, BPR-99513801, BPR-99505558, BPR-9951740 1. BPR-99505562, BPR-99517406, BPR-99505563, BPR-99517407, BPR-99550505, BPR-99550903, BPR-99550601, BPR -99550701, BPR-99550801, BPR-99551001, BPR-99521011, BPR-99521020, BPR-99504011, BPR-99516120, BPR-99504001, BPR-99500114, BPR-99504660, BPR-99518801, BPR-99500120, BPR-99516134, BPR-99500150, BPR-99506301. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM in HepG2 cell-based assays exhibits a relative inhibition rate of over 70% against APOC3 expression. Preferably, the assay is performed according to the method described in Example 2.

[0260] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401, BPR-99551143, BPR-99505557, BPR-99505551, BPR-99551152, BPR-99551206, BPR-99500101, BPR-99551156, BPR-99551205, BPR-99551153, BPR-99551201, BPR-99 551141, BPR-99517451, BPR-99517450, BPR-99517420, BPR-99521051, BPR-99521050, BPR- 99521060, BPR-99516174, BPR-99516101, BPR-99516150, BPR-99515301, BPR-99513801, BPR -99505558, BPR-99517401, BPR-99505562, BPR-99517406, BPR-99505563, BPR-99517407, BPR-99550505, BPR-99550903, BPR-99550601, BPR-99550701, BPR-99550801, BPR-99551001. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM exhibits a relative inhibition rate of over 80% against APOC3 expression in HepG2 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0261] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401, BPR-99551143, BPR-99505557, BPR-99505551, BPR-99505558, BPR-99517406, BPR-99505563. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 90% against APOC3 expression at a concentration of 0.1 nM in a HepG2 cell-based assay. Preferably, the assay is performed according to Example 2.

[0262] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401, BPR-99505557, BPR-99505551, BPR-99551143, BPR-99517451, BPR-99517450, BPR-99551156, BPR-99551201, BPR-99517420, BPR-99551152, BPR-99551206, BPR-99500150, BPR-99551153, BPR-99 551141, BPR-99521051, BPR-99516120, BPR-99521050, BPR-99521020, BPR-99521060, BPR-99516134, BPR-99521011, BPR-99516174. Preferably, the RNAi activator, at a concentration of 0.04 nM, exhibits a relative inhibition rate of over 70% against APOC3 expression in a HepG2 cell-based assay compared to the solvent control. Preferably, the assay is performed according to the method described in Example 2.

[0263] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401, BPR-99505557, BPR-99505551, BPR-99551143, BPR-99517451, BPR-99517450, BPR-99551156, BPR-99551201. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 80% against APOC3 expression at a concentration of 0.04 nM in HepG2 cell-based assays. Preferably, the determination is performed according to the method described in Example 2.

[0264] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99505554, BPR-99551401. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 90% against APOC3 expression at a concentration of 0.04 nM in a HepG2 cell-based assay. Preferably, the assay is performed according to Example 2.

[0265] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99517451, BPR-99517450, BPR-99516120, BPR-99505551, BPR-99551152, BPR-99551153, BPR-99505554, BPR-99551206, BPR-99551156, BPR-99505557, BPR-99551205, BPR-99516134, BPR-99551201, BPR-99517420, BPR-99504001, BPR-99516174, BPR-99516150, BPR-99513801, BPR-99504660, BPR-99521051, BPR-99515301. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.02 nM exhibits a relative inhibition rate of over 50% against APOC3 expression in HepG2 cell-based assays. Preferably, the determination is performed according to the method described in Example 2.

[0266] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99517451, BPR-99517450, BPR-99 516120, BPR-99505551, BPR-99551152, BPR-99551153, BPR-99505554, BPR-99551206, BPR-99551156, BPR-99505557, BPR-99551205, BPR-99516134, BPR-99551201, BPR-99517420, BPR-99504001, BPR-99516174. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.02 nM exhibits a relative inhibition rate of over 60% against APOC3 expression in HepG2 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0267] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99517451, BPR-99517450, BPR-99516120, BPR-99505551, BPR-99551152, BPR-99505554, and BPR-99505557. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 70% against APOC3 expression at a concentration of 0.02 nM in a HepG2 cell-based assay. Preferably, the assay is performed according to Example 3.

[0268] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99505554, BPR-99 518814, BPR-99551401, BPR-99551152, BPR-99516134, BPR-99517420, BPR-99551206, BPR-99516174, BPR-99505 557. BPR-99505551, BPR-99518850, BPR-99517451, BPR-99516150, BPR-99517413, BPR-99517450, BPR-99517433, BPR-99500150, BPR-99521051, BPR-99521050, BPR-99500120, BPR-99504001, BPR-99521060, BPR-99516111, BPR -99500114, BPR-99551143, BPR-99551153, BPR-99551141, BPR-99518820, BPR-99521011, BPR-99504660, BPR-99 516120, BPR-99513850, BPR-99518831, BPR-99513814, BPR-99521001, BPR-99521002, BPR-99513833, BPR-99521020, BPR-99500160, BPR-99504650, BPR-99513843, BPR-99513820, BPR-99517441, BPR-99500141, BPR-99551110. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM exhibits a relative inhibition rate of over 60% against APOC3 expression in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0269] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence that differs from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99505554, BPR-99518814, BPR-99551401, BPR-99551152, BPR-99516134, BPR-99517420, BPR-99551206, BPR-99516174, BPR-99505557, BPR-99505551, BPR-99518850, BPR-99517451, BPR-99516150, BPR-99517413, BPR-99517450, BPR-99517433, BPR-99500150, BPR-99521051, BPR-99521050, BPR-99500120, BPR-99504001, BPR-99521060, BPR-99516111, BPR-99500114, BPR-99551143, BPR-99551153, BPR-99551141, BPR-99518820, BPR-99521011, BPR-99504660, BPR-99516120, BPR-99513850, BPR-99518831, BPR-99513814, BPR-99521001, BPR-99521002, BPR-99513833, BPR-99521020, BPR-99500160, BPR-99504650, BPR-99513843, BPR-99513820, BPR-99551110. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM exhibits a relative inhibition rate of over 70% against APOC3 expression in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0270] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99505554, BPR-99518814, BPR-99551401, BPR-99551152, BPR-99516134, BPR-99517420, BPR-99551206, BPR-99516174, BPR-99505557, BPR-99 505551, BPR-99518850, BPR-99517451, BPR-99516150, BPR-99517413, BPR-99517450, B PR-99517433, BPR-99500150, BPR-99521051, BPR-99521050, BPR-99500120, BPR-99504 001, BPR-99521060, BPR-99516111, BPR-99500114, BPR-99551143, BPR-99551153, BPR-99551141, BPR-99518820, BPR-99521011, BPR-99504660, BPR-99516120, BPR-99551110. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM exhibits a relative inhibition rate of over 80% against APOC3 expression in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0271] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99505554, BPR-99518814, BPR-99 551401, BPR-99551152, BPR-99516134, BPR-99517420, BPR-99551206, BPR-99516174, BPR-99505557, BPR-99505551, BPR-99518850, BPR-99517451, BPR-99516150, BPR-99517413, BPR-99517450, BPR-99517433, BPR-99500150, BPR-99551110. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.1 nM achieves a relative inhibition rate of over 90% against APOC3 expression in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0272] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99517. 451. BPR-99517450, BPR-99551152, BPR-99551206, BPR-99516134, BPR-99505557, BPR-99521051, BPR-995 05551, BPR-99521050, BPR-99505554, BPR-99551401, BPR-99500120, BPR-99518814, BPR-99500150, BPR-99 500114, BPR-99551202, BPR-99551110, BPR-99551403, BPR-99551601, BPR-99551701, BPR-99505558, BPR- 99551801, BPR-99551501, BPR-99551901, BPR-99552101, BPR-99517420, BPR-99518850, BPR-99517433, BPR -99516174, BPR-99504001, BPR-99516150, BPR-99517413, BPR-99552001, BPR-99552201, BPR-99521060, BPR-99551143, BPR-99551153, BPR-99551141, BPR-99516111, BPR-99521011, BPR-99513850, BPR-99518820. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.04 nM in Huh7 cell-based assays exhibits a relative inhibition rate of over 60% against APOC3 expression. Preferably, the assay is performed according to the method described in Example 2.

[0273] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR -99551201, BPR-99517451, BPR-99517450, BPR-99551152, BPR-99551206, BPR-99516134, BPR- 99505557, BPR-99521051, BPR-99505551, BPR-99521050, BPR-99505554, BPR-99551401, BPR-99 500120, BPR-99518814, BPR-99500150, BPR-99500114, BPR-99551202, BPR-99551110, BPR-995 51403, BPR-99551601, BPR-99551701, BPR-99505558, BPR-99551801, BPR-99551501, BPR-99551 901, BPR-99552101, BPR-99517420, BPR-99518850, BPR-99517433, BPR-99516174, BPR-99504001, BPR-99516150, BPR-99517413, BPR-99552001, BPR-99552201, BPR-99521060, BPR-99551143. Preferably, relative to the solvent control, the RNAi activator at a concentration of 0.04 nM exhibits a relative inhibition rate of over 70% against APOC3 expression in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0274] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99517451, BPR-99517450, BPR-99551152, BPR-99551206, BPR-99516134, BPR-99505557, BPR-99521051, BPR-99505551, BPR-99521050, BPR-99505554, BPR-99551401, BPR-99500120, BPR-99518814, BPR-99500150, BPR-99500114, BPR-99 551202, BPR-99551110, BPR-99551403, BPR-99551601, BPR-99551701, BPR-99505558, BPR-99551801, BPR-99551501, BPR-99551901, BPR-99552101. Preferably, the RNAi activator, at a concentration of 0.04 nM, exhibits a relative inhibition rate of over 80% against APOC3 expression in a Huh7 cell-based assay compared to the solvent control. Preferably, the assay is performed according to the method described in Example 2.

[0275] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence that differs from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99517451, BPR-99517450, BPR-99551152, BPR-99551206, BPR-99551202, BPR-99551110, BPR-99551403, BPR-99551601, BPR-99551701, BPR-99505558, BPR-99551801. Preferably, the RNAi activator, relative to the solvent control, exhibits a relative inhibition rate of over 85% against APOC3 expression at a concentration of 0.04 nM in Huh7 cell-based assays. Preferably, the assay is performed according to the method described in Example 2.

[0276] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551156, BPR-99551201, BPR-99551202, BPR-99551110, BPR-99551403, BPR-99551601, BPR-99551701, BPR-99505558. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 90% against APOC3 expression at a concentration of 0.04 nM in a Huh7 cell-based assay. Preferably, the assay is performed according to Example 2.

[0277] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand each comprise a modified nucleotide sequence that differs from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551111, BPR-99551114, BPR-99551152, BPR-99551211, BPR-99551201, BPR-99551417, BPR-99551407, BPR-99551705, BPR-99551717, BPR-99551815, and BPR-99551905. Preferably, the RNAi activator, at a concentration of 0.02 nM, exhibits a relative inhibition rate of over 70% against APOC3 expression in a Huh7 cell-based assay compared to the solvent control. Preferably, the assay is performed according to the method described in Example 2.

[0278] In some embodiments, the present invention provides an RNAi activator comprising an antisense strand and a sense strand, wherein the antisense strand and sense strand each comprise a modified nucleotide sequence differing from a modified antisense strand and a modified sense strand selected from any of the following compounds by no more than 3, 2, 1, or 0 nucleotides, respectively; preferably, the RNAi activator comprises a modified antisense strand and a modified sense strand selected from the following compounds: BPR-99551110, BPR-99551601, BPR-99505558, BPR-99551403. Preferably, the RNAi activator, relative to a solvent control, exhibits a relative inhibition rate of over 70% against APOC3 expression at a concentration of 0.01 nM in a Huh7 cell-based assay. Preferably, the assay is performed according to Example 2.

[0279] In some preferred embodiments, the RNAi activator comprises a modified sense strand, a modified antisense strand, or preferably both a modified sense strand and a modified antisense strand of any of the compounds shown in Table 2 or Table 3.

[0280] In some preferred embodiments, the RNAi activator comprises a modified sense strand, a modified antisense strand, or preferably both a modified sense strand and a modified antisense strand selected from any of the compounds in Table B or Table C.

[0281] RNAi conjugates

[0282] In a third aspect, the present invention provides conjugate compounds formed by covalently linking an oligonucleotide double strand of RNAi according to the present invention to a non-nucleotide portion (conjugate portion). In this document, such conjugates are also referred to as "RNAi conjugates" or "siRNA conjugates".

[0283] The conjugation of the oligonucleotide duplex of the RNAi of the present invention to one or more non-nucleotide moieties can, for example, improve the pharmacological properties of the RNAi by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugated portion modulates or enhances the pharmacokinetic properties of the RNAi of the present invention by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular, the conjugation can direct the oligonucleotide to a specific organ, tissue, or cell type and thus enhance the effectiveness of the RNAi of the present invention in such organ, tissue, or cell type. Simultaneously, the conjugation can reduce the activity of the RNAi of the present invention in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

[0284] Regarding the conjugate moieties and conjugation modifications applicable to RNAi, descriptions are provided in Vajinder Kumar, Targeted delivery of oligonucleotides using multivalent protein–carbohydrate interactions, Cite this: Chem. Soc. Rev., 2023, 52, 1273; Rosemary Kanasty, Delivery materials for siRNA therapeutics, NATURE MATERIALS, VOL 12, NOVEMBER 2013; Wanyi Tai, Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211; and WO 93 / 07883 and WO 2013 / 033230, which are incorporated herein by reference.

[0285] In some embodiments, the conjugate portion used in the RNAi activator of the present invention may be selected from: antibodies, peptides, peptide mimics, aptamers, small chemical compounds, lipids, cell-penetrating peptide polymers, or nanoparticle conjugates.

[0286] In some embodiments, the RNAi of the conjugates of the present invention is conjugated to the desialyl glycoprotein receptor (ASGPR) ligand via chemical bonds or chemical groups.

[0287] In some embodiments, the conjugates of the present invention may comprise a suitable ligand-binding molecule as the conjugate portion. For example, as in International Patent Application WO 91 / 04753, oligonucleotides may be conjugated to ligand-binding molecules for therapeutic application to recognize cell surface molecules. Such ligand-binding molecules may comprise, for example, antibodies against cell surface antigens, antibodies against cell surface receptors, ligands having corresponding cell surface receptors, antibodies against such ligands, or antibodies recognizing a complex of said ligand and its receptor. Methods for conjugating ligand-binding molecules to oligonucleotides are detailed in WO 91 / 04753. Additionally, conjugation methods and methods for improving cellular uptake are also described in the following International Patent Applications: WO 9640961, WO 9964449, WO 9902673, WO 9803533, WO 0015265, and U.S. Patents 5856438 and 5138045.

[0288] In some embodiments, the chemical component that can be used as a conjugate moiety is a peptide, such as a poly(L-lysine) and an antennal foot transport peptide that can significantly increase cell permeability. Such conjugates are described by Lemaitre et al., "Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site," Proc. Natl. Acad. Sci. USA, 84:648-652, 1987; U.S. Patent Nos. 6,166,089 and 6,086,900.

[0289] In other embodiments, the conjugate portion of the RNAi activator of the present invention may be selected from sugars, cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0290] In some embodiments, for example, the conjugated moiety may include a lipid moiety, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553); bile acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioether, such as hexyl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533); an aliphatic chain, such as dodecyl glycol or undecyl residue (Saison-Behmoaras et al., EMBO). J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777); polyamines (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 14:969) Lett., 1995, 36:3651); palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229) or octadecylamine or hexano-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). In some embodiments, cholesterol conjugates are preferred because this moiety can enhance targeting to hepatocytes (sites of APOC3 expression).

[0291] In some embodiments, the conjugation moiety is or comprises a sugar moiety. The sugar conjugation moiety includes, but is not limited to, galactose, lactose, N-acetylgalactosamine, mannose, and mannose-6-phosphate. Sugar conjugations can be used to enhance delivery or activity in a range of tissues such as the liver and / or muscle. See, for example, EP 1495769, WO 99 / 65925, Yang et al., Bioconjug Chem (2009) 20(2):213-21; Zatsepin and Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

[0292] In other embodiments, preferably, the conjugated moiety of the RNAi activator of the present invention is a compound moiety capable of binding to the asialic acid glycoprotein receptor (ASGPR). For example, monovalent, divalent, and trivalent N-acetylgalactosamine and its derivatives conjugated moieties are all suitable for binding to ASGPR and are therefore suitable for use in the present invention. For examples of ASGPR-targeting conjugated moieties, see WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620 (these documents are incorporated herein by reference). Such conjugations can be used to enhance hepatic uptake of RNAi oligonucleotides while reducing their presence in the kidneys, thus increasing the liver / kidney ratio of the conjugated oligonucleotide compared to the same unconjugated form. Such oligonucleotide conjugates and their synthesis can also be found in the following literature: Manoharan’s comprehensive review, cited in Antisense Drug Technology, Principles, Strategies, and Applications, ed. STCrooke, Chapter 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

[0293] In some embodiments, the conjugate moiety is attached to the 5' and / or 3' terminal nucleotide of the sense strand of the RNAi of the present invention and optionally the 3' terminal nucleotide of the antisense strand, optionally linked by a thiophosphate group or a phosphate group. In some cases, the conjugate moiety may also be conjugated to the internal sequence of the RNAi oligonucleotide. In some embodiments, the conjugate moiety may be attached to a phosphate group, a 2′-hydroxyl group, or a base of the nucleotide. In other embodiments, the conjugate moiety may be attached to a 3′-hydroxyl group of the nucleotide, in which case the nucleotides are linked by a 2′-5′ phosphodiester bond. When the conjugate moiety is attached to the end of an RNAi (such as siRNA) oligonucleotide chain, the conjugate moiety is typically attached to a phosphate group of the nucleotide; when the conjugate moiety is attached to the internal sequence of an RNAi (such as siRNA) oligonucleotide, the conjugate moiety is typically attached to a sugar ring of the ribose or a base.

[0294] The conjugate portion can be directly linked to the oligonucleotide duplex of the RNAi of the present invention or linked via a linker portion (e.g., a adapter). In some embodiments of the invention, the RNAi conjugate of the present invention may optionally include a linker region located between the oligonucleotide duplex of the RNAi and the conjugate portion. In some embodiments, the linker is a biocleavable linker. Such a linker typically contains or is composed of physiologically unstable bonds that are cleavable under normal in vivo conditions in mammals or under conditions similar to those in mammals. The conditions include conditions present in mammalian cells or similar chemical conditions, such as pH, temperature, oxidative or reducing conditions, or concentrations of substances and salts. The conditions also include enzymatic activities present normally in mammalian cells, such as those from proteases, hydrolases, or nucleases. In some embodiments, the linker need not be biocleavable, but may have the primary function of covalently linking the conjugate portion to the oligonucleotide. Such a linker may contain repeating units, such as amino acid units or chain structures or oligomers of aminoalkyl groups. For example, the linker may be an aminoalkyl group, such as a C2–C36 aminoalkyl group, for example including C6 to C12 aminoalkyl groups. In some implementations, conjugates with such linkers can be separated from RNAi oligonucleotides by processing with the Dicer enzyme.

[0295] In some embodiments, the adapter may include a branching region. Hereinafter, the term "branching region" means a compound portion capable of covalently coupling two or more entities together. In some embodiments, adapters having branching regions can be used to conjugate multiple entities, such as N-acetylgalactosamine moieties, to oligonucleotides of the RNAi of the present invention. Adapters with branching regions that can be used for this purpose are known in the art and include, but are not limited to, amino acids (including natural and non-natural amino acids), peptides and their derivatives, sugar units and their derivatives, aromatic-substituted compounds and their derivatives, substituted hydrocarbon groups and their derivatives, triazole-containing derivatives, etc. See, for example, CN104651408A, CN113286888A, WO2015 / 173208, and WO2023 / 076451.

[0296] Exemplary desialyl glycoprotein receptor (ASGPR) ligand conjugates

[0297] In one embodiment of the invention, the oligonucleotide duplex of the RNAi of the present invention is preferably conjugated to a conjugation moiety that can be used to deliver the oligonucleotide duplex to the liver of a subject, for example, to increase the uptake of the oligonucleotide duplex by hepatocytes. ASGP-R is a hepatocyte-specific endocytic receptor that specifically recognizes and binds to ligand molecules having galactose or galactose derivatives as terminal glycosyl groups. It has been shown that galactose derivative modifications can be used to construct drug carriers that deliver drugs to hepatocytes via ASGP-R-mediated endocytosis. Therefore, in some embodiments, the conjugation moiety used for the RNAi of the present invention comprises an ASGP-R targeting moiety (also referred to herein as an "ASGPR ligand"). The ASPG-R targeting moiety can be selected from galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine (GalNAc), N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, and other galactose derivatives. In this document, the term "galactose derivative" includes galactose and galactose derivatives with an affinity for ASPG-R equal to or greater than that for galactose. Preferred galactose derivatives are N-acetylgalactosamine (GalNAc) or GalNAc derivatives. Other sugars with affinity for desialyl glycoprotein receptors may also be used, including but not limited to galactosamine, N-butyrylgalactosamine, and N-isobutyrylgalactosamine. The affinity of many galactose derivatives for desialyl glycoprotein receptors has been studied (see, for example, Jobst, ST and Drickamer, K. JB. C. 1996, 271, 6686) or using methods common in the art.

[0298] In some embodiments, the ASGPR ligand constituting the conjugate moiety is monovalent, containing a single targeting moiety capable of binding to ASGPR. In other embodiments, the ASGPR ligand constituting the conjugate moiety is polyvalent, containing multiple targeting moieties capable of binding to ASGPR. In some embodiments, the ligand is a monovalent galactose ligand, providing a single galactose derivative capable of serving as an ASGPR targeting moiety. In some embodiments, the ligand is a polyvalent galactose ligand, providing multiple galactose derivatives capable of serving as ASGPR targeting moieties. Hereinafter, when the galactose derivative is N-acetylgalactosamine (GalNAc) or a GalNAc derivative, the ligand is also referred to as a GalNAc ligand, for example, providing one, two, three, or four GalNAc or GalNAc derivative structural moieties, in a monovalent, divalent, trivalent, or tetravalent manner.

[0299] In some embodiments, after the siRNA molecule according to the invention forms an siRNA conjugate with a conjugation portion containing a galactose derivative (e.g., GalNAc) as a targeting group, the molar ratio of the siRNA molecule to the galactose derivative (e.g., GalNAc) in the siRNA conjugate can be any suitable ratio, such as 1:1, 1:2, 1:3 or 1:4.

[0300] In some embodiments, the conjugation portion is a monovalent ASGPR ligand comprising a single terminal galactose derivative sugar moiety capable of binding to ASGPR. In other embodiments, the conjugation portion is a polyvalent ASGPR ligand comprising a galactose cluster, such as a divalent, trivalent, or tetravalent ligand, wherein the galactose cluster may contain, for example, two, three, or four terminal galactose derivative sugar moieties capable of binding to ASGPR.

[0301] In this document, a galactose cluster refers to a molecule having multiple, for example, two to four, terminal galactose derivatives. For example, a preferred galactose cluster has three terminal galactosamines or galactosamine derivatives, each exhibiting an affinity for ASGPR, wherein the terminal galactose derivatives are linked to the molecule via their C-1 or C-6 carbons. Such galactose clusters are also referred to in the art as tri-antennatic galactoses, trivalent galactoses, and galactose trimers. It is known that tri-antennatic galactose derivative clusters can bind to ASGP-R with a greater affinity than binary or monoantennatic galactose derivative structures (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, Biol. Chem., 257, 939-945).

[0302] In some embodiments, the galactose cluster to be conjugated with the oligonucleotide may comprise two or preferably three galactose derivatives respectively linked to a central branch point. In some embodiments, the galactose derivatives are preferably linked to the branch point by means of a linker or spacer group. In some embodiments, the preferred spacer group is a flexible hydrophilic spacer group (US Patent 5,885,968; Biessen et al., J. Med. Chem. 1995, Vol. 39, pp. 1538-1546). A preferred flexible hydrophilic spacer group is a PEG spacer group. A preferred PEG spacer group is a PEG3 spacer group (three ethylene units). In some aspects, the branch point can be any small molecule that allows the conjugation of the two or three galactose derivatives and further allows the branch point to conjugate to the oligonucleotide. An exemplary branch point group is dilysine. A dilysine molecule contains three amino groups that can be linked to three galactose derivatives and a carboxyl reactive group that can be linked to the oligonucleotide. Another exemplary branch point group is a tetravalent linker based on trihydroxyalkylmethane, such as a tetravalent linker based on trihydroxymethylmethane with the following structure:

[0303] The linking group comprises three oxygen groups that can be linked to three galactose derivatives and a central branching carbon atom that can be linked to an oligonucleotide. In some embodiments, each galactose derivative (sugar moiety, such as GalNAc) in the galactose cluster can be linked to the oligonucleotide via a linker, such as a polyethylene glycol (PEG) linker, such as two, three, four, five, or six PEG linkers. In this case, the linker, such as the PEG portion, can form a spacer group between the sugar moiety of the galactose derivative and the branching group. See, for example, WO2015 / 173208.

[0304] In other embodiments, the sugar moiety (e.g., GalNAc) or sugar-connector moiety (e.g., sugar-PEG moiety) to be conjugated with the oligonucleotide may also be covalently linked (conjugated) to the oligonucleotide by means of a branching group or branching region, such as an amino acid or peptide suitably containing two or more amino groups (e.g., 3, 4, or 5), such as lysine, dilysine, trilysine, or tetralysine. In some embodiments, the trilysine molecule is optionally provided with four amino groups that can thereby connect a sugar conjugation group such as a galactose derivative (e.g., GalNAc) and a carboxyl reactive group that can thereby connect the trilysine to the oligonucleotide. In other embodiments, other conjugation moieties, such as lipophilic / hydrophobic moieties, may also be linked to the oligonucleotide via the aforementioned lysine residues.

[0305] In other embodiments, the galactose cluster to be conjugated with the oligonucleotide comprises a peptide linker connected to the oligonucleotide via a bimodal linker, such as a Tyr-Asp(Asp) tripeptide or an Asp(Asp) dipeptide. Additionally, alternative branching molecules may be selected from 1,3-bis-[5-(4,4′-dimethoxytriphenylmethoxy)pentylamino]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphorimide (Glen Research catalog number: 10-1920-xx), tri-2,2,2-[3-(4,4'-dimethoxytriphenylmethoxy)propoxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphorimide (Glen Research catalog number: 10-1922-xx), tri-2,2,2-[3-(4,4'-dimethoxytriphenylmethoxy)propoxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorimide and 1-[5-(4,4'-dimethoxy-triphenylmethoxy)pentylamide]-3-[5-fluorenmethoxy-carbonyl-oxy-pentylamide]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphorimide (Glen Research catalog number: 10-1925-xx). WO 2014 / 179620 and European application number 14188444.5 describe the generation of various GalNac conjugate moieties (the aforementioned literature is hereby incorporated by reference).

[0306] In some embodiments, the linking (conjugation) of the branch point to the oligonucleotide can be achieved via a linker or a spacer group. In some cases, the spacer group can be a flexible hydrophilic spacer. An example of such a flexible hydrophilic spacer group is a PEG spacer group or a C6 linker. In this case, a preferred spacer group is a PEG3 spacer group (three ethylene units). In other cases, the spacer group can be a linking group containing an N-acylpyrrolidine, such as the following linking portion:

[0307] In the embodiments described above involving galactose derivatives, in some aspects, preferably, the conjugate portion according to the invention comprises GalNAc (N-acetylgalactosamine) or a derivative thereof as an ASGPR ligand, such as a monovalent, divalent, trivalent, or tetravalent GalNAc ligand. Such a GalNAc conjugate portion can be used to target the RNAi compound of the invention to the liver.

[0308] In some embodiments, the RNAi conjugate according to the present invention comprises one or more GalNAc or GalNAc derivatives. The GalNAc or GalNAc derivative can be attached to the oligonucleotide of the RNAi via a linker, such as a divalent, trivalent, or tetravalent branched linker. In some embodiments, the GalNAc conjugate portion binds to the 3' end of the sense strand of the RNAi. In some embodiments, preferably, the GalNAc conjugate portion is attached to the 3' end of the sense strand of the RNAi oligonucleotide via the linker. In some embodiments, the GalNAc conjugate portion binds to the 5' end of the sense strand. In some embodiments, preferably, the GalNAc conjugate portion is attached to the 5' end of the sense strand of the RNAi oligonucleotide via the linker. In some embodiments, the GalNAc conjugate portion binds to the 3' end of the antisense strand. In some embodiments, preferably, the GalNAc conjugate portion is attached to the 3' end of the antisense strand of the RNAi oligonucleotide via the linker.

[0309] In some embodiments, GalNAc or a GalNAc derivative is linked to the RNAi activator of the present invention via a bivalent adapter. In other embodiments, GalNAc or a GalNAc derivative is linked to the RNAi activator of the present invention via a trivalent adapter. In still other embodiments, GalNAc or a GalNAc derivative is linked to the RNAi activator of the present invention via a tetravalent adapter.

[0310] In some embodiments, the RNAi conjugate of the present invention comprises a single GalNAc or GalNAc derivative attached to a double-stranded RNAi. Preferably, the single GalNAc or GalNAc derivative is attached to the 5' or 3' end of the positive strand of the double-stranded RNAi via a linker.

[0311] In some embodiments, the RNAi conjugates of the present invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) of GalNAc or GalNAc derivatives. In some cases, one or more of the GalNAc or GalNAc derivatives may be individually linked to the RNAi oligonucleotide via a linker, independently of any other GalNAc or GalNAc derivative. In other cases, any two or more of the GalNAc or GalNAc derivatives may be linked to the RNAi oligonucleotide in a tandem cluster via a common linker portion. Furthermore, RNAi conjugates comprising a plurality of GalNAc or GalNAc derivatives linked thereto individually and / or in a tandem cluster are also within the scope of the present invention. In some embodiments, two GalNAc or GalNAc derivatives are linked in tandem to one end (5' or 3') of the positive strand of the double-stranded RNAi, and one GalNAc or GalNAc derivative is independently linked to the other end (3' or 5') of the positive strand of the double-stranded RNAi. In other embodiments, two GalNAc or GalNAc derivatives are independently linked to the two ends of the sense strand of the double-stranded RNAi, and optionally, the double-stranded RNAi may also include one GalNAc or GalNAc derivative linked to the 3' end of the antisense strand. In other embodiments, three GalNAc or GalNAc derivatives are linked in tandem to one end (5' or 3' end) of the sense strand of the double-stranded RNAi.

[0312] In some embodiments, the present invention provides RNAi conjugates comprising a conjugate moiety linked to an RNAi double-stranded oligonucleotide of the present invention, wherein the conjugate moiety comprises a galactose ligand having the following structure:

[0313] Wherein, L represents a linker with or without a branching region, and (Gal)n represents n terminal galactose derivatives attached to the linker, wherein n is preferably 1-4, and the wavy line on the right indicates that the L portion is directly or via an additional linker to the oligonucleotide of the RNAi. In the embodiments described, the branching region may be dianticular, trianticular, or other multi-branched shapes. In some preferred embodiments, Gal represents GalNAc or a GalNAc derivative.

[0314] In some embodiments, the galactose ligand has the following structure:

[0315] Where n is an integer from 1 to 4, preferably 1, 2 or 3;

[0316] L A This indicates the use of galactose derivatives (Gal) to link L B Partial connective base,

[0317] L B It indicates a 2-4 valent linker based on monohydroxymethylmethane, dihydroxymethylmethane, or trihydroxymethylmethane, wherein the L... B via oxygen atoms and each of the L A Partially linked via ether bonds and attached to the RNAi oligonucleotide via a methane carbon atom (preferably via a linker). In some embodiments, preferably, the galactose ligand has the following structure:

[0318] Among them Gal and L A As defined above, and the Gal is connected to the L via the C1 oxygen atom of galactose. A Some are linked by ether bonds.

[0319] In the above-described galactose ligand embodiments, in some preferred aspects, the L... A The linker has the following structure, where the wavy line on the left indicates a connection to a galactose derivative (Gal) via the oxygen atom at the C1 position of the galactose, and the wavy line on the right indicates a connection to the aforementioned L... B Partial connection.

[0320] In the above-described galactose ligand embodiments, in some preferred aspects, the L... B Partially connected via base L C Linked to oligonucleotides. In some embodiments, the L... C This represents a connection base with the following structure, where the wavy line on the left indicates a connection with L. B Partial methane carbon atoms (i.e., branch point carbon atoms) are connected, and the wavy line on the right indicates a connection to an oligonucleotide (e.g., connected to siRNA via a phosphodiester bond or a phosphothiodiester bond):

[0321] In the above-described galactose ligand embodiments, preferably, Gal represents GalNAc or a GalNAc derivative.

[0322] In some embodiments, the present invention provides RNAi conjugates comprising a conjugate moiety linked to an RNAi double-stranded oligonucleotide of the present invention, wherein the conjugate moiety comprises a trivalent GalNAc ligand having the following structure:

[0323] In some embodiments, the present invention provides an RNAi conjugate comprising a conjugate moiety linked to an RNAi oligonucleotide of the present invention, wherein the conjugate moiety has an L96 structure as shown below:

[0324] The wavy line on the right indicates the connection to the siRNA oligonucleotide chain, preferably via a phosphodiester bond or a thiophosphate diester bond.

[0325] In other embodiments, the present invention provides RNAi conjugates comprising a conjugate moiety linked to an RNAi oligonucleotide of the present invention, wherein the conjugate moiety comprises a galactose ligand having the following structure:

[0326] Wherein, Gal represents a galactose derivative, X represents O or S, and n is an integer from 0 to 3, preferably n is 0, 1, or 2, and the wavy line on the left indicates a link to an oligonucleotide chain with RNAi, preferably via a phosphodiester bond or a phosphothiodiester bond. Preferably, the Gal is linked to the ligand via the 6-carbon atom of galactose. In some embodiments, the Gal preferably represents GalNAc or a GalNAc derivative. In some embodiments, the Gal represents a GalNAc derivative having the following structure:

[0327] in,

[0328] R G1 This indicates hydrogen, hydroxyl group, C1-C20 straight-chain or branched alkyl group, C2-C20 straight-chain or branched alkenyl group, -O-C1-C20 straight-chain or branched alkyl group, -S-C1-C20 straight-chain or branched alkyl group, -NH-C1-C20 straight-chain or branched alkyl group, -N-(C1-C20 straight-chain or branched alkyl)2, -O-C0-C8 straight-chain or branched alkylene group -C6-C20 aryl group, -S-C0-C8 straight-chain or branched alkylene group -C6-C20 aryl group, galactosyl or galactosamide group, or q represents an integer from 1 to 16, wherein the aryl group is optionally replaced by one or more C1 to C8 straight-chain or branched alkyl groups;

[0329] Preferably, the R G1The term represents hydrogen, hydroxyl, C1-C6 straight-chain or branched alkyl, C2-C6 straight-chain or branched alkenyl, -O-C1-C16 straight-chain or branched alkyl (e.g. -O-C1-C6 straight-chain or branched alkyl), -S-C1-C6 straight-chain or branched alkyl, -NH-C1-C6 straight-chain or branched alkyl, -N-(C1-C6 straight-chain or branched alkyl)2, -O-C0-C4 straight-chain or branched alkylene-phenyl, wherein the phenyl is optionally substituted with one or more C1-C4 straight-chain or branched alkyl groups;

[0330] R G1 Specific examples include, but are not limited to, one of the following structures:

[0331] In some preferred embodiments, the present invention provides RNAi conjugates, wherein the RNAi is siRNA, and the conjugate portion is a monovalent GalNAc ligand (e.g., a monovalent GalNAc ligand as described above). The monovalent GalNAc ligand may be conjugated singly or in multiples to the oligonucleotide double strand of the siRNA, such that the molar ratio of siRNA to GalNAc in the final conjugate is 1:1, 1:2, 1:3, or 1:4. In some embodiments, the RNAi conjugate of the present invention having a monovalent GalNAc ligand has the following structure:

[0332] (M')x'-(ON)-(N')y', where ON represents the oligonucleotide duplex of the RNAi, M' and N' independently represent monovalent ASGPR ligands (especially monovalent GalNAc ligands), and x' and y' are independently integers of 0, 1, 2, or 3, where x'+y' = 1 to 3. Preferably, M' and N' are linked to the positive strand of the RNAi, and "-" indicates that the ligand is coupled to the oligonucleotide by a chemical bond or chemical group. In some embodiments, x' = 1 and y' = 1, with M' and N' linked to the 5' and 3' ends of the positive strand of the RNAi, respectively. In some embodiments, x' = 2 and y' = 0, where two M's are linked in series to the 5' or 3' end of the positive strand of the RNAi. In some embodiments, x' = 2 and y' = 1, where two M's are linked in series to the 5' end of the positive strand of the RNAi, and N' is linked to the 3' end of the positive strand of the RNAi; or vice versa. In some implementations, x' = 2 and y' = 1, wherein the two M's are respectively attached to the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi, and N' is attached to the 3' end of the sense strand of the RNAi.

[0333] In some embodiments, preferably, M' and N' independently represent monovalent GalNAc ligands having the structure of the following formula (P):

[0334] R G1 This indicates hydrogen, hydroxyl group, C1-C20 straight-chain or branched alkyl group, C2-C20 straight-chain or branched alkenyl group, -O-C1-C20 straight-chain or branched alkyl group, -S-C1-C20 straight-chain or branched alkyl group, -NH-C1-C20 straight-chain or branched alkyl group, -N-(C1-C20 straight-chain or branched alkyl)2, -O-C0-C8 straight-chain or branched alkylene group -C6-C20 aryl group, -S-C0-C8 straight-chain or branched alkylene group -C6-C20 aryl group, galactosyl or galactosamide group, or q represents an integer from 1 to 16, wherein the aryl group is optionally replaced by one or more C1 to C8 straight-chain or branched alkyl groups;

[0335] Preferably, the R G1 The term represents hydrogen, hydroxyl, C1-C6 straight-chain or branched alkyl, C2-C6 straight-chain or branched alkenyl, -O-C1-C16 straight-chain or branched alkyl (e.g. -O-C1-C6 straight-chain or branched alkyl), -S-C1-C6 straight-chain or branched alkyl, -NH-C1-C6 straight-chain or branched alkyl, -N-(C1-C6 straight-chain or branched alkyl)2, -O-C0-C4 straight-chain or branched alkylene-phenyl, wherein the phenyl is optionally substituted with one or more C1-C4 straight-chain or branched alkyl groups;

[0336] In some preferred embodiments, the R G1 Represent one of the following structures:

[0337] In some preferred embodiments, the R G1 express

[0338] In some preferred embodiments, M' and N' have a P36 structure as shown in the following formula:

[0339] In some preferred embodiments, the present invention provides RNAi conjugates having structures of formula (Ia), (Ib), (Ic), (Id), (Ie), or (If).

[0340] in, The oligonucleotide double strand representing siRNA

[0341] Where SS-5' represents the 5' end of the sense strand of the siRNA, and SS-3' represents the 3' end of the sense strand of the siRNA.

[0342] Wherein, X is O or S, and R G1 As defined above, and preferably representing one of the following structures:

[0343] More preferably, the R G1 express

[0344] In some further preferred embodiments, the present invention provides an RNAi conjugate having the structure M'-(ON)-N', wherein M' and N' are respectively linked to the 5' and 3' ends of the positive strand of the RNAi, and preferably linked by phosphate thioester groups. Preferably, the RNAi conjugate has the following structure:

[0345] in, The oligonucleotide double strand representing siRNA

[0346] Here, SS-5' represents the 5' end of the sense strand of the siRNA.

[0347] Where X is O or S, and preferably S.

[0348] Among them, R G1 As defined above.

[0349] In some of the most preferred embodiments, the present invention provides RNAi conjugates having the structure shown below.

[0350] in, The oligonucleotide double strand representing siRNA

[0351] SS-5' represents the 5' end of the sense strand of the siRNA.

[0352] X is either O or S, and preferably S.

[0353] In some preferred embodiments, the present invention provides RNAi conjugates having the following structures:

[0354] M-(ON), where ON represents the oligonucleotide duplex of the RNAi, and M represents the trivalent GalNAc ligand. Preferably, M is attached to the 3' end of the positive strand of the RNAi.

[0355] Preferably, the trivalent GalNAc ligand has the following structure:

[0356] Where L A For the connection base as defined above,

[0357] More preferably, the trivalent GalNAc ligand has the following structure:

[0358] More preferably, the trivalent GalNAc ligand is linked to an oligonucleotide of RNAi via an L96 structure as described above.

[0359] In some of the most preferred embodiments, the present invention provides RNAi conjugates having the structure shown in formula (II):

[0360] in, This represents the oligonucleotide double strand of siRNA, where 3' represents the 3' end of the positive strand, and X is O or S.

[0361] In some embodiments, conjugates of the compounds in Tables 2 and B formed by formula (Ia), (Ib), (Ic), (Id), (Ie), (If), or (II), and conjugates of the compounds in Tables 3 and C formed by formula (Ib) or (II) are preferred. In some embodiments, conjugates of the compounds in Tables 2 and B formed by formula (Ic), (If), or (II), and conjugates of the compounds in Tables 3 and C formed by formula (II) are more preferred. In some embodiments, conjugates of the compounds in Table B formed by formula (Ic), (If), or (II), and conjugates of the compounds in Table C formed by formula (II) are particularly preferred. In these embodiments, optionally, the RNAi conjugate contains at least one (e.g., 1, 2, 3, or 4) phosphate thioester groups. In some embodiments, for the RNAi conjugates of formula (Ia), (Ib), (Ic), (Id), (Ie), or (If), at least one (e.g., two, three, or four) consecutive phosphate thioester groups are included at the 5' and 3' ends of the sense and antisense strands, respectively, for linking end-chain ligands (if present) and end-chain nucleotides. In other embodiments, for the RNAi conjugate of formula (II), at least one (e.g., two, three, or four) consecutive phosphate thioester groups are included at the 5' end of the sense strand and at the 5' and 3' ends of the antisense strand, respectively, for linking end-chain nucleotides.

[0362] In some embodiments, the RNAi conjugates according to this disclosure are particularly preferably compounds selected from Table 5.

[0363] Delivery of RNAi activators

[0364] The RNAi activators and RNAi conjugates of this disclosure can be delivered or introduced by any means known in the art (e.g., to cells in vitro, to test animals, or to humans). In some embodiments, the RNAi activators or RNAi conjugates of this disclosure are delivered to a subject, the delivery of which enables the RNAi to hybridize with cellular mRNA encoding APOC3 and suppress expression by inhibiting transcription. Examples of routes for delivering the RNAi activator include direct injection at a tissue site. Alternatively, the RNAi activator can be modified to target selected cells and then administered systemically. For systemic administration, in some embodiments, the RNAi molecule can be modified to specifically bind to a receptor or antigen expressed on the surface of selected cells. In some embodiments, the modification includes forming the aforementioned RNAi conjugates of this invention that target hepatocytes, for example, by conjugating the oligonucleotide duplex of the RNAi to a peptide or antibody that binds to a receptor on the surface of hepatocytes, or to an ASGPR ligand according to this invention. The nucleic acid molecules of this invention can also be delivered to cells using vectors commonly known in the art and, for example, those described in US20070111230 (the entire contents of which are integrated herein).

[0365] In this document, when referring to RNAi active agents, “delivery to cells” means a process that facilitates or enables the uptake or absorption of RNAi active agents into cells. Cellular uptake or absorption of RNAi active agents can occur through unassisted diffusion or active processes, or through the use of auxiliary agents or devices. The term is not limited to cells in vitro; it also includes “delivering” RNAi active agents to the cells of a living organism. In such cases, delivery to cells will include delivery to the organism. For in vivo delivery, RNAi active agents can be injected into tissue sites or administered systemically. In vivo delivery can also be performed using β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005 / 0281781. In vitro delivery to cells includes methods known in the art, such as electroporation and lipid transfection. Other means, including those described herein or known in the art, can also be used to deliver RNAi active agents. Methods known in the art include, but are not limited to: viral delivery (retroviruses, adenoviruses, lentiviruses, baculoviruses, AAVs); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC) or nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); and chemical modification of siRNA (LNA) to increase stability. Xia et al., 2002 Nat. Biotechnol. 20 and Devroe et al., 2002 BMC Biotechnol. 21:15 disclose the incorporation of siRNA into viral vectors. Furthermore, the RNAi active agents disclosed herein can also be delivered using other systems for delivering RNAi active agents, or through various methods to be discovered in the future and / or approved by the FDA or other regulatory agencies. In some embodiments, the RNAi active agents of this disclosure are formulated in suitable pharmaceutical compositions for delivery.

[0366] Pharmaceutical compositions of RNAi activators

[0367] As used herein, a “pharmaceutical composition” includes a pharmaceutically effective amount of one or more APOC3 RNAi activators (especially the RNAi conjugates of the present invention), a pharmaceutically acceptable carrier, and, optionally, other therapeutic agents that synergize with the RNAi activator. As used herein, “pharmaceuticalally effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to an amount of RNAi activator that effectively produces the desired pharmacological, therapeutic, or preventative outcome. For example, a given clinical treatment is considered effective if a measurable parameter associated with a disease or condition is reduced by at least 10%, and the therapeutically effective amount of a drug used to treat said disease or condition is the amount necessary to produce a reduction of at least 10% in said parameter. In this embodiment, a therapeutically effective amount of an APOC3-targeting RNAi activator can reduce serum APOC3 protein levels or serum triglyceride levels by at least 10%. In other embodiments, a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition exhibits a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, and the therapeutically effective amount of the drug used to treat said disease or condition is the amount necessary to produce a reduction of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, respectively. The term "drug-approved carrier" refers to a carrier for administering a therapeutically active agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof.

[0368] In some embodiments, the present invention provides pharmaceutical compositions comprising an RNAi active agent or RNAi conjugate (especially modified siRNA or siRNA conjugated to a GalNAc ligand) as the active ingredient and a pharmaceutically acceptable diluent, carrier, and / or excipient (e.g., PBS buffer, physiological saline, water). The purpose of the pharmaceutical compositions is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity. The pharmaceutically acceptable diluents, carriers, and / or excipients used in this invention include any suitable pharmaceutically acceptable diluents, carriers, and / or excipients known in the art.

[0369] In some embodiments, the RNAi active agent or RNAi conjugate according to the invention may be present in a non-buffered solution, preferably saline or water. In other embodiments, the RNAi active agent or RNAi conjugate according to the invention may be present in a buffered solution, preferably comprising acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof; more preferably, the buffered solution is phosphate-buffered saline (PBS). In some embodiments, the RNAi active agent or RNAi conjugate according to the invention is formulated as a subcutaneous formulation. In other embodiments, the RNAi active agent or RNAi conjugate according to the invention is formulated as an intravenous formulation.

[0370] Application of RNAi activators

[0371] Pharmaceutical compositions comprising an APOC3 RNAi active agent or RNAi conjugate targeting APOC3 of this disclosure may be administered via a variety of suitable routes of administration, including but not limited to buccal, inhalation (including blowing or deep inhalation), nasal, oral, parenteral, implantation, injection, or infusion (via epidural, intra-articular, intra-articular, intracapsular, intracardiac, intracranial, intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, subepidermal, transendothelial, transtracheal, transvascular, rectal, sublingual, local, and / or vaginal routes). Administration may be performed by injection, infusion, skin patch, or any other method known in the art. Formulations for administration may be powdered, atomized, aerosolized, granulated, or suitably prepared for delivery.

[0372] Pharmaceutical compositions comprising the APOC3 RNAi active agent or RNAi conjugate of this disclosure can be administered using medical devices known in the art. For example, in certain embodiments, the RNAi active agent can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of known implants and modules that can be used in the disclosure herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs via the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow-rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system with multiple compartments; and U.S. Patent No. 4,475,196, which discloses a permeable drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art. In some embodiments, pharmaceutical compositions comprising the APOC3 RNAi active agent or RNAi conjugate of the present disclosure can be formulated to ensure proper distribution in vivo.

[0373] Treatment and prevention of APOC3-related diseases or conditions

[0374] This disclosure provides the use of the RNAi active agent or RNAi conjugate of this disclosure in the treatment or prevention of APOC3-related diseases or conditions, wherein a therapeutically effective amount or a preventatively effective amount of the RNAi active agent or RNAi conjugate of this disclosure is administered to human and non-human animal subjects in need. In this document, “APOC3-related diseases or conditions” means any disease involving dysfunction of APOC3 levels, expression, and / or activity, and / or any disease that can be treated and / or alleviated by regulating APOC3 levels, expression, and / or activity, particularly cardiovascular disease, dyslipidemia, lipid metabolism disorders, chylomicronemia syndrome, hypertriglyceridemia, and / or pancreatitis.

[0375] The RNAi active agents or RNAi conjugates targeting APOC3 described herein can be formulated into pharmaceutical compositions that can be administered to humans and non-human animals. These compositions may include one or more of the RNAi active agents or RNAi conjugates disclosed herein, and optionally other therapeutic agents for treating APOC3-related diseases. They may be administered as part of early / preventative treatment or may be administered at a therapeutically effective dose to treat individuals already presenting with symptoms related to the disease or condition. The administerable pharmaceutical compositions include those of any of the embodiments of the present invention described above.

[0376] In some embodiments, the RNAi activator or RNAi conjugate used for this purpose may be any RNAi activator or any RNAi activator conjugate described in this disclosure. In some embodiments, the APOC3-related disease or condition includes dyslipidemia. In some embodiments, the APOC3-related disease or condition includes hyperlipidemia. In some embodiments, the hyperlipidemia includes hypertriglyceridemia. In some embodiments, the APOC3-related disease or condition includes pancreatitis. In some embodiments, the APOC3-related disease or condition includes lipid metabolism disorders. In some embodiments, the APOC3-related disease or condition includes familial partial lipodystrophy or chylomicronemia syndrome. In some embodiments, the APOC3-related disease or condition includes non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. In some embodiments, the APOC3-related disease or condition includes polycystic ovary syndrome, kidney disease, obesity, or type 2 diabetes (insulin resistance). In some embodiments, the APOC3-related disease or condition includes cardiovascular disease. Preferably, the cardiovascular disease includes hypertension, atherosclerosis, or coronary heart disease.

[0377] In some embodiments, the present invention also provides a method for treating pathological conditions at least partially mediated or associated with APOC3 expression, the method comprising administering a therapeutically effective amount of the disclosed RNAi active agent or RNAi conjugate targeting APOC3 to a subject in need.

[0378] In some embodiments, the subject is a human being, and the RNAi active agent or RNAi conjugate is administered subcutaneously or intravenously.

[0379] In some embodiments, the RNAi activator according to the invention can be used to reduce APOC3 expression levels in cells (e.g., mammalian cells, such as human cells). In some embodiments, the RNAi activator according to the invention comprises at least 15, at least 16, at least 17, or at least 18 or more consecutive nucleotides targeting APOC3 mRNA. In some embodiments, the RNAi activator according to the invention comprises two oligonucleotide chains capable of forming a double-stranded region of at least 19 or more nucleotides. In some embodiments, the two oligonucleotide chains each comprise a sequence motif that differs from the sense and antisense strand sequences of at least 18 consecutive nucleotides in Tables 1, 2, and 3 or Table AC by 0, 1, 2, or 3 nucleotides. In some embodiments, each chain of the RNAi activator according to the invention has a length of less than 30 nucleotides, for example, 18-23 nucleotides and / or 19-21 nucleotides. In some embodiments, the double-stranded RNAi activator according to the invention may have one or two blunt ends or one or two overhangs, such as 3' and / or 5' overhangs of 1, 2, 3, or 4 nucleotides (i.e., 1-4 nt). The double-stranded RNAi activator according to the invention may also optionally contain one or two 5' and / or 3' end modifications (i.e., 5' caps and / or 3' caps) and / or one or more modified nucleotides. In some embodiments, the double-stranded RNAi activator according to the invention is an RNAi conjugate according to the invention, said conjugate containing the GalNAc ligand according to the invention, particularly monovalent, divalent, or trivalent GalNAc ligands having a P36 and / or L96 structure.

[0380] In some embodiments, the RNAi activators or RNAi conjugates of this disclosure (especially modified siRNAs or siRNAs conjugated to a GalNAc ligand) and their pharmaceutical compositions are particularly useful for the treatment and / or prevention of APOC3-related diseases or conditions such as dyslipidemia and cardiovascular disease. The RNAi activators or RNAi conjugates are preferably RNAi conjugate molecules formed by formulas (Ia)-(If) and (II) from compounds selected from Tables 2 and 3 or Tables B and C, especially RNAi conjugates containing a GalNAc ligand formed by compounds from Tables B and C through formulas (Ia)-(If) and (II).

[0381] In some embodiments of the treatment and prevention provided by this invention, the subject is a mammal, such as a primate, rodent, or human. In some embodiments, administration of the GalNAc-siRNA of this invention or its pharmaceutical composition results in a decrease in serum APOC3 protein in the subject. It has been demonstrated that reducing serum APOC3 protein levels can effectively treat and / or prevent diseases or conditions caused by APOC3, including dyslipidemia (e.g., hypertriglyceridemia), cardiovascular diseases (e.g., atherosclerosis, coronary heart disease), lipid metabolism-related diseases (e.g., familial partial lipodystrophy, chylomicronemia syndrome), kidney disease, liver disease, obesity, type 2 diabetes (insulin resistance), etc.

[0382] The siRNA can be administered to the patient via any suitable route known in the art, including but not limited to: subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration. The dosage of the RNAi reagents and compositions disclosed herein can be determined based on the patient's weight, age, sex, disease severity, etc. Subjects can be given therapeutic doses of siRNA, such as 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg body weight, etc. Dosing frequency can be based on regularity, such as daily, weekly, every two weeks, every three weeks, every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, annually, or for longer periods, with repeated administration. Following the initial treatment regimen, treatment can be administered at lower frequencies, for example, after monthly administration for three months, administration can continue for six months, one year, or longer. Administration of siRNA agents can reduce APOC3 protein levels in, for example, a patient's cells, tissues, blood, urine, or other compartments by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0383] abbreviation:

[0384] In describing RNAi activators in this disclosure, including Tables 1 to 3, Tables A, B, and C of the above and the following examples, the following abbreviations are used:

[0385] Example

[0386] Example 1. siRNA preparation

[0387] Based on the human APOC3 mRNA sequence ID NM_000040.3 (sourced from the National Center for Biotechnology Information, NCBI database), 513 siRNAs were designed using bioinformatics, from which 139 siRNAs were selected for evaluation. Details are shown in Table 1.

[0388] Human APOC3 mRNA sequence (NM_000040.3, SEQ ID NO:361) 535bp

[0389] Table 1 provides the sequence motifs of the preferred siRNA duplexes; Tables 2 and 3 provide the modified forms of the siRNA duplexes shown in Table 1. In Tables 1, 2, and 3, "SS" represents the sense strand of the siRNA, and "AS" represents the antisense strand. The first six digits of the modified compound IDs in Tables 2 and 3 are the IDs of their corresponding unmodified compounds in Table 1; the last two digits indicate different modifications of the compound. Thus, for example, compound "BPR-99500101" in Table 2 and compound "BPR-99500102" in Table 3 are formed by introducing different modifications to the sequence motif of compound "BPR-995001" in Table 1. Table 1 also provides the target gene sequences of the siRNA duplexes and their starting positions in the hAPOC3 mRNA sequence (NM_000040.3, SEQ ID NO:361).

[0390] The hAPOC3 siRNAs listed in Tables 2 and 3 were synthesized using an oligonucleotide solid-phase synthesis process. Specifically, using a universal solid-phase synthesis support (UnyLinker™ loaded HL Solid Supports, Kinovate Life Sciences) as the support and phosphoramidite nucleoside monomers as the starting materials, the nucleoside monomers were sequentially linked from the 3'-5' direction according to the oligonucleotide arrangement using the phosphoramidite solid-phase synthesis method. Then, ammonolysis was used to obtain crude oligonucleotides. After purification, ultrafiltration, and lyophilization, the crude products yielded two single-stranded oligonucleotides that constitute the siRNA. Finally, the two single-stranded oligonucleotides were annealed to form a double-stranded siRNA with inverse complementary base pairing.

[0391] The solid-phase synthesis process mainly consists of the following four steps (a, b, c, d). These four steps constitute a cycle; the cycle is repeated, and different types of nucleoside phosphoramide monomers are added sequentially according to the oligonucleotide sequence until the desired sequence is synthesized; the 5'-DMTr group at the end of the oligonucleotide is removed, and the cyanoethyl-protected phosphate is removed using a diethylamine acetonitrile solution (20% (v / v)). The solid-phase reaction ends, and an oligonucleotide single chain linked to a solid-phase support is obtained.

[0392] a. Deprotection reaction: Under room temperature conditions (20-25℃), the protecting group DMTr (dimethoxytriphenylmethyl) on the carrier / nucleotide is removed with dichloroacetic acid to obtain an active hydroxyl group that can undergo coupling reaction. The deprotecting reagent is a toluene solution of dichloroacetic acid (3% (v / v)) or a dichloromethane solution of dichloroacetic acid (3% (v / v)).

[0393] b. Coupling reaction: The nucleotide phosphoramidite monomer and the activator are simultaneously added to the solid-phase synthesis column. The phosphoramidite group is activated and undergoes a coupling condensation reaction with the active hydroxyl group to generate a phosphite triester. The activator is a 0.6M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT).

[0394] c. Oxidation reaction: Under the action of the oxidant iodine water, the triphosphite generated in the previous coupling condensation reaction is converted into a stable triphosphate. The oxidant is a 0.04M iodine / water / pyridine solution, v(water):v(pyridine) = 1:9.

[0395] d. Capping reaction: The active hydroxyl groups that have not fully reacted during the coupling reaction are capped to prevent them from participating in subsequent reactions. The capping reagents are CapA (acetic anhydride / acetonitrile) and CapB (NMI:Py:acetonitrile = 2:3:5).

[0396] The solid-phase support containing the above-mentioned oligonucleotides was subjected to ammonolysis using an ammonolysis reagent (25%–28% concentrated ammonia solution). This process cleaved and separated the oligonucleotides from the support while simultaneously removing various protecting groups from the nucleoside bases. The resulting solution was concentrated to obtain crude oligonucleotides. The crude product was sent for testing, and the target molecular weight was determined using high-resolution liquid chromatography-mass spectrometry (LC-MS).

[0397] The crude oligonucleotides were purified by anion exchange chromatography, followed by desalting using a gel column (HiTrap™ Desalting). The desalted sense and antisense strands were mixed in an equimolar ratio, heated to 65°C, held for 30 minutes, and then allowed to cool naturally to room temperature. The two single strands formed a double-stranded structure, siRNA, through hydrogen bonding. Molecular weight was determined using ion-pair reversed-phase chromatography (IPRP-HPLC) and high-resolution liquid chromatography-mass spectrometry (LC-MS), confirming the successful preparation of the hAPOC3 siRNA shown in Table 2.

[0398] The meanings of the abbreviations used in the preparation embodiments of the present invention are shown in the table below:

[0399] Threonoside synthesis process:

[0400] The following diagram illustrates the synthesis process of compound 11. Compounds 2-11 are then synthesized according to the process flow.

[0401] Synthesis of compound 2:

[0402] Compound 1 (20.0 g, 105.15 mmol, 1.0 eq.) was dissolved in DCM (200 mL). Imidazole (17.9 g, 262.88 mmol, 2.5 eq.) was added to the reaction system, and the reaction was cooled to 0 °C and stirred continuously for 30 minutes. Then, tert-butyldiphenylchlorosilane (31.79 g, 115.67 mmol, 1.1 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was gradually restored to room temperature and allowed to proceed overnight at room temperature. The reaction was monitored by TLC, and compound 1 was found to be in complete reaction. Water was added to the reaction system, followed by extraction twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE / EA = 100 / 15) to give compound 2 (35.24 g, 82.29 mmol, 78.3% yield). ESI-MS: m / z 451.2 [M+Na]+

[0403] Synthesis of compound 3:

[0404] Compound 2 (38.2 g, 89.13 mmol, 1.0 eq.) was dissolved in DMF (300 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. NaH (5.35 g, 133.70 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, and after the addition was complete, the reaction system was stirred at 0 °C for 30 minutes. Benzyl bromide (22.87 g, 133.70 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 2 had reacted completely. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 3 (68.2 g), which was directly used in the next reaction step. ESI-MS: m / z 519.3 [M+H]+

[0405] Synthesis of compound 4:

[0406] Crude compound 3 (68.2 g) was dissolved in THF (400 mL) and stirred until completely dissolved. TBAF (1 M in THF, 100 mL, 100 mmol, 1.12 eq.) was added to the reaction mixture, and the mixture was stirred overnight at room temperature until compound 3 reacted completely. The reaction solution was concentrated under reduced pressure to remove the solvent, and the crude product was subjected to column chromatography (PE / EA = 10 / 3) to give compound 4 (22.07 g, 78.73 mmol, two-step yield 88.3%). 1H NMR (400MHz, DMSO-d6) δ7.43–7.23(m,5H),5.84(d,J=3.6Hz,1H),4.74(t,J=5.2Hz,1H),4.67(m,2H),4.5 4–4.47(d,J=12Hz,1H),4.10(m,1H),3.89(d,J=3.2Hz,1H),3.71–3.55(m,2H),1.39(s,3H),1.26(s,3H).

[0407] Synthesis of compound 5:

[0408] Compound 4 (22.07 g, 78.73 mmol, 1.0 eq.) was dissolved in DMF (200 mL) and stirred until homogeneous. The reaction mixture was cooled to 0 °C and stirred for 30 minutes. Bromododecane (23.55 g, 94.48 mmol, 1.2 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the reaction mixture was allowed to return to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 4 reacted completely. The reaction mixture was then slowly poured into ice water to quench the reaction completely. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude compound was subjected to column chromatography (PE / EA = 100 / 9) to give compound 5 (32.0 g, 71.37 mmol, 90.6% yield). 1H NMR(400MHz, DMSO-d6)δ7.39–7.24(m,5H),5.85(d,J=3.8Hz,1H),4.73–4.62(m, 2H),4.48(d,J=12.0Hz,1H),4.17(td,J=5.9,3.2Hz,1H),3.88(d,J=3.2Hz,1H),3 .62(dd,J=10.1,5.4Hz,1H),3.51(dd,J=10.1,6.4Hz,1H),3.46–3.30(m,2H),1. 47(m,2H),1.39(s,3H),1.24(d,J=11.6Hz,21H),0.89–0.81(m,3H).,ESI-MS:m / z 471.3[M+Na]+

[0409] Synthesis of compound 6:

[0410] Compound 5 (35.32 g, 78.73 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and acetic acid (120 mL) and acetic anhydride (40.19 g, 393.65 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (5 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for another 8 hours until compound 5 had completely reacted. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 6. The crude product 6 was subjected to column chromatography (PE / EA = 10 / 2) to give compound 6 (19.0 g, 38.59 mmol, 49% yield). 1H NMR (400MHz, DMSO-d6) δ7.36–7.25(m,5H),6.24(d,J=8.4,1H),5.19–5.15(m,1H),4.72–4.53(m,2H),4.39–4.09(m,2H),3.67–3 .46(m,2H),3.42–3.32(m,2H),2.04(s,3H),2.02(s,3H),1.50–1.42(m,2H),1.31–1.19(m,18H),1.23(t,J=6.8,3H).ESI-MS:m / z 493.3[M+H]+

[0411] Synthesis of compound 7:

[0412] Compound 6 (5.0 g, 10.15 mmol, 1.00 eq.) and uracil (2.28 g, 20.30 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (60 mL) was added and stirred to dissolve. Then, BSA (6.19 g, 30.45 mmol, 3.0 eq.) was added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (2.26 g, 10.15 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 6 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution to the reaction system. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 7. The crude product was subjected to column chromatography (PE / EA = 5 / 2) to give compound 7 (4.71 g, 8.65 mmol, 85.2% yield). 1HNMR(400MHz,DMSO-d6)δ11.37(s,1H),7.62(d,J=8.1Hz,1H),7.35–7.25(m,5H),5.91(d,J=2.1Hz,1H),5 .58(dd,J=8.1,2.2Hz,1H),5.22(t,J=1.9Hz,1H),4.71(d,J=12.0Hz,1H),4.54(d,J=12.0Hz,1H),4.26(m, 1H),4.12(dd,J=4.1,1.6Hz,1H),3.71(dd,J=10.6,4.5Hz,1H),3.64(dd,J=10.6,6.1Hz,1H),3.48–3.34(m ,2H),2.08(s,3H),1.50–1.43(m,2H),1.29–1.20(m,18H),1.18(t,J=7.1Hz,3H).ESI-MS:m / z545.3[M+H]+

[0413] Synthesis of compound 8:

[0414] Compound 7 (4.71 g, 8.65 mmol) was added to a 250 mL three-necked flask, and ultra-dry dichloromethane (50 mL) was added to the flask. The reaction system was cooled to -15 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 26 mL, 25.95 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 7 was completely reacted. The reaction was quenched at -15 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 8. The crude compound was subjected to column chromatography (PE / EA = 2 / 3) to obtain compound 8 (2.0 g, 4.4 mmol, 50.9% yield). ESI-MS: m / z 455.3 [M+H]+

[0415] Synthesis of compound 9:

[0416] Compound 8 (2.0 g, 4.40 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (30 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (7.45 g, 22.00 mmol, 5.0 eq.), silver nitrate (747 mg, 4.40 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.33 g, 44 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction was heated to 80 °C in an oil bath and allowed to proceed overnight. The reaction of compound 8 was confirmed to be complete by TLC and LCMS. The reaction was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 9. The crude product was subjected to column chromatography (PE / EA = 100 / 35) to give compound 9 (3.2 g, 4.23 mmol, 96% yield). ESI-MS: m / z 757.4 [M+H]+

[0417] Synthesis of compound 10:

[0418] Compound 9 (3.2 g, 4.23 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, followed by the addition of 50 mL of 7 M ammonia-methanol solution at room temperature. The reaction mixture was stirred at room temperature for 3 hours until compound 9 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude product 10. The crude product was subjected to column chromatography (PE / EA = 1 / 1) to obtain compound 10 (2.9 g, 4.06 mmol, 96% yield). 1H NMR(400MHz,DMSO-d6)δ11.28(s,1H),7.90(d,J=8.1Hz,1H),7.46–7.39(m,2H),7 .33–7.22(m,7H),6.90–6.78(m,4H),5.45(d,J=3.6Hz,1H),4.14(t,J=4.7Hz,1H) ,3.74(s,6H),3.68(dd,J=9.2,4.5Hz,1H),3.62–3.49(m,2H),3.49–3.39(m,3H), 1.58(m,2H),1.39–1.20(m,20H),0.85(t,J=6.7Hz,3H).ESI-MS:m / z715.4[M+H]+

[0419] Synthesis of compound 11:

[0420] The dried compound 10 (2.9 g, 4.06 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (1.05 g, 8.12 mmol, 2.0 eq.) and DMAP (99 mg, 0.81 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.44 g, 6.09 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 10 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 11. The crude product was purified by column chromatography (PE / EA = 1 / 1) to give compound 11 (3.16 g, 3.46 mmol, 85.2% yield). ¹H NMR (400 MHz, DMSO-d6) δ 11.35 (dd, J = 14.6, 2.2 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.47–7.36 (m, 2H), 7.33–7.23 (m, 7H), 6.89–6.83 (m, 4H), 5.71–5.51 (m, 2H), 4.34 (dt, J = 8.8, 4.7 Hz, 1H), 4.11–3.90 (m, 1H) ,3.84–3.76(m,1H),3.75(s,6H),3.69–3.36(m,6H),2.71(t,J=6.0Hz,1H),2.60–2.39(m,1H),1.62– 1.52(m,2H),1.42–1.20(m,20H),1.10(t,J=6.6Hz,6H),1.02(d,J=6.7Hz,6H),0.89–0.81(m,3H).31P NMR(162MHz,DMSO-d6)δ151.30,148.65.,ESI-MS:m / z 915.6[M+H]+

[0421] The following diagram illustrates the synthesis process of compound 16. Compounds 12-16 were synthesized according to the process flow.

[0422] Synthesis of compound 12:

[0423] Compound 10 (2.6 g, 3.64 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DMF (30 mL) was added and stirred to dissolve. Imidazole (991 mg, 14.56 mmol, 4.0 eq.) was added at room temperature, and the mixture was stirred for 10 minutes. Then, TBSCl (1.10 g, 7.28 mmol, 2.0 eq.) was slowly added in portions to the reaction system, and the reaction was carried out overnight at room temperature under nitrogen protection. TLC and LCMS analysis showed that compound 10 reacted completely, and the reaction was quenched with water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE / EA = 2 / 1) to obtain compound 12 (2.75 g, 3.32 mmol, 91.2% yield). ESI-MS: m / z 829.5 [M+H]+

[0424] Synthesis of compound 13:

[0425] Compound 12 (2.75 g, 3.32 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were then added to the reaction mixture and stirred until homogeneous. The reaction was cooled to 0–5 °C, and compound TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added in portions. After the addition was complete, the ice bath was removed, and the mixture was allowed to return to room temperature. The reaction was stirred overnight at room temperature. TLC analysis showed that compound 12 reacted completely. At room temperature, ammonia (20 mL) was added to the reaction mixture and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 13 (8.3 g, calculated as 100% yield). ESI-MS: m / z 828.6 [M+H]+

[0426] Synthesis of compound 14:

[0427] Crude compound 13 (8.3 g, 1.0 eq.) was added to a 100 mL round-bottom flask, along with pyridine (50 mL) and stirred until completely dissolved. The reaction mixture was cooled to 0 °C, and BzCl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise to the mixture while stirring at 0 °C for 1 hour until compound 13 was completely reacted. The entire reaction was carried out under nitrogen protection. The reaction mixture was then brought to room temperature and quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude compound was subjected to column chromatography (PE / EA = 1 / 1) to give compound 14 (2.35 g, 2.52 mmol, 76% overall yield in two steps). ESI-MS: m / z 931.5 [M+H]+

[0428] Synthesis of compound 15:

[0429] Compound 14 (2.35 g, 2.52 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized to alkalinity with triethylamine (17 mL) and then added to the above reaction system. The reaction was carried out overnight in a 40 °C oil bath under nitrogen protection. TLC and LCMS analysis showed that compound 14 reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE / EA = 1 / 1) to give compound 15 (1.57 g, 1.92 mmol, 76.2% yield). ESI-MS: m / z 818.5 [M+H]+

[0430] Synthesis of compound 16:

[0431] The dried compound 15 (1.57 g, 1.92 mmol) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred to dissolve it. DIPEA (496 mg, 3.84 mmol, 2.0 eq.) and DMAP (47 mg, 0.38 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was carried out under nitrogen purging protection. The reaction was cooled to approximately 0 °C, and CEP-Cl (682 mg, 2.93 mmol, 1.5 eq.) was slowly added dropwise. The reaction was carried out at this temperature under nitrogen protection for 1 hour until compound 15 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to give compound 16 (1.7 g, 1.67 mmol, 87% yield). ¹H NMR (400 MHz, DMSO-d6) δ 11.27 (d, J = 17.5 Hz, 1H), 8.46 (dd, J = 30.3, 7.5 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.55–7.44 (m, 2H), 7.39–7.13 (m, 9H), 6.87–6.82 (m, 4H), 5.71 (d, J = 25.0 Hz, 2H). 1H),4.23–3.90(m,3H),3.78–3.38(m,13H),2.74(dd,J=10.7,5.6Hz,1H),2.61–2.32(m,1H),1.61–1 .50(m,2H),1.38–1.19(m,20H),1.12–1.04(m,9H),0.96(d,J=6.7Hz,3H),0.83(t,J=6.6Hz,3H),31P NMR(162MHz,DMSO-d6)δ149.87,149.34,ESI-MS:m / z 1018.6[M+H]+

[0432] The following diagram illustrates the synthesis process of compound 21. Compounds 17-21 were synthesized according to the process flow.

[0433] Synthesis of compound 17:

[0434] Compound 6 (27 g, 54.81 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (250 mL) was added and stirred to dissolve. Then, BSA (33.45 g, 164.43 mmol, 3.0 eq.) and ABz (26.22 g, 109.62 mmol, 2.0 eq.) were added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (12.18 g, 54.81 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 6 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 17. The crude product was subjected to column chromatography (PE / EA = 1 / 1) to give compound 17 (25 g, 37.23 mmol, 67.9% yield).

[0435] Synthesis of compound 18:

[0436] Compound 17 (3.0 g, 4.47 mmol, 1.0 eq.) was added to a 250 mL three-necked flask, and ultra-dry dichloromethane (30 mL) was added as solvent. The reaction system was cooled to -10 °C and stirred at this temperature for 30 min. A 1.0 mol / L boron trichloride dichloromethane solution (13.4 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 17 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 18. The crude compound was subjected to column chromatography (PE / EA = 1 / 2) to obtain compound 18 (1.0 g, 1.72 mmol, 38.5% yield).

[0437] Synthesis of compound 19:

[0438] Compound 18 (1.0 g, 1.72 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (15 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (2.9 g, 8.6 mmol, 5.0 eq.), silver nitrate (0.29 g, 1.72 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (2.08 g, 17.2 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 18 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 19. The crude product was subjected to column chromatography (PE / EA = 2 / 3) to give compound 19 (1.2 g, 1.36 mmol, 72% yield). ESI-MS: m / z 884.5 [M+H]+

[0439] Synthesis of compound 20:

[0440] Compound 19 (1.2 g, 1.36 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (10 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol / L sodium methoxide solution (0.76 mL, 4.08 mmol, 3.0 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours until compound 19 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude product 20. The crude product was subjected to column chromatography to obtain compound 20 (0.82 g, 0.97 mmol, 73% yield). ESI-MS: m / z 842.4 [M+H]+

[0441] Synthesis of compound 21:

[0442] The dried compound 20 (0.82 g, 0.97 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and 10 mL of ultradry dichloromethane was added and stirred until completely dissolved. DIPEA (0.25 g, 1.94 mmol, 2.0 eq.) and DMAP (23.8 mg, 0.194 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (0.35 g, 1.46 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 20 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 21. The crude product was purified by column chromatography to give compound 21 (0.74 g, 0.71 mmol, 73.2% yield). ¹H NMR (400 MHz, DMSO-d⁶) δ 11.21 (d, J = 4.0 Hz, 1H), 8.71 (d, J = 5.9 Hz, 1H), 8.60 (d, J = 29.9 Hz, 1H), 8.04 (d, J = 7.6 Hz, 2H), 7.65 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 8.6 Hz, 2H), 7.21–7.12 (m, 7 H),6.83–6.71(m,4H),6.02(dd,J=36.3,3.4Hz,1H),4.78–4.31(m,2H),3.81–3.36(m,15H),2.67–2. 38(m,2H),1.62–1.48(m,2H),1.42–1.21(m,18H),1.12–0.99(m,9H),0.93–0.78(m,6H).ESI-MS:m / z 1042.6[M+H]+

[0443] The following diagram illustrates the synthesis process of compound 26. Compounds 22-26 were synthesized according to the process flow.

[0444] Synthesis of compound 22:

[0445] Compound 6 (20 g, 40.60 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (200 mL) was added and stirred to dissolve. Then, BSA (24.78 g, 121.8 mmol, 3.0 eq.) and GiBu (17.96 g, 81.2 mmol, 2.0 eq.) were added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (9.02 g, 40.6 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 6 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 22. The crude product was subjected to column chromatography (PE / EA = 1 / 1) to give compound 22 (18 g, 27.53 mmol, 67.8% yield). ESI-MS: m / z 654.5 [M+H]+

[0446] Synthesis of compound 23:

[0447] Compound 22 (5.0 g, 7.64 mmol, 1.0 eq.) was added to a 250 mL three-necked flask, and ultra-dry dichloromethane (50 mL) was added as solvent. The reaction system was cooled to -10 °C and stirred at this temperature for 30 min. A 1.0 mol / L boron trichloride dichloromethane solution (22.92 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 22 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 23. The crude compound was subjected to column chromatography (PE / EA = 1 / 2) to obtain compound 23 (2.6 g, 4.61 mmol, 60.3% yield). ESI-MS: m / z 564.3 [M+H]+

[0448] Synthesis of compound 24:

[0449] Compound 23 (2.6 g, 4.61 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (35 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (7.8 g, 23.05 mmol, 5.0 eq.), silver nitrate (0.8 g, 4.61 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.59 g, 46.1 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 23 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 24. The crude product was subjected to column chromatography (PE / EA = 2 / 3) to give compound 24 (3.18 g, 3.67 mmol, 79.6% yield). ESI-MS: m / z 866.6 [M+H]+

[0450] Synthesis of compound 25:

[0451] Compound 24 (3.18 g, 3.67 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol / L sodium methoxide solution (2.04 mL, 11.01 mmol, 3.0 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours until compound 24 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude compound 25. The crude compound was subjected to column chromatography to obtain compound 25 (2.48 g, 3.01 mmol, 82.0% yield). ESI-MS: m / z 824.5 [M+H]+

[0452] Synthesis of compound 26:

[0453] The dried compound 25 (2.0 g, 2.43 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (0.63 g, 4.86 mmol, 2.0 eq.) and DMAP (59.4 mg, 0.486 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (0.86 g, 3.65 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 25 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 26. The crude product was purified by column chromatography to give compound 26 (2.08 g, 2.06 mmol, 83.5% yield). ¹H NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 11.48 (s, 1H), 8.18 (d, J = 29.6 Hz, 1H), 7.52–7.11 (m, 9H), 6.93–6.84 (m, 4H), 5.72 (dd, J = 38.6, 4.8 Hz, 1H), 5.21–5.04 (m, 1H), 4.53 (t, J = 5.5 Hz, 1H), 3.81–3.69 (m, 8H). ,3.61–3.34(m,3H),3.30–3.23(m,2H),3.11(d,J=8.4Hz,1H),2.79–2.64(m,1H),2.59–2.48(m,2H),2. 46–2.35(m,1H),1.51(d,J=6.6Hz,2H),1.38–1.18(m,18H),1.13–0.98(m,13H),0.88–0.74(m,8H).31P NMR(162MHz,DMSO-d6)δ151.94,148.86,ESI-MS:m / z 1024.6[M+H]+

[0454] Example 2. Screening of cancer cell activity in vitro using siRNA

[0455] Using the HepG2 and Huh7 liver cancer cell lines, the inhibitory effect of siRNA transfection on APOC3 mRNA in HepG2 and Huh7 cells was investigated. For the HepG2 cell line, siRNA concentrations of 0.5 nM, 0.1 nM, 0.04 nM, and 0.02 nM were used; for the Huh7 cell line, siRNA concentrations of 0.1 nM, 0.04 nM, 0.02 nM, and 0.01 nM were used. Cells transfected with siRNA served as the test group, those transfected with PBS served as the negative control group, and those transfected with PC served as the positive control group. PC information is as follows:

[0456] The specific method is as follows: 5-10 μL of siRNA duplex was added to the transfection reagent Invitrogen Lipofectamine RNAiMAX Reagent (40-45 μL) (Invitrogen, 13778-150), mixed well, and incubated at room temperature for 20 min. Then, the mixture was transferred to a 96-well plate. Next, 150 μL of cell suspension containing 15,000 Huh7 cells (Nanjing Kebai Biotechnology Co., Ltd., CBP60202) or HepG2 cells (ATCC, HB-8065) ​​was added to each well. After incubating the cells in an incubator for 24 hours, the cells were harvested, and total RNA was extracted using the VAZYME FastPure Universal Plant Total RNA Isolation Kit V2 (VAZYME, RC112-01) according to the kit instructions. Reverse transcription was performed using the VAZYME HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (VAZYME, R312-02) according to the kit instructions. AceQ qPCR Master Mix (VAZYME, Q112-02) and APOC3 probe (TaqMan) were used. TM 4331182), GAPDH probe (TaqMan) TM (4326317E), GAPDH was used as an internal reference gene, and the relative expression level of APOC3 mRNA in cells was detected by qPCR.

[0457] ΔCT=APOC3 ct-GAPDH ct

[0458] ΔCt = ΔCT test group - ΔCT negative control group

[0459] APOC3 mRNA relative expression level = 2 -ΔΔCt

[0460] APOC3 mRNA relative inhibition rate (%) = (1 - APOC3 mRNA relative expression level in the test group) × 100%

[0461] The results of in vitro screening for HepG2 liver cancer cell activity are shown in Tables 4-1 to 4-4. Among the compounds tested, the maximum inhibition rate of APOC3 was 94.23% for 0.5 nM siRNA, 94.25% for 0.1 nM siRNA, 91.71% for 0.04 nM siRNA, and 78.67% for 0.02 nM siRNA.

[0462] Compounds with the same modification scheme located in the 3'UTR region of the human APOC3 mRNA sequence ID NM_000040.3 were selected from Table 4-2. Figure 1 shows the inhibition rate of these compounds against APOC3 in the HepG2 cell line at a concentration of 0.1 nM. BPR-99500101 and BPR-99551205 showed the best inhibition rate against APOC3.

[0463] The results of in vitro screening for Huh7 hepatocellular carcinoma cell activity are shown in Tables 4-5, 4-6, and 4-7. In Table 4-5, the maximum APOC3 inhibition rate was 96.77% for 0.1 nM siRNA and 94.21% for 0.04 nM siRNA. In Table 4-6, the maximum APOC3 inhibition rate was 92.03% for 0.04 nM siRNA and 75.77% for 0.01 nM siRNA. In Table 4-7, the maximum APOC3 inhibition rate was 80.18% for 0.02 nM siRNA.

[0464] Table 4-1

[0465] Table 4-2

[0466] Table 4-3

[0467] Table 4-4

[0468] Table 4-5

[0469] Table 4-6

[0470] Table 4-7

[0471] Example 3. Preparation of GalNAc-siRNA

[0472] P36 Synthesis Process:

[0473] Synthesis of Compound 57

[0474] Compound 100 (D-2-acetamido-2-deoxygalactose-1,3,4,6-tetraacetic acid ester, CAS: 3006-60-8) was purchased from Anaiji, product number: A022038. Compound 100 (30 g, 77.09 mmol) was dissolved in 300 mL of dry 1,2-DCE. The system was placed at 0 °C, and trimethylsilyl trifluoromethanesulfonate (21 mL, 115.64 mmol, 1.5 eq.) was added. The reaction system was stirred at 0 °C for 10 min under nitrogen protection, and then reacted in a 50 °C oil bath for 12 h. The reaction was monitored by TLC and LC-MS to ensure complete reaction of the starting material. The system was then placed at room temperature, and 4A molecular sieves were added and stirred for 30 min. Then, anhydrous 3-pentanol (12.7 mL, 115.64 mmol, 1.5 eq.) was added. The system was stirred at room temperature for 12 h under nitrogen protection. The reaction was monitored by TLC and LC-MS. After the reaction was complete, triethylamine was added until the system was neutral. The mixture was then extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1 to 1:2) to give a white solid compound 105 (19 g, two-step yield: 60%). MS (ESI): m / z calcd for C 19 H 31 NO9[M+H] + :418.20,found:418.21

[0475] Compound 105 (58 g, 139.568 mmol) was dissolved in 300 mL of dry NH3 / MeOH solution. The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was not separated and purified, and was directly added to the subsequent reaction to obtain compound 106. MS (ESI): m / z calcd for C 13 H 25 NO6[M+H] + :292.17,found:292.19.

[0476] Compound 106 (40.6 g, 139.57 mmol) was dissolved in dry pyridine (800 mL), and TBDPSCl (47 mL, 181.44 mmol, 2 eq.) and 4-dimethylaminopyridine (5.12 g, 41.87 mmol, 0.3 eq.) were added. The system was stirred at room temperature under nitrogen protection for 12 h, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, compound 107 was obtained, which was directly used for subsequent reactions without post-treatment. MS (ESI): m / z calcd for C 29 H 43 NO6Si[M+H] + :530.29,found:530.26.

[0477] The reaction system of compound 107 was placed at 0 °C, and benzoyl chloride (48 mL, 418.8 mmol, 2 eq.) was added dropwise, followed by the addition of 4-dimethylaminopyridine (6.8 g, 55.8 mmol, 0.4 eq.). The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was diluted with ethyl acetate and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain a white solid compound 108, which was directly used for the next step. MS (ESI): m / z calcd for C 43 H 51 NO8Si[M+H] + :738.34,found:738.33.

[0478] Compound 108 (103 g, 139.57 mmol) was dissolved in anhydrous tetrahydrofuran (800 mL), and triethylamine trihydrofluoric acid (114 mL, 697.85 mmol, 5.0 eq.) was added. The reaction system was stirred at 50 °C under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After about 12 h at room temperature, the reaction was complete. The reaction system was directly concentrated under reduced pressure to remove most of the solvent, diluted with ethyl acetate, and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1) to give a white solid compound 109 (147 g, yield from compound 105 in four steps: 65%). MS (ESI): m / z calcd for C 27 H 33 NO8[M+H] +:500.22,found:500.23.

[0479] Compound 109 (10 g, 20.031 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). The system was placed at 0 °C, and PPh3 (10.5 g, 40.062 mmol, 2 eq.) and DPPA (8.6 mL, 40.062 mmol, 2 eq.) were added. DIAD (7.9 mL, 40.062 mmol, 2 eq.) was added dropwise. The system was stirred at 0 °C under nitrogen protection for 4 h, and then the system was placed at room temperature for about 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1 to 3:1) to give a white solid compound 57 (4.7 g, 44%). 1 H NMR(400MHz,DMSO-d6)δ8.01–7.91(m,3H),7.75–7.68(m,3H),7.63–7.53(m,3H) ),7.40(t,J=7.8Hz,2H),5.62(d,J=3.4Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4 .77(d,J=8.5Hz,1H),4.26–4.15(m,2H),3.60–3.49(m,2H),3.32(d,J=3.7Hz,1 H),1.70(s,3H),1.61–1.42(m,4H),0.87(dt,J=17.7,7.4Hz,6H).MS(ESI):m / z calcd for C 27 H 32 N4O7[M+H] + :525.23,found:525.24.

[0480] Synthesis of compound P36a:

[0481] Compound 101 (50 g, 0.55 mol, 1.0 eq) was weighed and dissolved in 500 mL of acetonitrile. After purging with nitrogen, 167 g of triethylamine (1.65 mol, 3.0 eq) and 160 g of ethyl trifluoroacetate (1.1 mol, 2.0 eq) were added, and the mixture was reacted overnight at room temperature. After the reaction was completed by TLC monitoring, the solvent was removed by direct concentration under reduced pressure to obtain an oily crude product.

[0482] The crude oily product was dissolved in 400 mL of pyridine, purged with nitrogen, and cooled to 0 °C in an ice-water bath. 186 g (0.55 mol, 1.0 eq) of 4,4'-bismethoxytriphenylmethyl chloride was added in portions. After the addition was complete, the mixture was allowed to react at room temperature overnight. The reaction was quenched with water after TLC monitoring. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by column chromatography to give 102,233 g of the compound. The two-step yield was 86.9%.

[0483] Compound 102 (233 g, 0.47 mol, 1.0 eq) was dissolved in 1.5 L of methanol, and 53.4 g (0.95 mol, 2.0 eq) of potassium hydroxide (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column chromatography to obtain compound 103, 174 g, yield: 92.8%. 1 H NMR (400MHz, DMSO-d6) δ7.40(d,J=7.5Hz,2H),7.33–7.18(m,7H),6.88(d,J=8.5Hz,4H),4.75(s,1H),3.73(s,6H),3.56(p,J=6.1Hz,1H),2.9 4(dd,J=9.1,5.4Hz,1H),2.83(dd,J=9.0,6.1Hz,1H),2.69(dd,J=12.7,4.0Hz,1H),2.49–2.42(m,1H),1.97(d,J=81.8Hz,1.7H).MS(ESI):m / z calcd for C 24 H 27 NO4[MH] - :392.19,found:392.16.

[0484] Compound 104 (5.0 g, 43.5 mmol, 1.0 eq) was weighed and dissolved in 50 mL of acetonitrile. After purging with nitrogen, triethylamine (15.0 g, 130.5 mmol, 3.0 eq) and ethyl trifluoroacetate (14.1 g, 87.0 mmol, 2.0 eq) were added, and the mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was directly concentrated under reduced pressure to obtain a crude oil product (100% yield was directly used for the next reaction).

[0485] The crude oily product and compound 103 (17.1 g, 43.5 mmol, 1.0 eq) were dissolved in 100 mL of N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine (11.2 g, 87.0 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (19.8 g, 52.2 mmol, 1.2 eq) were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by column chromatography to give compound 110, 17.5 g. The two-step yield was 68.2%.

[0486] Compound 11017.5 g (29.8 mmol, 1.0 eq) was dissolved in 200 mL of methanol, and 3.4 g (59.7 mmol, 2.0 eq) of potassium hydroxide (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column purification to obtain compound 111, 13.8 g, yield: 95.1%.

[0487] Compound 111 (1.7 g, 3.5 mmol, 1.0 eq) and acetylacetic acid (0.4 g, 4.2 mmol, 1.2 eq) were weighed and dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen protection, 0.9 g (7.0 mmol, 2.0 eq) of N,N-diisopropylethylamine and 1.6 g (4.2 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to give compound 113, 1.2 g of white solid, yield: 60.9%.

[0488] Compound 113 (570 mg, 1.0 mmol, 1.0 eq) and compound 57 (524 mg, 1.0 mmol, 1.0 eq) were weighed and dissolved in 10 mL of methanol. Copper sulfate pentahydrate (500 mg, 2.0 mmol, 2.0 eq) and sodium ascorbate (400 mg, 2.0 mmol, 2.0 eq) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC until completion. After concentration, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain compound 114560 mg, yield: 51.2%.

[0489] Compound 114 (560 g, 0.51 mmol, 1.0 eq) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purging protection, 10 mL of anhydrous dichloromethane and 132 mg (1.02 mmol, 2.0 eq) of N,N-diisopropylethylamine were added dropwise using a syringe. Then, 181 mg (0.77 mmol, 1.5 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphine (CEP-Cl) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until it was complete. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to give compound P36a, 430 mg of white solid, yield: 65.2%. 1 H NMR (400MHz, DMSO-d6) δ7.99–7.91(m,3H),7.91–7.80(m,1H),7.77–7.63(m,4H),7. 59(t,J=7.8Hz,3H),7.46–7.36(m,4H),7.36–7.17(m,7H),6.87(ddd,J=8.7,6.1,2.2 Hz,4H),5.66(d,J=3.4Hz,1H),5.34(dt,J=11.1,3.7Hz,1H),4.65–4.52(m,2H),4.46 (d,J=12.1Hz,2H),4.28–4.13(m,2H),4.13–3.96(m,1H),3.87–3.75(m,1H),3.73(d, J=2.3Hz,7H),3.65–3.49(m,2H),3.40(d,J=7.4Hz,2H),3.31–3.23(m,2H),3.04(m,2 H),2.91–2.75(m,3H),2.64(td,J=5.7,2.2Hz,1H),2.57(d,J=8.2Hz,1H),2.07(s,2H ),1.92–1.80(m,1H),1.79–1.70(m,1H),1.68(s,3H),1.65–1.56(m,1H),1.52–1.25( m,5H),1.15(dd,J=12.7,6.7Hz,9H),1.03(dd,J=6.8,4.6Hz,3H),0.83–0.65(m,6H). 31 P NMR(162MHz,DMSO)δ148.32,148.22,148.05,147.87.MS(ESI):m / z calcd for C 70 H 86 N8O 14 P[MH] -:1293.60,found:1293.63.

[0490] P36 Solid Support Loading Examples

[0491] Compound 93 (420 mg, 0.35 mmol) and 4-dimethylaminopyridine (8.5 mg, 0.07 mmol) were added to a 100 mL single-necked flask. After nitrogen purging protection, 30 mL of anhydrous dichloromethane and succinic anhydride (70 mg, 0.7 mmol) were added, and the reaction was allowed to proceed overnight. The reaction was monitored by TLC until completion. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain 350 mg of compound P36b. 1 H NMR (400MHz, DMSO) δ8.02(d,J=9.3Hz,1H),7.95(d,J=7.4Hz,2H),7.84(t,J=17.3Hz,2H), 7.71(d,J=7.1Hz,3H),7.59(t,J=7.6Hz,3H),7.39(t,J=7.8Hz,2H),7.35(t,J=8.1Hz,2H) ,7.31(t,J=7.6Hz,2H),7.22(d,J=8.5Hz,5H),6.89(d,J=8.7Hz,4H),5.66(s,1H),5.35(d d,J=11.0,3.4Hz,1H),5.12–4.94(m,1H),4.62(t,J=7.0Hz,1H),4.58(t,J=8.4Hz,1H),4.5 1–4.42(m,2H),4.19(t,J=9.6Hz,2H),3.73(s,7H),3.46(s,1H),3.40–3.28(m,3H),3.22( ddd,J=22.7,12.7,6.3Hz,2H),3.04(d,J=5.1Hz,2H),2.80(t,J=7.6Hz,2H),2.54(d,J=7.2 Hz,3H),2.47–2.41(m,2H),1.88(dd,J=16.5,8.8Hz,1H),1.73(s,1H),1.68(s,4H),1.65– 1.54(m,2H),1.48–1.16(m,5H),0.79(t,J=7.3Hz,3H),0.73(t,J=7.4Hz,3H).MS(ESI):m / z calcd for C 65 H 74 N6O 16 [MH] - :1193.51,found:1193.63.

[0492] Compound P36b was coupled with an amino support (polystyrene resin (PS) or CPG powder) through a coupling reaction and a capping reaction to prepare a solid-phase synthesis support pre-loaded with P36. After the support loading was determined by analysis, it was used as a support for subsequent solid-phase synthesis to synthesize oligonucleotides with 3'-terminal conjugated ligands.

[0493] Example of L96 solid support loading

[0494] Following the process steps, a solid-phase synthesis support pre-loaded with L96 was prepared. After analyzing and determining the support loading, it was used as a support for subsequent solid-phase synthesis to perform base monomer coupling to synthesize oligonucleotides with 3'-terminal conjugated ligand L96.

[0495] GalNAc-siRNA Synthesis

[0496] 1. Following the solid-phase carrier loading process, L96 (GalNAc3) or P36b (ASGPR monovalent ligand) was coupled with an amino carrier (polystyrene resin or CPG powder) through a coupling reaction and a capping reaction to prepare a pre-loaded solid-phase synthetic carrier with L96 or P36b. The carrier loading was measured by analysis.

[0497] 2. Load a commercially available general-purpose solid-phase support or the aforementioned solid-phase synthesis support into a solid-phase reaction column. In a nucleic acid synthesizer, following the standard oligonucleotide solid-phase synthesis method, using modified nucleotide monomers, perform the dedimethoxytriphenylmethylation reaction, coupling reaction, oxidation reaction, and capping reaction as described in Example 1, cycling these four steps to synthesize oligonucleotide sequences coupled with L96 or P36 (modified SS chains). If the target product is modified with thiophosphate, a thiolation reaction is used instead of an oxidation reaction; that is, the oxidation reaction in step (c) of Example 1 is replaced with the following thiolation reaction: under the action of the thiolation reagent PADS / ADTT, the triphosphite generated in the previous coupling condensation is converted into a stable triphosphite. After synthesizing the desired sequence, the cyanoethyl protecting group is removed with 20% diethylamine, and the solid-phase support is dried by argon gas; the support is then deprotected by ammonolysis in concentrated ammonia at 55°C for 5 to 16 hours. After ammonia desorption and protection are completed, the carrier is removed by filtration; the ammonia hydrolysate is concentrated to remove ammonia water, and the remaining concentrated sample is sent for LC-MS analysis to confirm that the sample molecular weight is consistent with the theoretical molecular weight.

[0498] 3. The oligonucleotide conjugate samples coupled with L96 or P36 were purified by ion exchange on an AKTA Pure 150, followed by gel column desalting to obtain single-stranded samples that met the requirements.

[0499] 4. Using the general-purpose solid-phase synthesis support Nitto Phase HLUny Linker, antisense chains (modified AS chains) were synthesized according to the standard solid-phase synthesis method.

[0500] 5. Add the modified SS chain and the modified AS chain to the annealing container in an equimolar ratio, heat to 55°C, hold for about 30 minutes, and then allow to cool naturally to room temperature.

[0501] 6. The annealed double-stranded samples were dispensed into freeze-drying containers according to the required amount and then freeze-dried to obtain GalNAc-siRNA conjugates.

[0502] The L96-siRNA conjugate has the structure shown in formula (II) as defined above, wherein L96 is coupled to the 3' end of the sense strand.

[0503] The p36-siRNA conjugate has the structure of formulas (Ia) to (If) as defined above, wherein the 5' and / or 3' ends of the sense strand and optionally the 3' end of the antisense strand are coupled with the monovalent GalNAc derivative ligand described above. Similarly, R G1 Monovalent GalNAc derivative ligands of formula (P) as defined above, having one of the following structures, can also be used to form siRNA conjugates:

[0504] The monovalent GalNAc derivative ligands described herein are linked to oligonucleotides via phosphodiester bonds or thiophosphate diester bonds. For a description of such formula (P) monovalent GalNAc derivative ligands that can be used in this invention, see the applicant's co-pending Chinese application (CN202311826326.5). This application is hereby incorporated herein by reference in its entirety for the purposes of this invention.

[0505] Specifically, an exemplary RNAi conjugate is formed as follows:

[0506] The compounds in Tables 2 and 3 form conjugates by formula (Ia). The conjugate ID is the corresponding compound ID with the suffix ".1". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (Ia), and its ID is BPR-99500101.1.

[0507] The compounds in Tables 2, B, C and 3 form conjugates by formula (Ib). The conjugate ID is the corresponding compound ID with the suffix ".2". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (Ib), and its ID is BPR-99500101.2.

[0508] The compounds in Tables 2 and 3 form conjugates by formula (Ic). The ID of the conjugate is the corresponding compound ID with the suffix ".3". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (Ic), and its ID is BPR-99500101.3.

[0509] The compounds in Tables 2 and 3 form conjugates by formula (Id). The conjugate ID is the corresponding compound ID with the suffix ".4". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (Id), and its ID is BPR-99500101.4.

[0510] The compounds in Tables 2 and 3 form conjugates by formula (Ie). The conjugate ID is the corresponding compound ID with the suffix ".5". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (Ie), and its ID is BPR-99500101.5.

[0511] The compounds in Table 2 and Table B form conjugates using formula (If). The conjugate ID is the corresponding compound ID with the suffix ".6". For example, compound BPR-99500101 in Table 2 forms a conjugate using formula (If), and its ID is BPR-99500101.6.

[0512] The compounds in Tables 2, B, C and 3 form conjugates by formula (II). The conjugate ID is the corresponding compound ID with the suffix ".7". For example, compound BPR-99500101 in Table 2 forms a conjugate by formula (II), and its ID is BPR-99500101.7.

[0513] Using the conjugate formation method described above, GalNAc-siRNA conjugates were synthesized based on the siRNAs shown in Tables 2, B, C, and 3, after modification with phosphate thioesters. Compound information for these conjugates is shown in Table 5. The structure of each prepared GalNAc-siRNA conjugate was confirmed by LC-MS analysis, and its purity was determined by UV (260 nm) quantification and HPLC analysis.

[0514] Example 4. Detection of the free uptake activity of GalNac-siRNA conjugates in primary hepatocytes

[0515] On day 0, cryopreserved primary human hepatocytes (PHH) or primary cynomogus hepatocytes (PCH) are thawed and adjusted to a cell size of 6 × 10⁶ cells / year. 5 Cells were cultured at a density of 100 cells / ml, and 90 μl of each was seeded into 96-well plates. The culture medium was Invitro GROCP Medium (BioIVT, catalog number Z990005) containing 10% FBS and 1% P / S. Simultaneously, 10 μl of the tested GalNAc-siRNA was added to each well, and control wells (PBS negative control group) without GalNAc-siRNA were also included. After 48 hours of culture, cells were harvested, and total RNA was extracted using the VAZYME FastPure Universal Plant Total RNA Isolation Kit V2 (VAZYME, RC112-01) according to the kit instructions. Reverse transcription was performed using the VAZYME HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (VAZYME, R312-02) according to the kit instructions. AceQ qPCR Master Mix (VAZYME, Q112-02) and the APOC3 probe (TaqMan) were used. TM 4331182), GAPDH probe (TaqMan) TM (4326317E), GAPDH was used as an internal reference gene, and the relative expression level of APOC3 mRNA in cells was detected by qPCR.

[0516] ΔCT=APOC3 ct-GAPDH ct

[0517] ΔCt = ΔCT test group - ΔCT negative control group

[0518] APOC3 mRNA relative expression level = 2 -ΔΔCt

[0519] APOC3 mRNA relative inhibition rate (%) = (1 - APOC3 mRNA relative expression level in the test group) × 100%

[0520] The results of the assay for the free uptake activity of the conjugate in primary hepatocytes of monkeys are shown in Table 6.

[0521] Table 6. Results of the assay for the free uptake activity of the conjugate in primary monkey hepatocytes.

[0522] Example 5. hAPOC3 humanized mouse experiment

[0523] This embodiment describes the use of a single subcutaneous injection of the GalNAc-RNAi composition shown in Table 5, prepared in Example 3, into APOC3 humanized mice (B6-hAPOC3, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to investigate the ability of this drug composition to inhibit APOC3 gene expression in vivo and / or the knockdown effect on triglycerides (TG) in the blood. Specifically, male mice aged 6-8 weeks were placed in an acclimatization chamber at least one week in advance. Blood samples were collected during the acclimatization period (as pre-drug administration) for blood lipid and serum APOC3 protein detection. After acclimatization, the mice were randomly divided into groups of 3-6 animals each according to their body weight, blood lipid levels, and pre-drug serum APOC3 protein levels. AD05876 (derived from AD05876 in US10597657B2) was set as the positive control group, physiological saline as the negative control group, and the RNAi compound of this invention as the experimental group. The single-dose subcutaneous injection was administered at a dose of 1 mg / kg or 0.5 mg / kg, with an administration volume of 10 μL / g, on Day 0. Blood samples were collected at different time points after administration: Day 7, Day 14, Day 21, Day 28, and Day 35, for the quantitative detection of lipid-related indicators such as triglycerides (TG) and human APOC3 protein. Serum APOC3 protein was quantitatively detected using an ELISA kit (Thermo Fisher Scientific EHAPOC3), following the kit's instructions. The assay was performed using a fully automated biochemical analyzer. Lipid levels were measured using an Integra 400 (Roche Diagnostics) scanner. The formula for calculating the relative inhibition rate at each time point after drug administration in each animal is: Relative inhibition rate at each time point after drug administration = 100% × (Test results at each time point after drug administration / Corresponding test results before drug administration for the corresponding individual - 1).

[0524] The results showed that all experimental groups effectively knocked down serum hAPOC3 protein and TG levels in hAPOC3 humanized mice. The results are shown in Figures 2-6. Figures 2 and 3 show the inhibition rate of serum human APOC3 protein at different time points relative to pre-administration levels after a single subcutaneous injection of 1 mg / kg of the drug. Figure 4 shows the inhibition rate of serum human APOC3 protein at different time points after a single subcutaneous injection of 1 mg / kg of the drug, relative to pre-administration levels and 35 days post-administration. Figure 5 shows the inhibition rate of serum human APOC3 protein at different time points relative to the saline group after a single subcutaneous injection of 0.5 mg / kg of the drug. Figure 6 shows the inhibition rate of serum TG at different time points after a single subcutaneous injection of 0.5 mg / kg of the drug, relative to pre-administration levels and 35 days post-administration.

[0525] Example 6. Cynomolgus monkey drug efficacy experiment

[0526] This embodiment investigates the ability of the RNAi conjugate described in Example 5 to inhibit APOC3 gene expression in vivo by administering a single subcutaneous injection to non-human primates. Specifically, wild male cynomolgus monkeys were acclimatized beforehand, and blood samples were collected during this period (as pre-drug administration) for blood lipid and serum APOC3 protein levels. After acclimatization, the cynomolgus monkeys were randomly assigned to groups of 2-4 animals each based on body weight, blood lipid levels, and pre-drug administration serum APOC3 protein levels. AD05876 (derived from AD05876 in US10597657B2) served as the positive control group, saline as the negative control group, and the RNAi compound of this invention as the experimental group. The single subcutaneous injection dose was 1 mg / kg or 3 mg / kg, with an administration volume of 1 ml / kg, and the day of administration was Day 0. Blood samples were collected at different time points (Day 7, Day 14, Day 21, Day 28, and Day 35) after drug administration for quantitative detection of lipid-related indicators such as triglycerides (TG) and APOC3 protein. Serum APOC3 protein was quantitatively detected using an ELISA kit (Thermo Fisher Scientific EHAPOC3), following the kit's instructions. The assay was performed using a fully automated biochemical analyzer. Relevant blood lipids, such as triglyceride levels, were measured using the Integra 400 (Roche Diagnostics). The formula for calculating the relative inhibition rate at each time point after drug administration to the pre-administration level for each animal is: Relative inhibition rate at each time point after drug administration = 100% × (Result at each time point after drug administration / Corresponding pre-administration result for the corresponding individual - 1). The formula for calculating the relative inhibition rate of the animal to the saline group at each time point after drug administration is: Relative inhibition rate at each time point after drug administration = 100% × (Result at each time point after drug administration / Mean value of the corresponding saline group - 1).

[0527] The results showed that the experimental groups effectively knocked down serum APOC3 protein and TG levels in cynomolgus monkeys. Table 7 shows the mean inhibition rates of serum APOC3 protein and triglyceride levels relative to the saline group in spontaneously hypertriglyceridemia cynomolgus monkeys (n=4 per group), after a single subcutaneous injection of 3 mg / kg of the drug. Table 8 shows the mean inhibition rate of serum APOC3 protein levels relative to the pre-drug level in cynomolgus monkeys (n=3-4 per group), after a single subcutaneous injection of 1 mg / kg of the drug.

[0528] Table 7

[0529] Table 8

Claims

An RNAi active agent for inhibiting the expression of APOC3, wherein the RNAi active agent comprises an antisense strand and a sense strand forming a duplex region, wherein: the antisense strand comprises a nucleotide sequence complementary to, or a nucleotide sequence differing by no more than 3, 2, or 1 nucleotides from, at least 15 (preferably, at least 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleotides in a segment of a human APOC3 mRNA, and optionally, the sense strand comprises a nucleotide sequence identical to, or differing by no more than 3, 2, or 1 nucleotides from, at least 15 (preferably, at least 16, 17, 18, 19, 20, or 21) consecutive nucleotides in the corresponding segment of the mRNA, wherein the segment of the APOC3 mRNA is the segment corresponding to nucleotide positions 418-464 of SEQ ID NO:

361. The RNAi active agent according to claim 1, wherein the antisense strand comprises: (i) 18-23 consecutive nucleotides in the antisense strand nucleotide sequence of any one of the compounds selected from the group consisting of: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BPR-995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511, (ii) 18-23 consecutive nucleotides in the antisense strand nucleotide sequence of any of the compounds listed in Table A; or (iii) a nucleotide sequence differing by no more than 3, 2, or 1 nucleotide from the 18-23 consecutive nucleotides of (i) or (ii), wherein the antisense strand is 18-30 nucleotides in length, preferably, the antisense strand is 18-23 nucleotides in length. The RNAi active agent according to claim 1 or 2, the sense strand comprises: (i) 17-21 contiguous nucleotides in the sense strand nucleotide sequence of any of the compounds selected from the group consisting of: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, BPR-995056, BPR-995210, BPR-995055, BPR-995199, BPR-995174, BPR-995135, BPR-995138, BPR-995084, BPR-995161, BPR-995136, BPR-995063, BPR-995188, BPR-995511, (ii) 17-21 contiguous nucleotides in the sense strand nucleotide sequence of any of the compounds listed in Table A; or (iii) a nucleotide sequence differing by no more than 3, 2 or 1 nucleotides from said 17-21 contiguous nucleotides of (i) or (ii), wherein the sense strand is 17-28 nucleotides in length, preferably, the sense strand is 17-21 nucleotides in length. The RNAi agent according to any one of claims 1-3, wherein the antisense strand and the sense strand of the RNAi comprise, respectively, the antisense strand nucleotide sequence and the sense strand nucleotide sequence of any of the compounds selected from the group consisting of: BPR-995040, BPR-995069, BPR-995100, BPR-995028, BPR-995041, BPR-995017, BPR-995046, BPR-995104, BPR-995001, BPR-995166, B PR-995056, BPR-995210, BPR-995055, BPR- 995199, BPR-995174, BPR-995135, BPR-995 138, BPR-995084, BPR-995161, BPR-995136, The RNAi agent according to any one of claims 1-3, wherein the antisense and sense strands of the RNAi comprise, respectively, the antisense strand nucleotide sequence and the sense strand sequence of any of the compounds listed in Table A, or a nucleotide sequence differing by no more than 3, 2 or 1 nucleotid.es therefrom. An RNAi agent that inhibits expression of APOC3 in a cell, wherein the RNAi agent comprises an antisense strand and a sense strand that form a duplex region, wherein the antisense strand of the RNAi agent comprises: (i) 18-23 contiguous nucleotides in the antisense strand nucleotide sequence of any of the compounds listed in Table 1; or (i) 18-23 contiguous nucleotides in the antisense strand nucleotide sequence of the compounds listed in Table 1; or (ii) a nucleotide sequence differing by no more than 3, 2, or 1 nucleotides from the 18-23 contiguous nucleotides of (i), wherein the antisense strand is 18-30 nucleotides in length, preferably the antisense strand is 18-23 nucleotides in length. The RNAi agent according to claim 6, wherein the sense strand of the RNAi agent comprises: (i) 17-21 contiguous nucleotides from the sense strand nucleotide sequence of any one of the compounds listed in Table 1 ; or (ii) a nucleotide sequence differing by no more than 3, 2, or 1 nucleotides from the 17-21 contiguous nucleotides of (i), wherein the sense strand is 17-28 nucleotides in length, preferably the sense strand is 17-21 nucleotides in length. The RNAi agent according to any one of claims 6-7, wherein the antisense strand and the sense strand of the RNAi each comprise the antisense strand nucleotide sequence and the sense strand nucleotide sequence, respectively, of any one of the compounds listed in Table 1, or a nucleotide sequence differing by no more than 3, 2, or 1 nucleotides therefrom. The RNAi agent according to any one of claims 1-8, wherein the RNAi agent comprises a duplex with overhangs consisting of an antisense strand and a sense strand, preferably wherein the overhangs are 3' overhangs consisting of 1, 2, 3, or 4 nucleotides from the 3' most end of the antisense strand. The RNAi agent according to any one of claims 1-9, wherein the RNAi agent comprises at least one phosphorothioate backbone modification and / or at least one nucleotide modification, preferably substantially all or all nucleotides of the antisense strand and / or the sense strand are modified nucleotides. The RNAi agent according to any one of claims 1-10, wherein the RNAi agent comprises at least one 2'-modified nucleotide, wherein the 2'-modification is selected from the group consisting of 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O-N-methylacetamido (2'-O-NMA), preferably the 2'-modification is selected from the group consisting of 2'-deoxy, 21-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl. The RNAi agent according to any one of claims 1-11, wherein the RNAi agent comprises at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-deoxy ribonucleotides, unlocked nucleotides (UNA), locked nucleotides (LNA), threose nucleotides (TNA), and abasic nucleotides. The RNAi agent according to any one of claims 1-12, wherein the RNAi agent comprises a threose nucleotide, The RNAi agent according to any one of claims 1-13, wherein the RNAi agent comprises a 2'-O-methoxyethyl modified nucleotide. Preferably, the threose nucleoside is a 4' modified threose nucleoside having the following formula (A), wherein Base represents a natural or modified nucleobase, the natural nucleobases being A, T, C, G or U, and wherein R is an alkoxy group having 1-30 carbon atoms (e.g., 25, 23, 21, 19, 17, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 carbon atom), Preferably, the threose nucleoside is located at the 5' most end of the sense strand of the RNAi agent, more preferably R represents a straight chain alkyloxy group having 12 carbon atoms, and even more preferably the threose nucleoside is a 4' modified threose nucleoside having the structure of formula (A1), wherein Base is a natural nucleobase A, T, C, G or U. The RNAi active agent according to any one of claims 1 to 13, wherein: the sense strand is 21 nucleotides in length, and wherein: (a1) positions 7 and 9-11, or positions 9, 11 and 13, of the sense strand comprise 2'-fluoro modified nucleotides; (b1) position 1 of the sense strand comprises a threose nucleoside of Formula (A); and / or (c1) the sense strand comprises 2'-0-methyl modified nucleotides at at least 15, 16, 17 or 18 positions; wherein the positions are counted from the 5' end of the sense strand. The RNAi active agent according to any one of claims 1 to 14, wherein: the antisense strand is 23 nucleotides in length, and wherein: (a2) positions 14 and 16, or positions 2, 14 and 16, of the antisense strand comprise 2'-fluoro modified nucleotides and positions 5 and 7 comprise deoxyribonucleotides; (b2) position 15 of the antisense strand comprises a 2'-0-methoxyethyl modified nucleotide; and / or (c2) the antisense strand comprises 2'-0-methyl modified nucleotides at at least 16, 17, 18, 19 or 20 positions, wherein the positions are counted from the 5' end of the antisense strand, optionally, the antisense strand as defined in (a2) above further comprises (i) a 2'-fluoro modified nucleotide at position 12; and / or (ii) deoxyribonucleotides selected from positions 2, 9 and 12. The RNAi agent according to any one of claims 1-15, wherein the antisense strand of the RNAi agent comprises: (i) 18-23 modified contiguous nucleotides in a modified antisense strand nucleotide sequence selected from any one of the compounds listed in Table 2, Table 3, Table B or Table C; or (ii) a modified nucleotide sequence that differs by no more than 3, 2 or 1 nucleotide from the 18-23 modified contiguous nucleotides of (i). The RNAi agent according to any one of claims 1-16, wherein the sense strand of the RNAi agent comprises: (i) 17-21 modified contiguous nucleotides in a modified sense strand nucleotide sequence selected from any one of the compounds listed in Table 2, Table 4, Table B or Table C; or (ii) a modified nucleotide sequence that differs by not more than 3, 2 or 1 nucleotide from the 17-21 modified contiguous nucleotides of (i). The RNAi agent according to any one of claims claim 1-17, wherein the RNAi agent comprises a modified sense strand and a modified antisense strand of any one of the compounds shown in Table 2 or Table B, or a modified sense strand and a modified antisense strand that differs by not more than 3, 2 or 1 nucleotide therefrom, respectively, preferably the RNAi agent comprises a modified sense strand and a modified antisense strand of any one of compounds shown in Table B. The RNAi active agent according to any one of claims 1 to 17, wherein said RNAi active agent comprises a modified sense strand and a modified antisense strand selected from any one of the compounds of Table 3 or Table C, or a modified sense strand and a modified antisense strand differing therefrom by no more than 3, 2 or 1 nucleotides, respectively, Preferably, said RNAi active agent comprises a modified sense strand and a modified antisense strand of any one of the compounds of Table C. The RNAi active agent according to any one of claims 1 to 19, wherein said RNAi active agent comprises at least 1 phosphorothioate or methylphosphonate linkage, preferably wherein said antisense strand and said sense strand comprise 0, 1, 2 or 3 phosphorothioate linkages at the 5' end and / or at the 3' end, respectively, more preferably wherein: said antisense strand comprises 1 or two phosphorothioate linkages at the 5' end and at the 3' end, respectively; and said sense strand comprises 0, 1 or 2 phosphorothioate linkages at the 5' end and at the 3'end, respectively. Conjugate comprising an RNAi active agent according to any one of claims 1 to 20. The conjugate according to claim 21, wherein said conjugation occurs at the 5' and / or 3' end of the sense strand and optionally at the 3' end of the antisense strand of said RNAi, optionally, said conjugation is performed via a phosphorothioate linkage or a phosphate linkage. The conjugate according to any one of claims 21 to 22, wherein said RNAi is conjugated to an antibody, a peptide, a peptidomimetic, an aptamer, a small chemical compound, a lipidoid, a cell-penetrating peptide polymer or a nanoparticle. The conjugate according to any one of claims 21 to 23, wherein said RNAi is conjugated to an asialoglycoprotein receptor (ASGPR) ligand via a chemical bond or a chemical group. The conjugate according to claim 24, wherein said ASGPR ligand comprises one or more N-acetylgalactosamine (GalNAc) or derivative moieties thereof, preferably selected from a monovalent, divalent or trivalent GalNAc or ASGPR ligand. The conjugate according to any one of claims 21 to 25, wherein said RNAi conjugate has the structure shown below: M-(ON), wherein ON represents the oligonucleotide double strand of said RNAi, M represents a trivalent GalNAc ligand, preferably M is linked at the 3' end of the sense strand of said RNAi, and optionally, the 5' end of the sense strand of said RNAi has a threose nucleoside, in particular a 4' modified threose nucleoside of formula (A) as defined in claim 13, Preferably, the trivalent GalNAc ligand has the structure shown below: and more preferably, the RNAi conjugate has the structure of Formula II: wherein represents the double stranded oligonucleotide of said RNAi, 3' represents the 3' end of the sense strand, X is O or S. The conjugate according to any one of claims 21 to 25, wherein said RNAI conjugate has the structure shown below: M-(ON), wherein ON represents the oligonucleotid double strand of said RNAi, M represents a trivalent GalNAc ligand, preferably, M is linked at the 3' end of the sense strand of said RNAi, and, optionally, the 5' end of the sense strand of said RNAi has a threosenucleoside, in particular a 4' modified threose nucleoside of formula ( A ) as defined in claim 13, (M')x'-(ON)-(N')y', wherein ON represents the oligonucleotide double strand of said RNAi, M' and N' each independently represents a monovalent ASGPR ligand, and x' and y' independently are an integer of 0, 1, 2 or 3, wherein x' + y' = 1 to 3, and "-" represents the coupling of the ligand to the oligonucleotide via a chemical bond or chemical group; preferably, M' and N' are linked to the sense strand and optionally to the antisense strand of said RNAi. Conjugates according to claim 27, wherein M' and N' independently represent the structure of formula (P) below, or a racemate, stereoisomer, isotopically labeled form thereof: wherein: R G1 represents hydrogen, hydroxyl, C1-C20 straight chain or branched chain alkyl, C2-C20 straight chain or branched chain alkenyl, -O-C1-C20 straight chain or branched chain alkyl, -S-C1-C20 straight chain or branched chain alkyl, -NH-C1-C20 straight chain or branched chain alkyl, -N-(C1-C20 straight chain or branched chain alkyl)2, -O-C0-C8 straight chain or branched chain alkylene-C6-C20 aryl, -S-C0-C8 straight chain or branched chain alkylene-C6-C20 aryl, galactosyl or galactosamido, or q represents an integer of 1 to 16, wherein said aryl is optionally substituted by one or more C1-C8 linear or branched alkyl groups; Preferably, said R G1 represents hydrogen, hydroxyl, C1-C6 linear or branched alkyl, C2-C6 linear or branched alkenyl, -O-C1-C16 linear or branched alkyl (e.g. -O-C1-C6 linear or branched alkyl), -S-C1-C6 linear or branched alkyl, -NH-C1-C6 linear or branched alkyl, -N-(C1-C6 linear or branched alkyl)2, -O-C0-C4 linear or branched alkylene-phenyl, wherein said phenyl is optionally substituted by one or more C1-C4 linear or branched alkyl groups; More preferably, said R G1 represents one of the following structures: More preferably, said R G1 denotes the RNAi conjugate has the structure (M')x'-(ON)-(N')y', x' is 0, 1 or 2, y' is 0, 1 or 2, wherein x' + y' = 1 to 3, ON represents the oligonucleotide double strand of siRNA, M' and N' are linked to the 5' and 3' ends of the sense strand and optionally to the 3' end of the antisense strand of said RNAi via a phosphate or phosphorothioate group, Preferably, the RNAi conjugate has the structure shown in Formula Ic or Formula If: wherein represents the oligonucleotide double strand of siRNA, SS-5' represents the 5' end of the sense strand of siRNA, X is O or S, and preferably S, R G1 As defined in claim 28. Conjugate according to any one of claims 27-29, wherein M' and N' have the structure of P36 as shown in the following formula or a racemate, a stereoisomer, an isotopically labeled form thereof: wherein, when the above structure is located at the 5' end of the oligonucleotide chain, the 5' end of the structure is linked to hydrogen; when the above structure is located at the 3' end of the oligonucleotide chain, the 3' end of the structure is linked to hydrogen. the RNAi conjugate is selected from the group consisting of: conjugates of the compounds of Table 2 and Table B formed by formula (la), formula (lb), formula (lc), formula (Id), formula (le), formula (If) or formula (II), and conjugates of the compounds of Table 3 and Table C formed by formula (lb) or formula (II), preferably, the RNAi conjugate is selected from the group consisting of: conjugates of the compounds of Table 2 and Table B formed by formula (lc), formula (If) or formula (II), and conjugates of the compounds of Table 4 and Table D formed by formula (II), more preferably, the RNAi conjugate is selected from the group consisting of: conjugates of the compounds of Table B formed by formula (lc), formula (If) or formula (II), and conjugate of the compounds of Table C formed by formula (II), optionally wherein: for the RNAi conjugate of formula (la), formula (lb), formula (lc), formula (Id), formula (le) or formula (If), at least 1 (e.g. 2, 3 or 4) consecutive phosphorothioate groups are comprised at the 5' and 3' ends of the sense and antisense strands, respectively, for the linkage between the end-of-chain ligand (if present) and the end-of-chain nucleotides, for the RNAi conjugate of formula (II), at least 1 (e.g. 2, 3 or 4) consecutive phosphorothioate groups are comprised at the 5' end of the sense strand and at the 5' and 3' ends of the antisense strand, respectively, for the linkage between the end-of-chain nucleotides. the RNAi conjugate is: (i) selected from the group consisting of the RNAi conjugates of Table 5, or (ii) selected from the group consisting of the RNAi conjugates of Table 6. (ii) an RNAi conjugate that differs by no more than 3, 2, or 1 nucleotides on the antisense strand and the sense strand, respectively, from the RNAi conjugate of (i). A composition comprising the RNAi agent of any one of claims 1-20 or the RNAi conjugate of any one of claims 21-32 and optionally a pharmaceutically acceptable carrier. The composition of claim 33, wherein the RNAi agent or RNAi conjugate is present in a non-buffered solution, preferably the non-buffered solution is saline or water; or The double stranded RNAi agent or RNAi conjugate is present in a buffered solution, preferably the buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof, more preferably the buffered solution is phosphate buffered saline (PBS). A method for inhibiting expression of APOC3 in a cell, wherein the method comprises delivering to the cell the RNAi agent of any one of claims 1-20 or the RNAi conjugate any one of claims 21-32 or the composition of any one of claims 33-34, whereby expression of the APOC3 gene in the cell is inhibited, optionally the cell is an in vitro or ex vivo cell; Preferably, the delivery results in at least 50%, 60%, 70%, 80%, 90%, or 95% inhibition of expression of the APOC3 gene in the cell. The RNAi agent of any one of claims 1-20 or the RNAi conjugate of claims 21-32 or the composition of any one of claims 33-34 for use as a medicament or in the manufacture of a medicament, preferably wherein the medicament is for reducing APOC3 levels and / or expression in a subject, or for preventing or treating a disease or disorder mediated by APOC3; Preferably, the disease or disorder is selected from the group consisting of cardiovascular disease, dyslipidemia, disorders of lipid metabolism, familial partial lipodystrophy, chylomicronemia syndrome, hypertriglyceridemia, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, nephropathy, obesity, type 2 diabetes (insulin resistance), hypertension, atherosclerosis, and pancreatitis, preferably the disease or disorder is selected from the group consisting of cardiovascular disease, chylomicronemia syndrome, hypertriglyceridemia, and pancreatitis; Preferably, the medicament reduces APOC3 protein levels in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 90%; Preferably, the subject is a human; Preferably, the RNAi agent or RNAi conjugate is formulated as a subcutaneous or intravenous administration formulation.

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