Sirna conjugate targeting angptl4 and use thereof

Abstract

Provided are an siRNA conjugate targeting angiopoietin-like protein 4 (ANGPTL4) and use thereof. The siRNA comprises a sense strand and an antisense strand. The antisense strand comprises at least 17 contiguous nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence shown in Table 1, and the antisense strand has a length of 17-30 nucleotides. The sense strand has a length of 17-30 nucleotides, and is at least partially complementary to the antisense strand. The siRNA conjugate and a pharmaceutical composition provided in the present invention have good stability, excellent ANGPTL4 gene inhibitory activity, and satisfactory immunostimulatory properties, and can significantly reduce the concentration of ANGPTL4 protein at the animal level.

Description

A siRNA conjugate targeting ANGPTL4 and its applications

[0001] This application claims priority to Chinese patent application 2024116411708, filed on 2024 / 11 / 18. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to an siRNA that inhibits the expression of the angiopoietin-like protein 4 (ANGPTL4) gene, its conjugates, pharmaceutical compositions, and their use in the prevention and / or treatment of dyslipidemia-related diseases. Background Technology

[0003] Angiopoietin-like protein 4 (ANGPTL4) is a member of the angiopoietin-like protein family. It is a secreted protein primarily expressed in adipose and liver tissues before being secreted into the bloodstream. It contains a 406-amino acid sequence, mainly comprising two functional domains: an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain. It often exists as oligomers, glycosylated forms, and various isoforms; both domains are conserved domains of the angiopoietin family. In addition, ANGPTL4 contains one asparagine glycosylation site, one cAMP / cGMP-dependent protein kinase phosphorylation site, two protein kinase C phosphorylation sites, four myristylation sites, and four casein kinase II phosphorylation sites. Its expression is regulated by factors such as transforming growth factor-β (TGF-β), peroxisome proliferator-activated receptor δ (PPARδ), and hypoxia-inducible factor 1 alpha (HIF1α).

[0004] Increasing evidence suggests that ANGPTL4 plays a crucial role in the pathological changes of metabolic diseases such as atherosclerosis, type 2 diabetes, fatty liver, and obesity. In particular, it plays a key role in lipid metabolism in the blood and liver by inhibiting LPL enzyme activity. Abnormal lipid metabolism in the liver leads to the accumulation of TAG (triacylglycerol) and DAG (1,2-diacylglycerol), thereby activating the PKCε signaling pathway, inhibiting insulin receptor activation, and reducing hepatic insulin sensitivity. Decreased hepatic insulin sensitivity inhibits the conversion of glucose to glycogen and promotes glycogenolysis to glucose, leading to hyperglycemia. Several studies have shown that ANGPTL4 expression is related to lipid and carbohydrate metabolism in vivo.

[0005] Type 2 diabetes and its related complications have a high incidence rate worldwide. According to the International Diabetes Federation, approximately 537 million adults currently have diabetes (10%), and this number is projected to rise to 643 million by 2023 and 783 million by 2045, with 90% of these cases being type 2 diabetes. Despite the increasing availability of new medications for type 2 diabetes in recent years, clinical glycemic control and patient adherence remain unsatisfactory. Furthermore, existing type 2 diabetes medications require frequent dosing, often daily or even with meals, further complicating patient adherence.

[0006] Compared to traditional drugs, siRNAs exhibit poor stability and are easily degraded by nucleases when administered systemically. Furthermore, it is necessary to explore ways to further enhance activity while avoiding off-target effects, immune stimulation, and cytotoxicity. Therefore, developing more candidate siRNAs that are stable in the blood, possess good biological activity, exhibit low cytotoxicity, and can effectively inhibit ANGPTL4 gene expression for extended periods has become an urgent problem to solve. Patent applications WO2023044458A1 and WO2022261005A1 disclose siRNAs capable of inhibiting ANGPTL4 gene expression; however, the inhibitory effect of these siRNAs needs further improvement. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, this invention provides an siRNA conjugate for inhibiting ANGPTL4 gene expression. The siRNA conjugate comprises a sense strand and an antisense strand, wherein the antisense strand is associated with SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 8 9, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 1 The nucleotide sequence of any one of the following nucleotides differs by no more than 4 nucleotides: at least 17 consecutive nucleotides, wherein the antisense strand is 17 to 30 nucleotides in length; and the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

[0008] In some embodiments of the present invention, the justice chain is related to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 1 The nucleotide sequence of at least 17 consecutive nucleotides differing by no more than 4 nucleotides from any one of the following: 44, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244.

[0009] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides in length; the sense strand is 19 to 25 nucleotides in length.

[0010] In some embodiments of the present invention, the antisense strand is 19 to 23 nucleotides in length; the sense strand is 19 to 21 nucleotides in length.

[0011] In some embodiments of the present invention, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments of the present invention, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments of the present invention, the antisense strand is 19 nucleotides long and the sense strand is 19 nucleotides long.

[0012] In some embodiments of the invention, the siRNA conjugate comprises one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotide overhangs. In some embodiments of the invention, the overhangs may be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the invention, the overhangs are located at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0013] In some embodiments of the present invention, the antisense strand 3' end of the siRNA conjugate has a two-nucleotide overhang.

[0014] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA conjugate has two nucleotide overhangs, and the overhangs are UU, GG, CU, and AA.

[0015] In some embodiments of the invention, the siRNA conjugate has blunt ends. In some embodiments of the invention, the siRNA has at least one blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand).

[0016] In some embodiments of the present invention, the siRNA conjugate has two blunt ends.

[0017] In some embodiments of the present invention, the antisense strand of the conjugate is associated with SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151 The nucleotide sequences shown in 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, and 245 differ by no more than 4 nucleotides, no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide, or are identical to them.In some embodiments of the present invention, the positive chain of the conjugate is associated with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 15 The nucleotides represented by 0, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, and 244 differ by no more than 4 nucleotides, no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide, or are the same as them.

[0018] In some embodiments of the present invention, the sense strand and antisense strand have a mismatch of no more than 3 nucleotides; in some embodiments of the present invention, the sense strand and antisense strand have a mismatch of no more than 2 nucleotides; in some embodiments of the present invention, the sense strand and antisense strand have a mismatch of no more than 1 nucleotide; in some embodiments of the present invention, the sense strand and antisense strand are completely complementary.

[0019] In some embodiments of the present invention, the siRNA conjugate contains at least one modifying nucleotide.

[0020] In some embodiments of the present invention, all nucleotides in the sense and / or antisense strands of the siRNA conjugate are modified nucleotides or nucleotide analogs.

[0021] In some embodiments of the present invention, the modified nucleotide or nucleotide analogue is selected from: 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-cleaved nucleotide analogue, 2'-fluoroarabinonucleotide, 2'-methoxyethylnucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, 3'-methoxynucleotide, 2'-allyl-modified nucleotide, nucleotide containing a thiophosphate group, nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate, nucleotide containing a 5'-phosphate mimic, diol-modified nucleotide, debaseted nucleotide, morpholinonucleotide, threonucleotide, locked nucleotide, unlocked nucleotide, glycerol nucleotide, and base-modified nucleotide.

[0022] In some embodiments of the present invention, the 5' end and 3' end of the positive chain each independently contain one or two thiophosphate groups.

[0023] In some embodiments of the present invention, the 5' end and 3' end of the antisense chain each independently contain one or two thiophosphate groups.

[0024] In some embodiments of the present invention, the 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups, and the 5' end and 3' end of the antisense chain each independently contain one or two thiophosphate groups.

[0025] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all linked by thiophosphate groups.

[0026] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide.

[0027] In some embodiments of the present invention, the antisense strand is 23 nucleotides long, and the sense strand is 21 nucleotides long; the 5'-1st nucleotide of the antisense strand is a (E)-vinyl phosphate and a 2'-methoxy modified nucleotide, the 5'-2nd, 14th, and 16th nucleotides are 2'-fluorinated modified nucleotides, the 5'-3rd to 4th, 6th, 8th to 13th, 15th, 17th to 21st, and 22nd to 23rd nucleotides are 2'-methoxy modified nucleotides, the 5'-5th nucleotide is a deoxyribonucleotide modified nucleotide, the 5'-7th nucleotide is a 2'-methoxy or deoxyribonucleotide modified nucleotide, and the 5'-1st nucleotide is a nucleoside. The acid and the 2nd nucleotide, the 2nd nucleotide and the 3rd nucleotide, the 21st and 22nd nucleotides, and the 22nd and 23rd nucleotides are linked by thiophosphate groups; the 1st to 7th and 12th to 21st nucleotides at the 5' end of the positive strand are 2'-methoxy modified nucleotides, the 8th nucleotide at the 5' end is a 2'-methoxy or 2'-fluorine modified nucleotide, the 9th to 10th nucleotides at the 5' end are 2'-fluorine modified nucleotides, the 11th nucleotide at the 5' end is a deoxyribonucleotide or a 2'-fluorine modified nucleotide, and the 1st nucleotide at the 5' end is linked by thiophosphate groups.

[0028] The present invention also provides siRNA conjugates obtained by conjugating siRNA with a delivery vector molecule; preferably, the delivery vector contains N-acetylgalactosamine.

[0029] In some embodiments of the present invention, the siRNA conjugate differs from the nucleotide sequences of the sense and / or antisense strands of any of the conjugate molecules shown in Table 1 by no more than 4 nucleotides, no more than 3 nucleotides, no more than 2 nucleotides, or no more than 1 nucleotide.

[0030] In some embodiments of the present invention, the siRNA conjugate is selected from any of the conjugate molecules shown in Table 1.

[0031] The present invention also provides a pharmaceutical composition comprising any of the above-described siRNA conjugates and a pharmaceutically acceptable carrier.

[0032] The present invention also provides the use of any of the above-described siRNA conjugates and / or the above-described pharmaceutical compositions in the preparation of medicaments for treating and / or preventing pathological conditions or diseases associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression.

[0033] Further, the pathological condition or disease is a disease related to dyslipidemia or a disease related to dysglucose; more preferably, the disease related to dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris or atherosclerosis.

[0034] The present invention also provides a method for treating and / or preventing pathological conditions or diseases associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression, the method comprising administering to a subject in need a therapeutically or preventively effective amount of any of the above-described siRNA conjugates and / or the above-described pharmaceutical compositions. In some embodiments, the pathological condition or disease is a disease associated with dyslipidemia or a disease associated with dysglucose abnormalities. In some embodiments, the disease associated with dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

[0035] The present invention also provides any of the above-described siRNA conjugates and / or the above-described pharmaceutical compositions for treating and / or preventing pathological conditions or diseases associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression. In some embodiments, the pathological condition or disease is a disease associated with dyslipidemia or a disease associated with dysglucose abnormalities. In some embodiments, the disease associated with dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

[0036] The siRNA conjugates and pharmaceutical compositions disclosed herein exhibit good stability, excellent ANGPTL4 gene inhibitory activity, satisfactory cytotoxicity and immunostimulatory properties, and can significantly reduce blood lipid levels.

[0037] The sequence of the ANGPTL4 gene targeted by siRNA in this invention is shown in SEQ ID NO:1:

[0038] SEQ ID NO:1 (ANGPTL4 gene)

[0039] In this invention, "siRNA" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains the ability to reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNA can function through RNA interference mechanisms (e.g., by interacting with the mRNA interference pathway mechanism in mammalian cells (RNA-induced silencing complex RISC)) or any other mechanism or pathway. While the term siRNA drug as used in this invention is considered to function primarily through RNA interference mechanisms, the siRNA drug is not limited to any particular pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of this invention consists of an oligonucleotide chain having at least a partial complementarity to the mRNA that is the target. In some embodiments, the siRNA drug of this invention is double-stranded and consists of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0040] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0041] In this invention, a "delivery carrier" refers to a chemical portion of siRNA covalently linked to siRNA and affecting the targeting, activity, cellular distribution, cellular uptake, or stability of oligonucleotides. Conjugation of siRNA to one or more delivery carriers can improve the pharmacological properties of siRNA. In some embodiments, the delivery carrier portion modifies or enhances the pharmacokinetic properties of oligonucleotides by improving their cellular distribution, bioavailability, metabolism, excretion, permeability, or cellular uptake. Specifically, the delivery carrier can target oligonucleotides to specific organs, tissues, or cell types, thereby enhancing the effectiveness of the oligonucleotides in those organs, tissues, or cell types. Simultaneously, the conjugate can be used to reduce the activity of oligonucleotides in non-target cell types, tissues, or organs, such as off-target activity or activity in non-target cell types, tissues, or organs.

[0042] In this invention, unless otherwise specified, the capital letter I indicates the base composition of the base-modified nucleotide, and the base is... (Inosine); mI represents Inosine with a methoxy-substituted ribose 2'-position; m6A indicates the base composition of the modified nucleotide, with the bases being... The uppercase letter X indicates the base composition of a base-modified nucleotide, where the base is... The capital letter B indicates the base composition of a base-modified nucleotide, where the base is... Unless otherwise specified, all of the above nucleotides containing special bases have a methoxy group substituted at the 2'-position of the ribose.

[0043] In this invention, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, where the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. In this invention, to satisfy the above hybridization ability requirements, the "complementary" sequence may also include or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairs or Hoogstein base pairs. Correspondingly, in this invention, unless otherwise specified, "mismatch" means that in the siRNA double-stranded molecule, the bases at corresponding positions are not paired in a complementary manner.

[0044] In this invention, unless otherwise specified, "difference in nucleotide sequence" refers to a change in the type of base (A, U, G, C) of nucleotides at the same or corresponding positions compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or the nucleotide is dT, dC, dG, etc., then a difference in nucleotide sequence is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, then a difference in nucleotide sequence is not considered to exist at that position. For example, if a nucleotide base in the original nucleotide sequence is U, and the nucleotide at the same or corresponding position is dT or a nucleotide modified with other bases (such as I, m6A, X, B), then a difference in nucleotide sequence is not considered to exist at that position.

[0045] The term "sense strand" refers to the nucleotide sequence on an RNA molecule that carries the information of the amino acids that encode the protein. It is also called the coding strand, sense strand, or positive strand, while the other nucleotide sequence that is complementary to it is called the antisense strand.

[0046] The term "antisense strand" refers to a nucleotide sequence in the mRNA of the target gene that is substantially anticomplementary or substantially anticomplementary to the antisense strand.

[0047] In this invention, unless otherwise specified, the term "pharmaceutical acceptable" means that the carrier, transporter, diluent, excipient and / or the salt / ester / hydrate formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0048] In this invention, unless otherwise specified, the term "inhibition" refers to the down-regulation of target gene expression due to siRNA-mediated mRNA degradation. "Down-regulation" refers to a decrease in target gene expression level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, or even 100%, compared to the absence of siRNA treatment. A 100% decrease in target gene expression level means that there is no detectable level of target gene expression.

[0049] In some embodiments of the present invention, the siRNA may also contain modified nucleotides as needed, wherein the modified nucleotides do not cause a significant weakening or loss of the siRNA's function in inhibiting ANGPTL4 gene expression. Currently, there are various ways in the art to modify siRNA, including, for example, backbone modification (such as phosphate group modification), ribose group modification, and base modification (Watts, JK, G.F. Deleavey, and M.J. Damha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20): p. 842-55).

[0050] In some embodiments of the present invention, at least one nucleotide in the sense or antisense strand of the siRNA is a modified nucleotide, for example, a ribose group and optionally a phosphate group modified nucleotide group, but not limited thereto.

[0051] In some embodiments of the present invention, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides or nucleotide analogs.

[0052] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-cleaved nucleotide analog, 2'-fluoroarabinonucleotide, 2'-methoxyethylnucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, 3'-methoxynucleotide, 2'-allyl-modified nucleotide, nucleotide containing a thiophosphate group, nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate, nucleotide containing a 5'-phosphate mimic, diol-modified nucleotide, debaseted nucleotide, morpholinonucleotide, locked nucleotide (LNA), unlocked nucleotide (UNA), threononucleotide (TNA), or glycerol nucleotide (GNA), but the present invention is not limited thereto.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 4 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0068] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 5 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0069] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0070] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 5th, 7th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 7th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0075] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0076] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0077] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0078] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0079] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxynucleotides, the 12th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0080] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0081] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0082] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 23 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0083] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 1 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0084] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0085] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0086] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0087] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0088] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 6 is 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides.

[0089] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0090] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein the 14th position of the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxynucleotides, the 12th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein the 8th, 9th, and 10th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0091] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein the antisense strand is located at 3-10 positions (e.g., positions 2, 14, 16, 2, 5, 14, 16, 2, 4, 6, 14, 16, 2, 6, 10, 14, 16, 2, 6, 12, 14, 16, 2, 5, 10, 14, 16, 2, 3, 12, 14, 16, 2, 9, 12, 14, 16, 2, 6, 8, 9, 14, 16, 2, 3, 5, 12, 14, 16, 2, 8, 9, 12, 14, 16). The siRNA has 2'-fluoronucleotides at positions 2, 7, 9, 12, 14, 16, 2, 4, 6, 8, 10, 14, 16, 18, 20, and 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, with the remaining positions being 2'-methoxynucleotides. The siRNA has a positive strand length of 20 nucleotides, wherein the 8th, 9th, and 10th positions at the 5' end of the positive strand are 2'-fluoronucleotides, optionally one position (preferably the 1st position at the 5' end) has a threonucleotide, optionally one position (preferably the 6th position at the 5' end) is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotide conjugates.

[0092] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein the antisense strand has 2'-fluoronucleotides at the following positions: positions 2, 14, 16 at the 5' end, or positions 2, 5, 14, 16, or positions 2, 6, 14, 16, or positions 2, 6, 10, 14, 16, or positions 2, 6, 12, 14, 16, or positions 2, 5, 10, 14, 16, or positions 2, 3, 12, 14, 16, or positions 2, 9, 12, 14, 16, or positions 2, 6, 8, 9, 14, 16, or positions 2, 3, 5, 12, 14, 16. The siRNA has a positive strand length of 20 nucleotides, wherein the 8th, 9th, and 10th positions at the 5' end of the positive strand are 2'-fluoronucleotides, and optionally the 1st position at the 5' end is a threonucleotide, or optionally the 6th position at the 5' end is a 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides.

[0093] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein the antisense strand has 2'-fluoronucleotides at the following positions: positions 2, 14, 16, or positions 2, 5, 14, 16, or positions 2, 4, 6, 14, 16, or positions 2, 6, 10, 14, 16, or positions 2, 6, 12, 14, 16, or positions 2, 5, 10, 14, 16, or positions 2, 3, 12, 14, 16, or positions 2, 9, 12, 14, 16, or positions 2, 6, 8, 9, 14 The siRNA has a positive strand length of 20 nucleotides, with the 8th, 9th, and 10th positions at the 5' end being 2'-fluoronucleotides and the remaining positions being 2'-methoxynucleotides.

[0094] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0095] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 20th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0096] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0097] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 14th position of the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxynucleotides, the 12th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0098] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified by a thiophosphate group. That is, a sulfur atom replaces the non-bridging oxygen atom in the phosphodiester bond, thereby replacing the phosphodiester bond with a thiophosphate diester bond.

[0099] In some embodiments of the present invention, the 5' end and 3' end of the sense chain each independently contain 0, 1, or 2 thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain 1 or 2 thiophosphate groups.

[0100] In some embodiments of the present invention, at least one of the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are linked by a thiophosphate group; preferably, at least four are linked by thiophosphate groups; in some embodiments of the present invention, at least six are linked by thiophosphate groups; in some embodiments of the present invention, all eight are linked by thiophosphate groups.

[0101] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and at positions 2 and 3, of the 5' end of the positive strand are linked by phosphate thioester groups.

[0102] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and positions 2 and 3 at the 5' end of the positive strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, and positions 2 and 3 at the 3' end are linked by thiophosphate groups.

[0103] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by thiophosphate groups, and the nucleotides at positions 2 and 3 at the 5' end are linked by thiophosphate groups.

[0104] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all linked by thiophosphate groups.

[0105] In some embodiments of the present invention, the positive strand may include one or more capping residues or portions, referred to as "capping residues". A "capping residue" is a non-nucleotide compound or other portion that can be incorporated into one or more ends of a nucleotide sequence of siRNA. In some embodiments of the present invention, the capping residues are present at the 5' end, the 3' end, or both the 5' end and the 3' end of the positive strand.

[0106] In some embodiments of the present invention, an inverse debasing residue (invAb) is added as a capping residue. See F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16. In some embodiments of the present invention, the 5' end and / or 3' end of the positive strand may contain more than one inverse debasing deoxyribose moiety as a capping residue.

[0107] In some embodiments of the present invention, one or more inverse debase residues (invAb) are added to the 3' end of the positive strand. In some embodiments of the present invention, one or more inverse debase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the present invention, one or more inverse debase residues may be inserted between the delivery vector portion and the nucleotide sequence of the siRNA positive strand. In some embodiments of the present invention, one or more inverse debase residues are included at or near one or more ends of the siRNA positive strand. The inverse debase residues (invAb) are selected from the structures shown in the following structural formulas (i)-(iv):

[0108] Structure (i): invAb is located at the 3' end of the oligonucleotide;

[0109] Structure (ii): invAb is located at the 5' end of the oligonucleotide;

[0110] Structure (iii): invAb is located inside the oligonucleotide; and

[0111] Structural formula (iv): invAb is located at both ends of the oligonucleotide;

[0112] Where X = O or S.

[0113] In some embodiments of the present invention, one or more inverse abase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the present invention, one or more inverse abase residues may be inserted between the delivery vector portion and the nucleotide sequence of the siRNA positive strand. In some embodiments of the present invention, one or more inverse abase residues (invAb) are added to both the 3' and 5' ends of the positive strand simultaneously.

[0114] Reverse debasing residues can be linked via covalent bonds between phosphate esters, thiophosphate esters, or other nucleosides.

[0115] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0116] Where Base is a base A, U, G, C, T or other nucleotide bases.

[0117] Where Base is a base A, U, G, C, T or other nucleotide bases.

[0118] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide.

[0119] In some embodiments of the present invention, the siRNA contains at least one base-modified nucleotide.

[0120] In some embodiments of the present invention, the bases of the base-modified nucleotide are selected from the following structures:

[0121] In some embodiments of the present invention, the base-modified nucleotides are located at positions 5, 6, 7, and 8 of the siRNA antisense strand.

[0122] In some embodiments of the present invention, the base-modified nucleotide is located at the single-stranded nucleotide overhang in the siRNA.

[0123] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the first nucleotide of the siRNA antisense strand overhang.

[0124] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the second nucleotide of the siRNA antisense strand overhang.

[0125] The present invention also provides siRNA conjugates obtained by conjugating the above-mentioned siRNA with conjugating molecules.

[0126] In this invention, unless otherwise specified, "conjugation" refers to the covalent connection between two or more chemical parts; "conjugated compound" refers to a compound formed by the covalent connection between various chemical parts; and "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts to siRNA. It should be noted that the chemical parts can be directly attached to the siRNA.

[0127] In some embodiments of the present invention, the delivery vector is linked to siRNA.

[0128] In some embodiments of the present invention, the delivery vector is independently or simultaneously attached to the 3' or 5' end of the siRNA's positive strand.

[0129] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugating molecule to obtain an siRNA conjugate. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugating molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site between the siRNA and the conjugating molecule can be at the 3' or 5' end of the siRNA's sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site between the siRNA and the conjugating molecule can also be within the internal sequence of the siRNA.

[0130] The pharmaceutically acceptable delivery carrier may be a delivery carrier commonly used in the field of siRNA drug delivery, such as, but not limited to, one or more of the following delivery carriers or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; or receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0131] In some embodiments of the present invention, the delivery carrier contains N-acetylgalactosamine.

[0132] In some embodiments of the present invention, the delivery vector may be directly attached to the 3' end of the siRNA positive strand. In some embodiments of the present invention, the delivery vector may be directly attached to the 5' end of the siRNA positive strand. In some embodiments of the present invention, the delivery vector may be attached to the 3' end of the siRNA positive strand. In some embodiments of the present invention, the delivery vector may be attached to the 5' end of the siRNA positive strand.

[0133] In some embodiments of the present invention, the delivery vector comprises N-acetylgalactosamine, which is covalently linked to the 3' end of the siRNA sense strand.

[0134] In some embodiments of the present invention, the delivery carrier portion is GalNAc(L96) and has the following structure:

[0135] In some embodiments of the present invention, GalNAc(L96) is linked to the 3' end of the siRNA positive strand.

[0136] In some embodiments of the present invention, GalNAc(L96) is linked to the 5' end of the siRNA positive strand.

[0137] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 3' end of the siRNA's positive strand.

[0138] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 5' end of the siRNA's positive strand.

[0139] In some embodiments of the present invention, the delivery carrier portion is Ser(GN) and has the following structure:

[0140] In some embodiments of the present invention, Ser(GN) is attached to the 3' end of the siRNA positive strand. In some embodiments of the present invention, Ser(GN) is attached to the 5' end of the siRNA positive strand.

[0141] In some embodiments of the present invention, Ser(GN) is simultaneously linked to both the 3' and 5' ends of the siRNA positive strand.

[0142] In some embodiments of the present invention, the GalNAc delivery carrier LP-GalNAc structure (attached to the 5' end of the positive chain) is as follows:

[0143] In some embodiments of the present invention, the delivery carrier portion is an XY-GalNAc (attached to the end of the justice chain 3'), having the following structure:

[0144] In some embodiments of the invention, the structure of other delivery carrier portions used (connected to the end of the justice chain 5') is shown below:

[0145] In some embodiments of the invention, the structure of other delivery carrier portions used (connected to the end of the justice chain 3') is as follows:

[0146] This application provides a small interfering RNA (siRNA) formulation targeting ANGPTL4, which can specifically bind to ANGPTL4 mRNA, disrupt the normal translation template function of ANGPTL4 mRNA, thereby preventing the translation of ANGPTL4 protein, thereby relieving the inhibition of LPL enzyme activity, reducing the content of TAG and DAG, reducing the PKCε signaling pathway, improving insulin sensitivity, and improving the metabolism of blood lipids and blood glucose.

[0147] The siRNA that inhibits ANGPTL4 gene expression in this invention can be used to prepare drugs that effectively prevent and / or treat pathological conditions or diseases (such as diseases related to dyslipidemia and glucose metabolism) associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression. Attached Figure Description

[0148] Figure 1 shows the inhibitory effect of conjugate 34 on hepatic ANGPTL4 mRNA in cynomolgus monkeys. Detailed Implementation

[0149] Those skilled in the art will recognize that the siRNA described in this invention can be obtained using conventional siRNA preparation methods (e.g., solid-phase synthesis and liquid-phase synthesis), both of which are commercially available custom-made services. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the siRNA described in this invention using appropriately modified nucleotide monomers. Methods for preparing appropriately modified nucleotide monomers are well known to those skilled in the art, and commercially available monomers are also available.

[0150] Example 1: siRNA Synthesis

[0151] The synthesis process is briefly described as follows: For the sense and antisense strands of the siRNA sequence of the present invention, as well as the sense and antisense strands of the modified double strands, CPG is used as a solid-phase carrier; GalNAc-modified CPG is used as the starting cycle for the synthesis of the sense strand, and universal CPG is used as the starting cycle for the synthesis of the antisense strand.

[0152] Using a YB-192S synthesizer, a solid-phase synthesis of phosphoramidite was performed, starting with a solid support and sequentially linking nucleoside monomers in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0153] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0154] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C or room temperature (25 °C) for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. After cooling to room temperature, the crude sequence was obtained by ethanol precipitation.

[0155] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0156] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The product was then freeze-dried to obtain the target product.

[0157] Table 1: Sequences of modified siRNA conjugates

[0158] In this diagram, uppercase letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter d indicates that the nucleotide adjacent to the right of d is a deoxyribonucleotide; lowercase letter m indicates that the nucleotide adjacent to the right of m is a 2'-methoxy modified nucleotide; the symbol f indicates that the nucleotide adjacent to the right of f is a 2'-fluorine modified nucleotide; gn indicates that the nucleotide adjacent to the right of gn is a glycerol nucleotide (GNA); tn indicates that the nucleotide adjacent to the right of tn is a threonucleotide (TNA); ln indicates that the nucleotide adjacent to the right of ln is a ribose ring 2',4'-locked nucleotide; mI represents inosine with methoxy substitution at the 2' position of the ribose; the symbol * indicates that the two nucleotides adjacent to * are linked by a thiophosphate group; the letter eVP indicates that the nucleotide adjacent to the right of eVP is a vinyl phosphate modified nucleotide; and invAb represents a reverse debasement residue. GalNAc(L96) refers to the GalNAc(L96) region where the linker-targeting ligand is attached.

[0159] The following are the structural characterization methods and results of the conjugates in Table 2:

[0160] Representative LC-MS test method: When the test sample is subjected to denaturing IP·RP-LC detection, the complementary double strands are untied into single strands (sense and antisense strands). Then, the parent ions of the sense and antisense strands are fragmented by tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the software CONFIRM Sequence. The sequence of the test sample is consistent with the theoretical sequence, that is, the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.05%. The results are shown in Table 2.

[0161] Table 2: Molecular weight (MW) of modified siRNA conjugates

[0162] Example 2: In vitro activity assay of ANGPTL4 siRNA - psiCHECK

[0163] To screen for ANGPTL4 siRNA, the human ANGPTL4 cDNA sequence (accession number NM_139314.3) was cloned from a commercially available mammalian expression vector (oriene, rockville, md) into a commercially available reporter-based selection plasmid (psiCHECK-2, Promega), which produces Renilla luciferase / ANGPTL4 fusion mRNA. psiCHECK screening was performed in 293T cells (Nanjing Kebai) to detect siRNA activity. 293T cells were seeded at 20,000 cells / well in 96-well plates; the ANGPTL4 siRNA of this invention was transfected at two concentrations: 50 ng ANGPTL4-psiCHECK 2 plasmid per well and 0.3 μL lipofectamine 2000 per well. PBS treatment was used as a control. After culturing at 37°C and 5% CO2 for 24 h, ANGPTL4 gene knockout activity was determined using a Dual-Glo luciferase assay system (Promega, E2920). Each conjugate was subjected to 3–4 independent transfection assays. Gene knockout was determined by measuring Renilla luciferase PBS normalized to constitutively expressed firefly luciferase levels (Table 3).

[0164] Table 3. Results of in vitro activity tests of the conjugates

[0165] Experimental results show that the siRNA conjugate of the present invention has good in vitro inhibitory activity against ANGPTL4 gene expression.

[0166] Example 3: Detection of ANGPTL4 RNAi in vitro activity - free uptake by human primary hepatocytes

[0167] After resuscitation, human primary hepatocytes were diluted with culture medium to a density of 600,000 cells / mL. Different concentrations of conjugates were added to 96-well collagen plates at 10 μL / well, followed by 90 μL / well of human primary hepatocytes (54,000 cells / well). A PBS control group was also included. The plates were incubated at 37°C with 5% CO2 for 48 hours. After 48 hours, the culture medium was removed, and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy96 Kit (QIAGEN-74182) according to the kit instructions. cDNA was then synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323-01) according to the kit instructions. Real-time PCR was performed using the ΔΔCt assay on an Applied Biosystems-QuantStudio 7Flex real-time PCR system (Table 4).

[0168] Table 4: Results of Free Uptake Test in Human Primary Hepatocytes

[0169] Experimental results show that the siRNA conjugate of the present invention has excellent activity in inhibiting ANGPTL4 gene expression in human primary liver cells.

[0170] Example 4: Activity assay of ANGPTL4 RNAi in AAV8-hANGPTL4 overexpressing mice

[0171] A 6-7 week old mouse overexpression model was established by tail vein injection of AAV8-hANGPTL4. Two weeks after injection, mice were randomly divided into groups of four according to their body weight. On Day 1, each group received a subcutaneous injection of the conjugate at a dose of 1 mg / kg, while an equal volume of PBS was injected as a control. On Day 8, all mice were euthanized by CO2 inhalation, and their livers were collected. The hANGPTL4 mRNA level in the liver was detected by qPCR to evaluate the knockdown effect of different conjugates on the target gene (Table 5).

[0172] Table 5: Inhibition of hepatic hANGPTL4 mRNA by conjugates in AAV8-hANGPTL4 overexpressing mice

[0173] The results showed that the above conjugates could significantly reduce the expression level of hANGPTL4 mRNA in the liver of AAV8-hANGPTL4 mouse model, with an inhibition rate of 41% to 77% after a single subcutaneous injection of 1 mg / kg.

[0174] Example 5: In vivo activity test of ANGPTL4 overexpression mice constructed by high-pressure tail vein injection of HDI.

[0175] Six- to seven-week-old mice were randomly divided into groups of 3 to 4 mice each based on their body weight. On Day 1, each group received a subcutaneous injection of the conjugate at a dose of 3 mg / kg, with an equal volume of PBS injected as a control. On Day 4 or Day 21, all mice received a tail vein injection containing 8% of their body weight of hANGPTL4 plasmid DNA solution within 5 seconds. On Day 5 or Day 22 (24 hours after ANGPTL4 plasmid injection), all mice were euthanized by CO2 inhalation, and their livers were collected. The hANGPTL4 mRNA level in the liver was detected by qPCR to evaluate the knockdown effect of different conjugates on the target gene (Table 6).

[0176] Table 6: Inhibition of hepatic hANGPTL4 mRNA by conjugates in HDI-hANGPTL4 mice

[0177] The results showed that the above conjugates could significantly reduce the expression level of hANGPTL4 mRNA in the liver of the HDI-hANGPTL4 mouse model, with an inhibition rate of 41% to 91% after a single subcutaneous injection of 3 mpk, and the inhibition of hepatic hANGPTL4 mRNA by the conjugates lasted for up to three weeks after administration.

[0178] Example 6: Activity test of the conjugate in cynomolgus monkeys

[0179] Three healthy male cynomolgus macaques were selected, and liver samples were collected from baseline (before administration) via liver biopsy. After 1–2 weeks of recovery, conjugate 34 was administered via a single subcutaneous injection at a dose of 10 mg / kg. Liver samples were collected by liver biopsy on days 14, 28, and 56 post-administration. The relative expression levels of hepatic ANGPTL4 mRNA at different time points were detected using qPCR (compared to pre-administration levels), as shown in Figure 1. The results showed that a single subcutaneous administration of conjugate 34 at 10 mg / kg significantly inhibited ANGPTL4 mRNA in the liver of cynomolgus macaques, and this inhibition persisted until day 56, with a maximum inhibition rate of approximately 90% (Day 56).

[0180] In summary, the siRNA and its conjugates of this invention exhibit good to excellent in vitro ANGPTL4 gene expression inhibitory activity, effectively inhibiting ANGPTL4 mRNA levels in various cell lines, demonstrating satisfactory immunostimulatory activity, no significant off-target effects, and significantly reducing ANGPTL4 mRNA expression at the animal level (ANGPTL4 humanized mice and cynomolgus monkeys).

Claims

1. An siRNA conjugate for inhibiting the expression of an ANGPTL4 gene, characterized by, The siRNA conjugate comprises a sense strand and an antisense strand; wherein the antisense strand comprises the same as SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 8 9, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 1 The nucleotide sequence of at least 17 consecutive nucleotides differing by no more than 4 nucleotides from any one of the following: 57, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245; the antisense strand is 17 to 30 nucleotides in length; the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

2. The siRNA conjugate according to claim 1, characterized in that, The antisense strand is 19–27 nucleotides long; the sense strand is 19–25 nucleotides long. Preferably, the antisense strand is 19-23 nucleotides in length; the sense strand is 19-21 nucleotides in length. More preferably, The antisense strand is 23 nucleotides long, and the sense strand is 21 nucleotides long; or The antisense strand is 22 nucleotides long, and the sense strand is 20 nucleotides long; or The antisense strand is 21 nucleotides long, and the sense strand is 21 nucleotides long; or The antisense strand is 21 nucleotides long, and the sense strand is 19 nucleotides long; or The antisense strand is 19 nucleotides long, and the sense strand is 19 nucleotides long.

3. The siRNA conjugate according to claim 1, wherein The antisense strand of the conjugate comprises the following sequences: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 The nucleotide sequences shown in 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, and 245 differ by no more than 3 nucleotides.

4. The siRNA conjugate according to any one of claims 1 to 3, characterized in that, The siRNA conjugate has a mismatch of no more than 3 nucleotides between the sense and antisense strands; Preferably, the sense strand and the antisense strand have a mismatch of no more than one nucleotide; More preferably, the justice chain and the antisense chain are completely complementary.

5. The siRNA conjugate according to any one of claims 1 to 4, wherein The siRNA conjugate contains at least one modifying nucleotide.

6. The siRNA conjugate according to claim 5, wherein All nucleotides in the sense and / or antisense strands of the siRNA conjugate are modified nucleotides or nucleotide analogs; Preferably, the modified nucleotide or nucleotide analogue is selected from: 2'-methoxynucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-cleaved nucleotide analogue, 2'-fluoroarabinonucleotide, 2'-methoxyethylnucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, 3'-methoxynucleotide, 2'-allyl-modified nucleotide, nucleotide containing a thiophosphate group, nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate group, nucleotide containing a 5'-phosphate mimic, diol-modified nucleotide, debased nucleotide, morpholinonucleotide, threonucleotide, locked nucleotide, unlocked nucleotide, glycerol nucleotide, and base-modified nucleotide.

7. The siRNA conjugate according to any one of claims 1 to 6, wherein, The 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain one or two thiophosphate groups.

8. The siRNA conjugate according to claim 7, characterized in that, The nucleotides at positions 1 and 2 of the 5' end of the sense strand, positions 2 and 3 of the 5' end of the sense strand, positions 1 and 2 of the 3' end of the antisense strand, positions 2 and 3 of the 3' end of the antisense strand, positions 1 and 2 of the 5' end of the antisense strand, and positions 2 and 3 of the 5' end of the antisense strand are all linked by thiophosphate groups.

9. The siRNA conjugate according to any one of claims 1 to 8, wherein, The first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide.

10. The siRNA conjugate of any one of claims 1-9, characterized in that, The siRNA conjugate is obtained by conjugating siRNA with a delivery vector; preferably, the delivery vector contains N-acetylgalactosamine, more preferably, the delivery vector contains GalNAc(L96).

11. The siRNA conjugate according to claim 10, characterized in that, The siRNA conjugate is selected from any of the conjugate molecules shown in Table 1.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the siRNA conjugate of claim 9 or 10 and a pharmaceutically acceptable carrier.

13. Use of the siRNA conjugate of any one of claims 1-11 and / or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression; Preferably, the pathological condition or disease is a disease related to dyslipidemia or a disease related to dysglucose; more preferably, the disease related to dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

14. A method of treating and / or preventing a pathological condition or a disease associated with overexpression of the Angiopoietin-like protein 4 (ANGPTL4) gene, characterized in that, The method includes administering a therapeutic or preventative amount of the siRNA conjugate as described in any one of claims 1-11 and / or the pharmaceutical composition as described in claim 12 to a subject in need; Preferably, the pathological condition or disease is a disease related to dyslipidemia or a disease related to dysglucose; more preferably, the disease related to dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

15. The siRNA conjugate of any one of claims 1-11 and / or the pharmaceutical composition of claim 12 for the treatment and / or prevention of pathological conditions or diseases associated with angiopoietin-like protein 4 (ANGPTL4) gene overexpression; Preferably, the pathological condition or disease is a disease related to dyslipidemia or a disease related to dysglucose; more preferably, the disease related to dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

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