ANGPTL4 targeting siRNA and conjugate and application thereof

Abstract

The invention provides siRNA (small interfering Ribonucleic Acid) targeting an ANGPTL4 (Angiopoietin-like protein 4) as well as a conjugate and application of the siRNA, and belongs to the technical field of biological medicines. The siRNA contains a positive-sense strand and an antisense strand, the antisense strand comprises at least 17 continuous nucleotides, the difference between the nucleotide sequences shown in SEQ ID NO: 2-SEQ ID NO: 124 and the nucleotide sequences shown in SEQ ID NO: 2-SEQ ID NO: 124 is not more than 4 nucleotides, and the length of the antisense strand is 17-30 nucleotides; the length of the positive-sense strand is 17-30 nucleotides, and the positive-sense strand is at least partially complementary to the antisense strand. The siRNA, the siRNA conjugate and the pharmaceutical composition provided by the invention have good stability, excellent ANGPTL4 gene inhibition activity and satisfactory immunostimulation, and can be used for remarkably reducing the concentration of ANGPTL4 protein at an animal level.

Description

siRNA targeting ANGPTL4 and its conjugate and use Technical Field

[0001] The present invention relates to an siRNA for inhibiting the expression of angiopoietin-like protein 4 (ANGPTL4) gene, a conjugate thereof, a pharmaceutical composition and use thereof in preventing and / or treating diseases related to dyslipidemia. Background Art

[0002] Angiopoietin-like 4 (ANGPTL4) is a member of the angiopoietin-like protein family and a secreted protein primarily expressed in adipose and liver tissues before being secreted into the blood. It contains a 406-amino acid sequence, primarily comprising two functional domains: an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain. It often exists as oligomers, glycosylated, and in various isoforms; both domains are conserved among angiopoietin family members. ANGPTL4 also contains an asparagine glycosylation site, a cAMP / cGMP-dependent protein kinase phosphorylation site, two protein kinase C phosphorylation sites, four myristoylation 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 receptorδ (PPARδ), and hypoxia-inducible factor 1 alpha (HIF1α).

[0003] Currently, increasing evidence indicates that the biological function of ANGPTL4 plays a key role in the pathological changes of metabolic diseases such as atherosclerosis, type 2 diabetes, fatty liver disease, and obesity. In particular, it can play 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), which activates the PKCε signaling pathway, resulting in the inhibition of insulin receptor activation and reduced liver insulin sensitivity. Reduced liver insulin sensitivity inhibits the conversion of blood glucose into glycogen, promotes the dissimilarity of glycogen to glucose, and leads to increased blood glucose. Several studies have shown that ANGPTL4 expression is associated with lipid and carbohydrate metabolism in the body.

[0004] Type 2 diabetes and its related complications are highly prevalent worldwide. According to the International Diabetes Federation, approximately 537 million adults (10%) currently have diabetes, a figure projected to rise to 643 million by 2023 and 783 million by 2045, 90% of whom suffer from type 2 diabetes. In my country, the 2020 edition of the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in my country" reports that the incidence of type 2 diabetes has risen to 11.2%. Despite the recent introduction of new drugs to treat type 2 diabetes, clinical blood sugar control and patient compliance remain suboptimal. A 2020 survey published in the British Medical Journal showed that the treatment rate for type 2 diabetes in China was 49%, while only 49.4% of patients met the glycated hemoglobin (HbA1c) target. Furthermore, existing type 2 diabetes medications require frequent dosing, with most requiring daily or even every meal, leaving room for improvement in patient compliance.

[0005] Compared to traditional drugs, siRNA has poor stability and is easily degraded by nucleases when administered systemically. Furthermore, efforts are needed to further improve activity while avoiding side effects such as off-target effects, immune stimulation, and cytotoxicity. Therefore, developing more candidate siRNAs that are stable in the blood, have good biological activity, low cytotoxicity, and can inhibit ANGPTL4 gene expression in a long-term manner has become an urgent issue that needs to be addressed. Patent applications WO2023044458A1 and WO2022261005A1 disclose siRNAs that can inhibit ANGPTL4 gene expression. However, the inhibitory effect of these siRNAs needs to be further improved.

[0006] Summary of the Invention

[0007] In order to solve the above-mentioned problems existing in the prior art, the present invention provides an siRNA for inhibiting the expression of the ANGPTL4 gene, wherein the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO:2 to SEQ ID NO:124, and 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.

[0008] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides in length; and the sense strand is 19 to 25 nucleotides in length.

[0009] In some embodiments of the present invention, the antisense strand is 19 to 23 nucleotides in length; and the sense strand is 19 to 21 nucleotides in length.

[0010] In some embodiments of the present invention, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments of the present invention, the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments of the present invention, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments of the present invention, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments of the present invention, the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.

[0011] In some embodiments of the present invention, the siRNA comprises one or more single-stranded nucleotide overhangs. For example, an overhang of 1, 2, 3, or 4 nucleotides. In some embodiments of the present invention, the overhang can be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the present invention, the overhang is present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0012] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has an overhang of 2 nucleotides.

[0013] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has a 2-nucleotide overhang, and the overhang is UU or GG.

[0014] In some embodiments of the invention, the siRNA 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).

[0015] In some embodiments of the invention, the siRNA has two blunt ends.

[0016] In some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:124 by no more than 4 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:124 by no more than 3 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:124 by no more than 2 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:124 by no more than 1 nucleotide; in some embodiments of the present invention, the antisense strand is any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:124.

[0017] In some embodiments of the present invention, the sense strand and the antisense strand have no more than 3 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 2 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 1 nucleotide mismatch; in some embodiments of the present invention, the sense strand and the antisense strand are fully complementary.

[0018] In some embodiments of the present invention, the sequence of the siRNA is selected from the sequences of duplex 1 to duplex 123.

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

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

[0021] Preferably, the modified nucleotide or nucleotide analogue is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogues, 2'-fluoroarabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, threose nucleotides, locked nucleotides, unlocked nucleotides, glycerol nucleotides or base-modified nucleotides.

[0022] In some embodiments of the invention, the 5' and 3' ends of the sense strand independently comprise 1 or 2 phosphorothioate linkages; and / or the 5' and 3' ends of the antisense strand independently comprise 1 or 2 phosphorothioate linkages.

[0023] Preferably, the nucleotides at positions 1 and 2 at the 5' end of the sense chain, the nucleotides at positions 2 and 3 at the 5' end of the sense chain, the nucleotides at positions 1 and 2 at the 3' end of the antisense chain, the nucleotides at positions 2 and 3 at the 3' end of the antisense chain, the nucleotides at positions 1 and 2 at the 5' end of the antisense chain, and the nucleotides at positions 2 and 3 at the 5' end of the antisense chain are all linked by phosphorothioate groups.

[0024] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate.

[0025] In some embodiments of the present invention, the antisense strand is 23 nucleotides in length, and the sense strand is 21 nucleotides in length; the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate and 2'-methoxy, the second, 14th, and 16th nucleotides at the 5' end are 2'-fluorine-modified nucleotides, the third to fourth, sixth, eighth to 13th, fifteenth, seventeenth to twenty-first, and twenty-second to twenty-third nucleotides at the 5' end are 2'-methoxy-modified nucleotides, the fifth nucleotide at the 5' end is a deoxyribonucleotide-modified nucleotide, the seventh nucleotide at the 5' end is a 2'-methoxy- or deoxyribonucleotide-modified nucleotide, and the first nucleoside at the 5' end is a nucleotide modified with 2'-methoxy or deoxyribonucleotide. The nucleic acid and the second nucleotide, the second nucleotide and the third nucleotide, the 21st and the 22nd nucleotide, and the 22nd and the 23rd nucleotide are connected by phosphorothioate groups; the 1st to 7th and the 12th to 21st nucleotides at the 5' end of the sense chain are 2'-methoxy modified nucleotides, the 8th nucleotide at the 5' end is a 2'-methoxy or 2'-fluorine modified nucleotide, the 9th to the 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 and the 2nd nucleotides and the 2nd and the 3rd nucleotides at the 5' end are connected by phosphorothioate groups.

[0026] The present invention also provides a siRNA conjugate obtained by conjugating any of the above-mentioned siRNAs with a conjugation molecule; preferably, the delivery vector contains N-acetylgalactosamine.

[0027] In some embodiments of the present invention, the siRNA conjugate is selected from conjugate 1 to conjugate 634, and conjugate 637 to conjugate 667.

[0028] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned siRNAs and / or any of the above-mentioned siRNA conjugates and a pharmaceutically acceptable carrier.

[0029] The present invention also provides use of any of the above siRNAs and / or any of the above siRNA conjugates and / or the above pharmaceutical compositions in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with overexpression of the angiopoietin-like protein 4 (ANGPTL4) gene.

[0030] Furthermore, the pathological condition or disease is a disease related to dyslipidemia or a disease related to dysglycemia; further 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.

[0031] The siRNA, siRNA conjugates and pharmaceutical compositions provided by the present disclosure have good stability, excellent ANGPTL4 gene inhibitory activity, satisfactory cytotoxicity and immunostimulatory properties, and can significantly reduce blood lipid levels.

[0032] The sequence of the ANGPTL4 gene targeted by the siRNA of the present invention is shown in SEQ ID NO: 1:

[0033] SEQ ID NO: 1 (ANGPTL4 gene)

[0034] In the present invention, "siRNA" refers to an oligonucleotide molecule containing RNA or RNA-like (e.g., chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNA can act through an RNA interference mechanism (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) of mammalian cells), or other arbitrary mechanisms or pathways. Although it is believed that the term siRNA drug used in the present invention mainly acts through the RNA interference mechanism, the siRNA drug is not limited to or restricted to any specific 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 the present invention is composed of an oligonucleotide chain that is at least partially complementary to the target mRNA. In some embodiments, the siRNA drug of the present invention is double-stranded and consists of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0035] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0036] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, including modified and unmodified nucleotides; lowercase letter m indicates that the nucleotide adjacent to the right of the identifier m is a 2'-methoxy nucleotide; lowercase letter f indicates that the nucleotide adjacent to the right of the identifier f is a 2'-fluoro nucleotide; lowercase letter d indicates that the nucleotide adjacent to the right of the identifier d is a 2'-deoxy nucleotide; gn indicates that the nucleotide adjacent to the right of the identifier gn is a glycerol nucleotide (GNA); tn indicates that the nucleotide adjacent to the right of the identifier tn is a threose nucleotide (TNA); the symbol * indicates that the two nucleotides adjacent to the left and right of the symbol * (or between the nucleotide and the delivery vector portion) are linked by phosphorothioate groups; eVP indicates that the nucleotide adjacent to the right is a (E)-vinyl phosphate-modified nucleotide; invAb indicates an inverted abasic residue; GalNAc(L96) indicates that the delivery vector GalNAc(L96) is conjugated to the site. Ser(GN) indicates that the delivery vector Ser(GN) is conjugated to the site.

[0037] In the present invention, " delivery vehicle " refers to that by being covalently linked to siRNA and by affecting the targeting, activity, cellular distribution, cellular absorption or the chemical part of stability of oligonucleotide, the conjugation of siRNA to one or more delivery vehicles can improve the pharmacological properties of siRNA. In certain embodiments, the delivery vehicle part is to modify or enhance the pharmacokinetic properties of oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability or cellular uptake of oligonucleotide. In particular, the delivery vehicle can target the oligonucleotide to a specific organ, tissue or cell type, thereby enhancing the effectiveness of the oligonucleotide in the organ, tissue or cell type. Simultaneously, the conjugate can be used to reduce the activity of the oligonucleotide in non-target cell types, tissues or organs, for example, off-target activity or activity in non-target cell types, tissues or organs.

[0038] In the present invention, unless otherwise specified, the capital letter I represents the base composition of the base-modified nucleotide. (Inosine); mI is Inosine with methoxy substituted at the 2'-position of ribose; m6A represents the base composition of base-modified nucleotides, the base is The capital letter X represents the base composition of the base-modified nucleotide. The capital letter B represents the base composition of the base-modified nucleotide, the base is Unless otherwise specified, the above nucleotides containing special bases all have a methoxy group substituted at the 2'-position of ribose.

[0039] In the present 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 generally have no more than 5, 4, 3, or 2 mismatched base pairs, while retaining the ability to hybridize under relevant conditions. In addition, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. In the present invention, when meeting the above hybridization ability requirements, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogstein base pairs. Correspondingly, in the present invention, unless otherwise specified, "mismatch" means that the bases at corresponding positions in the siRNA duplex molecule are not paired in a complementary form.

[0040] In the present invention, unless otherwise specified, "difference in nucleotide sequence" refers to a change in the base type (A, U, G, C) of the nucleotide at the same or corresponding position compared to the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, the nucleotide base at the same or corresponding position is changed to U, C, G or the nucleotide is dT, dC, dG, etc., it is considered that there is a difference in nucleotide sequence at that position. It should be noted here that, when compared to the original nucleotide sequence, the nucleotide at the same or corresponding position differs only in whether there is a modification or modification type, it is not considered that there is a difference in nucleotide sequence at that position. For example, when one nucleotide base in the original nucleotide sequence is U, the nucleotide at the same or corresponding position is dT or other base-modified nucleotides (such as I, m6A, X, B), it is not considered that there is a difference in nucleotide sequence at that position.

[0041] The term "sense strand" refers to the strand on the RNA molecule that carries the nucleotide sequence encoding protein amino acid information, which is called the sense strand, also known as the coding strand, sense strand or positive strand, and the other nucleotide sequence that is complementary to it is the antisense strand.

[0042] The term "antisense strand" refers to a nucleotide sequence in the mRNA expressed by the target gene that is substantially reverse complementary or essentially reverse complementary to a nucleotide sequence having the same length as the antisense strand.

[0043] In the present invention, unless otherwise specified, the term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient and / or the salt / ester / hydrate formed therefrom are generally chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form and physiologically compatible with the receptor.

[0044] In the present invention, unless otherwise specified, the term "inhibit" refers to the down-regulation of target gene expression due to siRNA-mediated degradation of target gene mRNA. The "down-regulation" refers to a decrease in target gene expression by 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%, relative to the absence of siRNA treatment. A 100% decrease in target gene expression means no detectable level of target gene expression.

[0045] In some embodiments of the present invention, the siRNA may further contain modified nucleotides as needed, provided that the modified nucleotides do not significantly weaken or abolish the siRNA's ability to inhibit ANGPTL4 gene expression. Currently, there are a variety of methods available in the art for modifying siRNA, including backbone modifications (e.g., phosphate group modifications), ribose group modifications, and base modifications (Watts, JK, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13(19-20): p. 842-55).

[0046] In some embodiments of the present invention, at least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide, for example, the modified nucleotide is a nucleotide group in which the ribose group and optionally the phosphate group are modified, but not limited thereto.

[0047] In some embodiments of the present invention, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides or nucleotide analogs.

[0048] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogs, 2'-fluoroarabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides (LNA), unlocked nucleotides (UNA), threose nucleotides (TNA) or glycerol nucleotides (GNA), but the present invention is not limited thereto.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, 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'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, 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'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 4 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

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

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0068] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0069] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 5, 7, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0070] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0075] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, the 2nd, 5th, and 7th positions are 2'-deoxy nucleotides, the 12th position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0076] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0077] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0078] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 23rd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0079] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0080] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0081] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0082] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0083] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0084] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 6th position is a 2'-deoxy nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0085] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 8th, 9th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0086] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, positions 2, 5, and 7 are 2'-deoxy nucleotides, position 12 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0087] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has a nucleotide sequence at positions 3-10 (e.g., 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 a 2'-fluoro nucleotide 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), and the remaining positions are 2'-methoxy nucleotides. The positive strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the positive strand are 2'-fluoro nucleotides, and optionally a threose nucleotide is present at one position (preferably the first position at the 5' end), and optionally a 2'-deoxynucleotide is present at one position (preferably the sixth position at the 5' end), and the remaining positions are 2'-methoxy nucleotide conjugates.

[0088] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides at the following positions: 2, 14, 16, or 2, 5, 14, 16, or 2, 6, 14, 16, or 2, 6, 10, 14, 16, or 2, 6, 12, 14, 16, or 2, 5, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 9, 12, 14, 16, or 2, 6, 8, 9, 14, 16, or 2, 3, 5, 12, 14, 16, or The siRNA has a positive strand length of 20 nucleotides, wherein positions 8, 9, and 10 at the 5' end of the positive strand are 2'-fluoro nucleotides, optionally having a threose nucleotide at the 1st position of the 5' end, or optionally having a 2'-deoxynucleotide at the 6th position of the 5' end, and the remaining positions are 2'-methoxy nucleotides.

[0089] In some embodiments of the invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the antisense strand has 2'-fluoro nucleotides at the following positions: 2, 14, 16, or 2, 5, 14, 16, or 2, 4, 6, 14, 16, or 2, 6, 10, 14, 16, or 2, 6, 12, 14, 16, or 2, 5, 10, 14, 16, or 2, 3, 12, 14, 16, or 2, 9, 12, 14, 16, or 2, 6, 8, 9, 14, 16, or 2, 6, 8, 9, 14, 16, or 2, 7, 9, 12, 14, 16, or 2, 8, 9 ...6, 12, 14, 16, or 2, 6, 12, 14, 16, or 2, 6, 10, 14, 16, or 2, 6, 12, 14, 16, or 2, , 16; or 2, 3, 5, 12, 14, 16; or 2, 8, 9, 12, 14, 16; or 2, 7, 9, 12, 14, 16; or 2, 4, 6, 8, 10, 14, 16, 18, 20; or 2, 4, 5, 6, 8, 10, 12, 14, 16, 18), and the remaining positions are 2'-methoxy nucleotides. The sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0090] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0091] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 20th position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0092] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0093] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, positions 2, 5, and 7 are 2'-deoxy nucleotides, position 12 is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0094] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified with a phosphorothioate group, that is, a non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond.

[0095] In some embodiments of the invention, the 5' and 3' ends of the sense strand independently contain 0, 1 or 2 phosphorothioate linkages; and / or the 5' and 3' ends of the antisense strand independently contain 1 or 2 phosphorothioate linkages.

[0096] In some embodiments of the present invention, at least one of the nucleotides between the 1st and 2nd positions at the 5' end of the sense chain, between the 2nd and 3rd positions at the 5' end of the sense chain, between the 1st and 2nd positions at the 3' end of the sense chain, between the 2nd and 3rd positions at the 3' end of the sense chain, between the 1st and 2nd positions at the 3' end of the antisense chain, between the 2nd and 3rd positions at the 3' end of the antisense chain, between the 1st and 2nd positions at the 5' end of the antisense chain, and between the 2nd and 3rd positions at the 5' end of the antisense chain is linked by a thiophosphate group; preferably, at least four of the nucleotides are linked by thiophosphate groups; in some embodiments of the present invention, at least six of the nucleotides are linked by thiophosphate groups; in some embodiments of the present invention, all eight of the nucleotides are linked by thiophosphate groups.

[0097] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 at the 5' end of the sense strand are linked by phosphorothioate groups.

[0098] In some embodiments of the present invention, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 5' end of the sense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 3' end are linked by phosphorothioate groups.

[0099] In some embodiments of the present invention, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 at the 3' end of the antisense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 at the 5' end are linked by phosphorothioate groups.

[0100] In some embodiments of the present invention, the nucleotides between the 1st and 2nd positions at the 5' end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 5' end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3' end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 3' end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3' end of the antisense chain, the nucleotides between the 2nd and 3rd positions at the 3' end of the antisense chain, the nucleotides between the 1st and 2nd positions at the 5' end of the antisense chain, and the nucleotides between the 2nd and 3rd positions at the 5' end of the antisense chain are all linked by phosphorothioate groups.

[0101] In some embodiments of the present invention, the sense strand may include one or more blocking residues or moieties, referred to as "blocking residues." A "blocking residue" is a non-nucleotide compound or other moiety that can be incorporated into one or more ends of the nucleotide sequence of the siRNA. In some embodiments of the present invention, the blocking residue is present at the 5' end, the 3' end, or both the 5' end and the 3' end of the sense strand.

[0102] In some embodiments of the present invention, an inverted abasic 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 sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

[0103] In some embodiments of the present invention, one or more reverse abasic residues (invAb) are added to the 3' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues can be inserted between the nucleotide sequence of the delivery vehicle portion and the siRNA sense strand. In some embodiments of the present invention, one or more reverse abasic residues are included near or at one or more ends of the siRNA sense strand. The reverse abasic residues (invAb) are selected from the following structures:

[0104] Wherein, X=O or S.

[0105] In some embodiments of the invention, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments of the invention, one or more inverted abasic residues can be inserted between the delivery vehicle portion and the nucleotide sequence of the siRNA sense strand. In some embodiments of the invention, one or more inverted abasic residues (invAb) are added to both the 3' and 5' ends of the sense strand.

[0106] Inverted abasic residues can be linked via phosphate, phosphorothioate, or other internucleoside covalent bonds.

[0107] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0108] Wherein, Base is base A, U, G, C, T or other nucleotide bases.

[0109] Wherein, Base is base A, U, G, C, T or other nucleotide bases.

[0110] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate.

[0111] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0112] In some embodiments of the present invention, the base of the base-modified nucleotide is selected from the following structures:

[0113] In some embodiments of the present invention, the base-modified nucleotides are located at positions 5, 6, 7, and 8 of the antisense strand of the siRNA.

[0114] In some embodiments of the present invention, the base-modified nucleotides are located at single-stranded nucleotide overhangs in the siRNA.

[0115] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the first nucleotide of the overhang of the siRNA antisense strand.

[0116] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the second nucleotide of the overhang of the siRNA antisense strand.

[0117] The present invention also provides a siRNA conjugate obtained by conjugating the above siRNA with a conjugation molecule.

[0118] In the present invention, unless otherwise specified, "conjugation" refers to the covalent attachment of two or more chemical moieties to each other; "conjugate" refers to a compound formed by covalent attachment of chemical moieties; and "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties to siRNA. It should be noted that each chemical moiety can be directly attached to the siRNA.

[0119] In some embodiments of the invention, the delivery vector is linked to the siRNA.

[0120] In some embodiments of the present invention, the delivery vector is independently or simultaneously linked to the 3' end or the 5' end of the sense strand of the siRNA.

[0121] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugate molecule to obtain an siRNA conjugate. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugate molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site of the siRNA to the conjugate molecule can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site of the siRNA to the conjugate molecule can also be within the internal sequence of the siRNA.

[0122] The pharmaceutically acceptable delivery carrier can be a delivery carrier conventionally used in the field of siRNA administration, such as, but not limited to, one or more of the following delivery carriers or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); or receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialoglycosylated residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.

[0123] In some embodiments of the present invention, the delivery vehicle comprises N-acetylgalactosamine.

[0124] In some embodiments of the present invention, the delivery vector can be directly connected to the 3' end of the siRNA sense strand. In some embodiments of the present invention, the delivery vector can be directly connected to the 5' end of the siRNA sense strand. In some embodiments of the present invention, the delivery vector can be directly connected to the 3' end of the siRNA sense strand. In some embodiments of the present invention, the delivery vector can be connected to the 5' end of the siRNA sense strand.

[0125] In some embodiments of the present invention, the delivery vector comprises N-acetylgalactosamine, which is covalently linked to the 3' end of the sense strand of the siRNA.

[0126] In some embodiments of the invention, the delivery vector portion is GalNAc(L96) having the following structure:

[0127] In some embodiments of the invention, GalNAc(L96) is linked to the 3' end of the sense strand of the siRNA.

[0128] In some embodiments of the invention, GalNAc(L96) is linked to the 5' end of the sense strand of the siRNA.

[0129] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (invAb) at the 3' end of the siRNA sense strand.

[0130] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (invAb) at the 5' end of the sense strand of the siRNA.

[0131] In some embodiments of the present invention, the delivery vehicle portion is Ser(GN) having the following structure:

[0132] In some embodiments of the invention, Ser(GN) is linked to the 3' end of the sense strand of the siRNA. In some embodiments of the invention, Ser(GN) is linked to the 5' end of the sense strand of the siRNA.

[0133] In some embodiments of the present invention, Ser (GN) is simultaneously connected to the 3' end and the 5' end of the siRNA sense strand. In some embodiments of the present invention, the GalNAc delivery vector LP-GalNAc structure (connected to the 5'-end of the sense strand) is as follows:

[0134] In some embodiments of the present invention, the delivery vector portion is XY-GalNAc (attached to the 3'-end of the sense strand) having the following structure:

[0135] or

[0136] In some embodiments of the present invention, the structure of the other delivery vector moiety used (attached to the 5'-end of the sense strand) is as follows:

[0137] In some embodiments of the present invention, the structure of the other delivery vector moiety used (attached to the 3'-end of the sense strand) is as follows:

[0138] The present application provides a small interfering RNA (siRNA) preparation targeting ANGPTL4, which can specifically bind to ANGPTL4 mRNA, destroy the normal translation template function of ANGPTL4 mRNA, thereby preventing it from translating ANGPLT4 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 blood lipid and blood glucose metabolism.

[0139] The siRNA for inhibiting ANGPTL4 gene expression of the present invention can be used to prepare drugs for effectively preventing and / or treating pathological conditions or diseases associated with overexpression of angiopoietin-like protein 4 (ANGPTL4) gene (eg, diseases associated with abnormal blood lipid and blood glucose metabolism). BRIEF DESCRIPTION OF THE DRAWINGS

[0140] FIG1 is a schematic diagram of a solid phase synthesis route for synthesizing the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex using a solid phase carrier.

[0141] FIG2 is a flow chart of the synthesis process of the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex using a solid phase carrier.

[0142] FIG3 . Inhibition of liver hANGPTL4 mRNA by the conjugate in HDI-hANGPTL4 mice. DETAILED DESCRIPTION

[0143] It is known to those skilled in the art that the siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis), wherein both solid phase synthesis and liquid phase synthesis are commercially available. It is also clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications are well known to those skilled in the art, and commercial monomers are also available on the market.

[0144] Example 1: siRNA synthesis

[0145] For the sense and antisense strands of the siRNA sequences of the present invention and the sense and antisense strands of the modified duplexes, solid phase supports were used to initiate strand synthesis; a solid phase support modified with GalNAc(L96) was used as the starting cycle for sense strand synthesis, and a universal solid phase support (Primer support 5G unylinker 350) was used as the starting cycle for antisense strand synthesis ( FIG1 ). The structure of the GalNAc(L96)-modified solid phase support (L96-PS) is as follows:

[0146] Primer support 5G unylinker 350 carrier structure is as follows:

[0147] Here, ● represents a PS polystyrene solid phase support.

[0148] Using a YB-192S synthesizer and a phosphoramidite triester solid phase synthesis method, starting with a solid phase support, nucleoside monomers were sequentially connected in the 3'-5' direction to perform sequence synthesis on a synthesis scale of 0.2 μmol.

[0149] The process flow is shown in Figure 2.

[0150] Process description: Oligo synthesis starts with a PS solid phase support and uses a 3'-0-(2-cyanoethyl)phosphoramidite / 4,4'dimethoxytrityl (dimethoxytrityl, DMT) group protection method to assemble oligonucleotide chains on a PS solid phase support. Each synthesis cycle includes 5'-hydroxyl deprotection, coupling, capping and oxidation (thiolation). Each coupling reaction is carried out by activating the appropriate phosphoramidite monomer and reacting it with the free 5'-hydroxyl group of the protected nucleotide or oligonucleotide fixed on the support. According to the sequence information, the corresponding crude oligonucleotide single-strand PS-Oligo is synthesized cyclically, and the crude oligonucleotide single chain is cleaved from the solid phase support and the relevant protecting groups are removed. The final product is then purified by preparative chromatography, desalted by ultrafiltration, annealed, and freeze-dried.

[0151] (1) Synthesis procedure

[0152] Includes the following units:

[0153] 1) Deblocking: The 5'-OH end of the ribonucleotide is protected with a DMT group (di-p-methoxytrityl). In the first step of the synthesis, trichloroacetic acid (TCA) is used to remove the DMT protecting group from the solid phase support and the ribonucleotide, so that the 5'-OH end of the naked ribonucleotide can be coupled with a new base.

[0154] 2) Coupling: Nucleotide monomers are mixed with an activating reagent and react with the PS-Oligo in the synthesis column. The activating reagent donates a proton to the nitrogen atom of the diisopropylamide on the 3'-phosphate, forming a phosphoramidite tetrazole reactive intermediate. Upon contact with the PS-Oligo, the phosphoramidite tetrazole undergoes a nucleophilic reaction with the 5'-hydroxyl group, resulting in coupling and tetrazole removal, extending one nucleotide.

[0155] 3) Capping: Since the coupling efficiency cannot reach 100%, in order to prevent unsuccessfully coupled PS-Oligo from continuing to the next step of coupling, a capping reagent is used to cap the 5'-hydroxyl group of PS-Oligo.

[0156] 4) Oxidation or thiolation: After the coupling reaction, the nucleotide is linked to the oligonucleotide on the PS carrier via a phosphite bond (trivalent phosphorus). This phosphite bond is unstable and easily hydrolyzed by acid and base. The oxidizing agent oxidizes the trivalent phosphorus to pentavalent phosphorus. Thiolation refers to the reaction of the trivalent phosphorus in the phosphite bond with a thiolation agent under weak alkaline conditions to form a phosphorus-sulfur bond.

[0157] 5) VP protecting group removal and aminolysis

[0158] Transfer the synthesized PS-oligo from the synthesis column to a centrifuge tube. Prepare a stripping agent in a volume ratio of 3:2:100 = TMS-I:pyridine:DCM. Add the stripping agent to the centrifuge tube and react for 30 minutes. Prepare a 1:1 solution of TEA / acetonitrile, add 2-mercaptoethanol (final concentration 2M), mix thoroughly, and add to the reaction to terminate the reaction. Remove the supernatant and wash the PS-oligo twice for 5 minutes. After washing, add aminolysis solution (2-mercaptoethanol (2M) / 28% ammonia water) and proceed with aminolysis for 10 hours. After aminolysis, remove the ammonia by vacuum centrifugation and purify.

[0159] (2) Purification

[0160] The diluted sample after ammoniolysis was purified by anion column exchange chromatography, and the liquid phase system adopted high performance liquid chromatography. The chromatographic conditions were as follows.

[0161] Chromatographic column: PS-15Q10*250mm

[0162] Flow rate: 4ml / min

[0163] Detection wavelength: 260nm

[0164] Ion column preparation purification gradient program:

[0165] Mobile phase: Phase A: 10mM NaOH solution (PH=10) Phase B: 10mM NaOH solution (PH=10) + 2M NaCl

[0166] After preparation, the samples were confirmed by mass spectrometry and purity was confirmed.

[0167] (3) Ultrafiltration

[0168] Dissolve the purified sample in 4 ml of PBS, transfer it to a 1K ultrafiltration tube, and centrifuge it at 5000 rpm for 45 minutes. Use a Nanodrop to detect the presence of the sample in the tube. If the sample is detected at 260 nm, the ultrafiltration membrane is damaged and the tube should be replaced and ultrafiltration repeated. If the sample is not detected at 260 nm, add 4 ml of RNase-free water and ultrafiltration again. Repeat the ultrafiltration three times, each for 45 minutes.

[0169] (4) Annealing

[0170] After ultrafiltration, dilute the sense and antisense strands to 3 mg / ml, then mix them in a 1:1 molar ratio. Heat the water bath to 90°C and place the mixed sense and antisense strands in the water bath for 30 minutes. Then, close the water bath and allow the mixture to cool to room temperature for 16 hours. A 10 μL sample was collected for HPLC analysis.

[0171] The sense strands and antisense strands of other siRNA sequences of the present invention and the sense strands and antisense strands of modified duplexes are prepared using similar methods.

[0172] Table 1. Sense and antisense strand sequences of unmodified siRNA duplexes

[0173] Part of the siRNA sequence is chemically modified and conjugated to the delivery vector. The siRNA conjugate is as follows:

[0174] Table 2 Sequences of modified siRNA conjugates

[0175] The following are the structural characterization methods and results of the conjugates in Table 2:

[0176] Representative LC-MS testing method: When the test sample is subjected to denaturing IP·RP-LC, the complementary double strands are unwound into single strands (sense and antisense strands). The positive and antisense strand precursor ions are then fragmented by gas phase tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the CONFIRM Sequence software. The sequence of the test sample is consistent with the theoretical sequence, i.e., the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.5 parts per million. The results are shown in Table 3.

[0177] Table 3 Molecular weight (MW) of modified siRNA conjugates

[0178] Example 2: ANGPTL4 siRNA in vitro activity detection - psiCHECK

[0179] Bioinformatics analysis was used to screen candidate ANGPTL4 siRNA sequences for cross-reactivity between humans and non-human primates, thereby identifying potential ANGPTL4 inhibitors of the present invention. To screen for target siRNAs, the human ANGPTL4 cDNA sequence (accession number NMs139314.3) was cloned from a commercially available mammalian expression vector (Oreene, Rockville, MD) into a commercially available reporter-based screening plasmid (PsiCheck 2 (Promega, Madison, WI)), which produces a Renilla luciferase / ANGPTL4 fusion mRNA. PsiCheck screening was performed by assaying siRNA activity in 293T cells (Nanjing Kebai). 293T cells were seeded at 20,000 cells / well in 96-well plates. The ANGPTL4 siRNAs of the present invention were transfected at two concentrations: 50 ng of ANGPTL4-PsiCheck 2 plasmid per well and 0.3 μL of Lipofectamine 2000 per well. After 24 hours of incubation at 37°C in a 5% CO2 environment, ANGPTL4 gene knockdown activity was determined using a dual-luciferase reporter assay system (Promega, E2920). Three to four independent transfections were performed for each duplex. Gene knockdown was determined by measuring Renilla luciferase levels normalized to constitutively expressed firefly luciferase (Tables 4-8).

[0180] Table 4. Results of in vitro duplex activity test

[0181] Table 5. Results of in vitro duplex activity test

[0182] Table 6. Results of in vitro activity test of conjugates

[0183] Table 7. Results of in vitro activity test of conjugates

[0184] Table 8. Results of in vitro activity test of conjugates

[0185] The experimental results show that the siRNA duplex or siRNA conjugate of the present invention has good activity in inhibiting ANGPTL4 gene expression in vitro.

[0186] It can be further seen from the data in Table 5 that, compared with duplex 124 (the duplex reported in the prior art WO2023044458A1), the duplex 34 of the present invention has significantly improved activity in inhibiting ANGPTL4 gene expression.

[0187] Further, according to the data in Table 8, it can be seen that compared with conjugate 636 (the conjugate reported in the prior art WO2023044458A1), the activity of conjugates 370 and 372 of the present invention in inhibiting ANGPTL4 gene expression is significantly improved.

[0188] Example 3. ANGPTL4 RNAi in vitro activity detection - U138-MG cell transfection

[0189] U138-MG cells (ATCC) were seeded into 24-well plates at 40,000 cells / well and cultured at 37°C in a 5% CO2 environment for 16 h before transfection. siRNA and lipofectamine RNAiMax (Invitrogen) were co-transfected; after culture at 37°C in a 5% CO2 environment for 24 h, RNA was extracted using the MolPure Magnttic Tissue / Cell Total RNA Kit (Yisheng; cat: 18600ES60). cDNA synthesis was performed using the gDNA removal cDNA synthesis kit from Quanshijin (Beijing Quanshijin Biotechnology Co., Ltd.; Beijing; China Cat#AE311-03). ΔΔCt determination was performed in ABIQuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex or conjugate was tested in 3-4 independent transfections; each transfection was performed in triplicate (Tables 9-10). PC c, known to have an ANGPTL4 gene inhibitory effect, was used as a positive control.

[0190] Table 9. Duplex in vitro activity test results

[0191] Table 10. Conjugate IC50 test results

[0192] The experimental results show that the siRNA duplex or siRNA conjugate of the present invention has excellent activity in inhibiting ANGPTL4 gene expression in vitro.

[0193] Further according to the data in Table 10, it can be seen that compared with conjugate PC c (the conjugate reported in the prior art WO2022261005A1), conjugate 60, conjugate 65, conjugate 79, conjugate 82, conjugate 89, conjugate 91, conjugate 149, conjugate 150, conjugate 151, conjugate 121, conjugate 327, conjugate 119, conjugate 145, conjugate 331, conjugate 332, conjugate 333, conjugate 339, conjugate 340, conjugate 311 and conjugate 164 of the present invention have significantly improved activity in inhibiting ANGPTL4 gene expression.

[0194] Example 4. ANGPTL4 RNAi in vitro activity assay - free uptake by human and cynomolgus monkey primary liver cells

[0195] After the human and cynomolgus monkey liver primary cells were recovered, the cells were diluted with culture medium in proportion to adjust the density to 600,000 cells / mL. Different concentrations of conjugate were added to a 96-well collagen plate at 10 μL / well, and 90 μL / well of human liver primary cells or cynomolgus monkey liver primary cells (54,000 cells / well) were added to the plate. A PBS control group was also set up. After plating, the plates were placed in a 5% CO2, 37°C incubator and cultured for 48 hours. After 48 hours, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy 96 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 instructions. Real-time fluorescence PCR (Tables 11-14) was performed using the ΔΔCt assay in an Applied Biosystems-QuantStudio 7 Flex real-time fluorescence PCR system. PC c, which is known to have an ANGPTL4 gene inhibitory effect, was used as a positive control.

[0196] Table 11. Free uptake test results of human primary liver cells

[0197] Table 12. Free uptake test results of human primary liver cells

[0198] Table 13. Free uptake test of primary cynomolgus monkey liver cells

[0199] Table 14. Free uptake test of primary cynomolgus monkey liver cells

[0200] The experimental results show that the siRNA conjugate of the present invention has excellent activity in inhibiting ANGPTL4 gene expression in human and cynomolgus monkey primary liver cells.

[0201] Example 5. In vivo activity testing of ANGPTL4 RNAi in humanized ANGPTL4 (hANGPTL4) mice

[0202] 6-8 week-old male hANGPTL4 humanized mice (provided by Jiangsu Jicui) were randomly divided into groups based on body weight, with 6-8 mice per group. On Day 1, PBS, PC c 3 mpk, PC c 9 mpk, conjugate 65 3 mpk, and conjugate 82 3 mpk were subcutaneously injected. Liver samples were collected from 3-4 mice per group on Day 8 (D8) and Day 22 (D22) after administration. Liver hANGPTL4 mRNA levels were measured by QPCR, and the knockdown effects of the different conjugates on the target gene were compared (Table 15). A single subcutaneous administration of conjugate 65 3 mpk and conjugate 82 3 mpk resulted in 84% and 93% inhibition of hANGPTL4 mRNA in the liver on Day 8, respectively, which was superior to the 78% inhibition of conjugate 65 3 mpk. A single subcutaneous administration of conjugate 82 3 mpk also achieved a superior inhibition of 90% inhibition of conjugate 65 3 mpk on Day 8. At D22, conjugates 65 and 82 (3 mpk) still significantly inhibited hANGPTL4 mRNA expression in the liver, with inhibition rates of 43% and 71%, respectively. Conjugates PC c (3 mpk) and 9 mpk exhibited inhibition rates of 40% and 42%, respectively. These results demonstrate that conjugates 65 and 82 exhibited superior inhibitory activity and persistence against the target gene compared to conjugate PC c.

[0203] Table 15. Inhibition of liver hANGPTL4 mRNA expression by the conjugates in humanized ANGPTL4 mice

[0204] 6-8 week old male hANGPTL4 humanized mice (provided by Biocytogen) were randomly divided into groups of 3 based on body weight. On Day 1, conjugates 82, 121, 368, and 344 were administered subcutaneously at a dose of 3 mpk. Liver samples were collected from each group on Day 8 and Day 22 after administration, following an overnight fast. Liver hANGPTL4 mRNA levels were measured by QPCR to assess the knockdown effect of the different conjugates on the target gene (Table 16). Results demonstrated that conjugates 82, 121, 368, and 344 significantly and persistently inhibited ANGPTL4 expression in humanized mouse livers. After a single subcutaneous administration of 3 mpk, the maximum inhibition rates during the efficacy observation period (D22) were 68%, 65%, 80%, and 69%, respectively.

[0205] Table 16. Inhibition of liver hANGPTL4 mRNA expression by the conjugates in humanized ANGPTL4 mice

[0206] Example 6: In vivo activity testing of ANGPTL4 RNAi in hANGPTL4 overexpressing mice injected with HDI at high pressure in the tail vein

[0207] Six- to seven-week-old mice were randomly divided into groups based on body weight, with 3 to 5 mice per group. On Day 1, the conjugates were subcutaneously injected at a dose of 3 or 10 mpk, along with an equal volume of PBS as a control. On Day 4, all mice were injected via the tail vein with a hANGPTL4 plasmid DNA solution (8% of their body weight) over 5 seconds. On Day 5 (24 hours after ANGPTL4 plasmid injection), all mice were euthanized by CO2 inhalation, and livers were harvested for hANGPTL4 mRNA levels to assess the knockdown effect of the different conjugates on the target gene (Table 17, Figure 3).

[0208] Table 17. Inhibition of liver hANGPTL4 mRNA expression by the conjugates in HDI-hANGPTL4 mice

[0209] The results showed that conjugates 82, 121, 370, 371, 372, 373, 629 and 630 could significantly reduce the expression level of hANGPTL4 mRNA in the liver of the HDI-hANGPTL4 mouse model, with inhibition rates of 57% to 88% after a single subcutaneous injection of 3 mpk.

[0210] Example 7: In vivo activity test of conjugate 82 in common cynomolgus monkeys

[0211] During the acclimatization period, two male cynomolgus monkeys were screened based on body weight, food intake, temperature, electrocardiogram (ECG), blood pressure, hematology, and blood biochemistry levels. Baseline liver samples were collected via liver puncture (Day 7). After one week of recovery, the animals received a single subcutaneous injection of conjugate 82 (10 mpk) on Day 1. Liver samples were collected via liver puncture on Days 15 and 29. Liver ANGPTL4 mRNA levels were measured by QPCR (Table 18). Results showed that conjugate 82 significantly reduced ANGPTL4 mRNA expression in the liver of cynomolgus monkeys, with inhibition rates of 53% to 78%, which persisted for four weeks.

[0212] Table 18. Inhibition of ANGPTL4 mRNA in cynomolgus monkey liver by conjugate 82

[0213] Example 8: In vivo efficacy of conjugate 82 in a hyperlipidemic cynomolgus monkey model

[0214] Three hypertriglyceridemia (plasma triglyceride levels >1 mmol / L) cynomolgus monkeys were selected from a population of cynomolgus macaques fed a high-fat diet. They received a single subcutaneous injection of the conjugate 82 (10 mpk) on Day 1. Plasma triglyceride levels were monitored weekly following administration to assess the effects of LDR01035233 on blood lipids (Table 19). The results demonstrated that the conjugate 82 (10 mpk) significantly reduced plasma triglyceride levels in the hyperlipidemia cynomolgus monkey model, with a 46.87% reduction compared to baseline, which was sustained for four weeks.

[0215] Table 19. Efficacy of Conjugate 82 in the Hypertriglyceridemia Cynomolgus Monkey Model

[0216] Example 9: ANGPTL4 RNAi immunogenicity test

[0217] according to Freshly isolated and pooled human PBMCs were transfected with siRNA and the control compound polyIC (polyinosinic-polycytidylic acid) according to the Transfection Kit (Thermo-L3000-015) instructions. The final cell count was 20,000 cells per well. After incubation at 37°C in a 5% CO2 incubator for 24 hours, cell supernatants were collected and assayed for IFN alpha, IL-6, and TNF alpha levels (Cytokine Kit Thermo-PPX-03-MXU64WY). By comparing cytokine levels with those in control wells, the fold change for each siRNA was calculated to assess the cytokine-inducing effect of siRNA on human PBMCs (Table 20).

[0218] The experimental results showed that the induction effects of the control polyIC, unmodified (naked) siRNA and GS9688 on the three cytokines IFN alpha, IL-6 and TNF alpha at the tested concentrations were in line with expectations.

[0219] For IFN alpha, the reference conjugates PC c and conjugate 82 showed a slight induction effect at a transfection concentration of 100 nM, with fold changes of 3.50 and 4.62, respectively. At a transfection concentration of 10 nM, neither showed a significant induction effect (fold change less than 3). Conjugate 164 also showed no significant induction effect at either 100 nM or 10 nM. For IL-6, the reference conjugate PC c showed a slight induction effect at 10 nM, while conjugate 82 and conjugate 164 showed no significant induction effect at either 100 nM or 10 nM. For TNF alpha, conjugate 82 showed no significant induction effect at 100 nM, but a slight, but dose-independent, effect at 10 nM. Conjugate 164 showed no significant induction effect at either 100 nM or 10 nM.

[0220] Table 20. In vitro immunogenicity test results of ANGPTL4 RNAi

[0221] The above results indicate that conjugates 82 and 164 of the present invention have very low immunostimulatory properties.

[0222] Example 10. ANGPTL4 RNAi off-target analysis - free uptake RNAseq in human primary hepatocytes

[0223] After recovery of primary human liver cells, cells were diluted proportionally with culture medium to a density of 670,000 cells / mL. Conjugate 82 of varying concentrations was added to a 24-well collagen plate at 50 μL / well. 450 μL / well of primary human liver cells was then added to the plate. A PBS control group was also set up. After plating, the plates were incubated in a 5% CO2, 37°C incubator for 48 hours. After 48 hours, the culture medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN-74182) according to the kit instructions. 1 μg of extracted total RNA was used to prepare libraries using the Ribo-off rRNA Depletion Kit (Human / Mouse / Rat) (Vazyme N406-02), the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme NR604-02), and the VAHTS RNA Multiplex Oligos Set 1-Set 2 for Illumina (Vazyme N323 / N324). All samples were sequenced on a second-generation sequencer (NovaSeq 6000, Illumina) and compared with the sequencing results of the blank sample to identify genes significantly downregulated after RNAi treatment (log2 Fold Change ≤ -1, p-adjust < 0.05). cDNA was then synthesized using the HiScript III RT SuperMix for qPCR (with gDNA wiper) (Vazyme-R323-01) according to the manufacturer's instructions. Significantly down-regulated genes were validated by real-time PCR using the ΔΔCt assay in an Applied Biosystems-QuantStudio 7Flex Real-Time PCR System.

[0224] Sequencing analysis of RNA samples from human primary hepatocytes treated with Conjugate 82 confirmed significantly downregulated genes by real-time PCR. The results showed that only ANGPTL4 mRNA levels were significantly reduced. This suggests that Conjugate 82 has no significant off-target effects in human primary hepatocytes and that its inhibition of ANGPTL4 is highly specific.

[0225] In summary, the siRNA and its conjugates of the present invention have good to excellent in vitro ANGPTL4 gene expression inhibitory activity, can effectively inhibit the level of ANGPTL4 mRNA in multiple cell lines, have satisfactory immunostimulatory properties, have no obvious off-target effects, can significantly reduce the expression of ANGPTL4 mRNA at the animal level, and can significantly reduce the triglyceride level in the hyperlipidemia animal model.

Claims

1. A siRNA for inhibiting ANGPTL4 gene expression, characterized in that: The siRNA comprises a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence shown in SEQ ID NO:2 to SEQ ID NO:124, and 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 according to claim 1, characterized in that: The antisense strand is 19 to 27 nucleotides long; the sense strand is 19 to 25 nucleotides long; Preferably, the antisense strand is 19 to 23 nucleotides in length; the sense strand is 19 to 21 nucleotides in length; More preferably, The antisense strand is 23 nucleotides in length, and the sense strand is 21 nucleotides in length; or The antisense strand is 22 nucleotides in length, and the sense strand is 20 nucleotides in length; or The antisense strand is 21 nucleotides in length, and the sense strand is 21 nucleotides in length; or The antisense strand is 21 nucleotides in length, and the sense strand is 19 nucleotides in length; or The antisense strand is 19 nucleotides in length, and the sense strand is 19 nucleotides in length.

3. The siRNA according to claim 1, characterized in that: The antisense strand differs from any nucleotide sequence shown in SEQ ID NO:2 to SEQ ID NO:124 by no more than 3 nucleotides; Preferably, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO: 2 to SEQ ID NO: 124 by no more than 1 nucleotide; More preferably, the antisense strand sequence is any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO:

124.

4. The siRNA according to any one of claims 1 to 3, characterized in that: The sense strand and the antisense strand have no more than 3 nucleotide mismatches; Preferably, the sense strand and the antisense strand have no more than 1 nucleotide mismatch; More preferably, the sense strand is fully complementary to the antisense strand.

5. The siRNA according to claim 1, characterized in that: The sequence of the siRNA is selected from the sequence of duplex 1 to duplex 123.

6. The siRNA according to any one of claims 1 to 5, characterized in that: The siRNA contains at least one modified nucleotide.

7. The siRNA according to claim 6, characterized in that: All nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs; Preferably, the modified nucleotide or nucleotide analog is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split ring nucleotide analogs, 2'-fluoroarabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, threose nucleotides, locked nucleotides, unlocked nucleotides, glycerol nucleotides or base-modified nucleotides.

8. The siRNA according to any one of claims 1 to 7, characterized in that: The 5' end and 3' end of the sense strand independently contain 1 or 2 phosphorothioate linkages; and / or the 5' end and 3' end of the antisense strand independently contain 1 or 2 phosphorothioate linkages; Preferably, the 1st and 2nd nucleotides at the 5' end of the sense chain, the 2nd and 3rd nucleotides at the 5' end of the sense chain, the 1st and 2nd nucleotides at the 3' end of the antisense chain, the 2nd and 3rd nucleotides at the 3' end of the antisense chain, the 1st and 2nd nucleotides at the 5' end of the antisense chain, and the 2nd and 3rd nucleotides at the 5' end of the antisense chain are all linked by phosphorothioate groups.

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

10. The siRNA according to any one of claims 1 to 5, characterized in that: The antisense strand is 23 nucleotides long, and the sense strand is 21 nucleotides long; the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate and 2'-methoxy, the second, 14th, and 16th nucleotides at the 5' end are 2'-fluorine modified nucleotides, the third to fourth, sixth, eighth to 13th, 15th, 17th to 21st, and 22nd to 23rd nucleotides at the 5' end are 2'-methoxy modified nucleotides, the fifth nucleotide at the 5' end is a deoxyribonucleotide modified nucleotide, the seventh nucleotide at the 5' end is a 2'-methoxy or deoxyribonucleotide modified nucleotide, the first nucleotide and the second nucleotide at the 5' end are 2'-fluorine modified nucleotides, The nucleotides at positions 1 to 7, 12 to 21, and 2'-23 of the sense chain are connected by phosphorothioate groups; the nucleotides at positions 1 to 7 and 12 to 21 of the 5' end are 2'-methoxy-modified nucleotides, the nucleotide at position 8 of the 5' end is a 2'-methoxy- or 2'-fluorine-modified nucleotide, the nucleotides at positions 9 to 10 of the 5' end are 2'-fluorine-modified nucleotides, the nucleotide at position 11 of the 5' end is a deoxyribonucleotide or a 2'-fluorine-modified nucleotide, and the nucleotides at positions 1 and 2, and nucleotides at positions 2 and 3 of the 5' end are connected by phosphorothioate groups.

11. An siRNA conjugate obtained by conjugating the siRNA according to any one of claims 1 to 10 with a delivery vector; preferably, the delivery vector comprises N-acetylgalactosamine, and more preferably, the delivery vector comprises GalNAc(L96).

12. The siRNA conjugate according to claim 11, characterized in that: The siRNA conjugate is selected from conjugate 1 to conjugate 634 and conjugate 637 to conjugate 667.

13. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the siRNA according to any one of claims 1 to 9 and / or the siRNA conjugate according to claims 10 to 12 and a pharmaceutically acceptable carrier.

14. Use of the siRNA according to any one of claims 1 to 9 and / or the siRNA conjugate according to claims 10 to 12 and / or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating and / or preventing a pathological condition or disease associated with overexpression of the angiopoietin-like protein 4 (ANGPTL4) gene.

15. The use according to claim 14, characterized in that: The pathological condition or disease is a disease associated with dyslipidemia or a disease associated with dysglycemia; further preferably, the disease associated with dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, heart disease, myocardial infarction, angina pectoris or atherosclerosis.

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