SiRNA for inhibiting ANGPTL3 gene expression and application thereof

Abstract

The invention provides siRNA (small interfering Ribonucleic Acid) for inhibiting expression of an ANGPTL3 gene and application of the siRNA. Specifically, the invention provides a siRNA (small interfering Ribonucleic Acid) and a siRNA conjugate for inhibiting the expression of an ANGPTL3 (Angiopoietin-like 3) gene, a pharmaceutical composition of the siRNA and a method for reducing the expression of the ANGPTL3 gene by using the siRNA, the siRNA conjugate and the pharmaceutical composition of the siRNA. The siRNA, the siRNA conjugate and the pharmaceutical composition of the siRNA can be used for preventing and / or treating diseases or symptoms mediated by the ANGPTL3 gene.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to a siRNA for inhibiting the expression of ANGPTL3 (angiopoietin-like protein 3) gene and an application thereof. Background Art

[0002] Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like protein family that is involved in regulating lipid metabolism. The protein encoded by the ANGPTL3 gene is produced only in the liver and then secreted into the circulatory system. In the liver, ANGPTL3 is produced exclusively by hepatocytes, and its expression is regulated by hydroxysterol-activated liver X receptors (LXRs). Studies have shown that factors that may inhibit ANGPTL3 mRNA expression in vivo and / or in vitro include PPARδ, statins, insulin, leptin, thyroid hormones, and lipopolysaccharides. Studies have shown that the biological effects of ANGPTL3 are manifested in two aspects: regulating angiogenesis and lipid metabolism. It has a weak induction effect on angiogenesis, but a significant regulatory effect on lipid metabolism, and can regulate lipid metabolism by inhibiting lipoprotein lipase (LPL) activity.

[0003] The most successful ANGPTL3-related drug is the marketed antibody drug evinacumab, but its dosage is too high and is not widely used. Studies have shown that people with ANGPTL3 loss-of-function (LOF) mutations and / or reduced plasma ANGPTL3 levels have a reduced risk of CVD. It was found that circulating ANGPTL3 concentrations in healthy control subjects were lower than those in heart attack subjects. In addition, individuals who completely lacked ANGPTL3 showed no signs of atherosclerosis compared with control subjects with an average total atherosclerotic plaque burden of approximately 39%. Therefore, treatments that reduce ANGPTL3 plasma levels may be an important advance in cardiovascular treatment for individuals at risk for ischemic heart disease who have elevated LDL-C and plasma triglycerides, which generally reflect elevated plasma remnant cholesterol. Existing drugs that can lower both TG and LDL-C are less effective, and ANGPTL3 target drugs that can lower both TG and LDL-C are more effective, but the existing drugs for this target are antibody drugs, and their clinical dosages are very high, which limits their use in the field of cardiovascular disease.

[0004] Therefore, there is an urgent need in the art to develop a drug that can effectively and specifically inhibit ANGPTL3. Summary of the invention

[0005] The present invention provides a drug that can effectively and specifically inhibit ANGPTL3.

[0006] In a first aspect of the present invention, a siRNA for inhibiting the expression of angiopoietin-like protein 3 (ANGPTL3) gene is provided, wherein the siRNA comprises a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide.

[0007] wherein the antisense strand comprises at least 19 consecutive nucleotides that differ from any antisense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides,

[0008] The sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides of complementarity with the antisense strand.

[0009] In another preferred example, the sense strand comprises at least 19 consecutive nucleotides that differ from any sense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides.

[0010] In another preferred embodiment, the antisense strand has 19-23 bases matching and complementarity with the human ANGPTL3 transcript (NM_000253.4).

[0011] In another preferred embodiment, the lengths of the sense strand and the antisense strand are independently 19-23 nucleotides.

[0012] In another preferred embodiment, the antisense strand has 2 nucleotides protruding at the 3' end relative to the sense strand.

[0013] In another preferred embodiment, the antisense strand is a sequence selected from the following: SEQ ID NO: 2m, the value of m is any positive integer selected from 1 to 47, and m is not equal to 6, 30 and 46.

[0014] In another preferred embodiment, the sense strand is a sequence selected from the following: SEQ ID NO: 2n-1, wherein the value of n is any positive integer selected from 1 to 47, and n is not equal to 6, 30 and 46.

[0015] In another preferred example, the antisense strand comprises any one of the antisense strand nucleotide sequences shown in Table A, and the sense strand comprises any one of the sense strand nucleic acid sequences shown in Table A.

[0016] In another preferred embodiment, the siRNA is selected from any siRNA sequence code in Table A.

[0017] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown in the siRNA sequence code in the following Table A:

[0018] Table A

[0019]

[0020]

[0021] In another preferred embodiment, the sense strand and antisense strand are shown as SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0022] In another preferred embodiment, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0023] In another preferred embodiment, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

[0024] In another preferred embodiment, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, phosphorothioate modified nucleotides, or a combination thereof.

[0025] In another preferred embodiment, the 5' end of the sense strand contains 1 or 2 phosphorothioate-modified nucleotides; and / or the 5' end and 3' end of the antisense strand each independently contain 1 or 2 phosphorothioate-modified nucleotides.

[0026] In another preferred example, the antisense strand comprises any one of the antisense strand nucleotide sequences shown in Table B, and the sense strand comprises any one of the sense strand nucleic acid sequences shown in Table B.

[0027] In another preferred embodiment, the sense strand and antisense strand are selected from the sense strand and antisense strand shown in the siRNA sequence code in Table B:

[0028] Table B

[0029]

[0030]

[0031]

[0032] Among them, the letters mG are 2'-O-methylguanylic acid, mA is 2'-O-methyladenylic acid, mU is 2'-O-methyluridylic acid, and mC is 2'-O-methylcytidylic acid; 2FG is 2'-fluoroguanylic acid, 2FA is 2'-fluoroadenylic acid, 2FU is 2'-fluorouridylic acid, and 2FC is 2'-fluorocytidylic acid; the letter S indicates that the two nucleotides adjacent to the letter S are connected by thiophosphate groups; dA is 2'-deoxyadenylic acid, dC is 2'-deoxycytidylic acid, dG is 2'-deoxyguanylic acid, and dT is 2'-deoxythymidylic acid.

[0033] In another preferred embodiment, the siRNA is selected from any siRNA sequence code in Table B.

[0034] In another preferred embodiment, the siRNA is selected from the siRNAs shown in the following siRNA sequence codes in Table B: L3-02, L45-01 or L5N-03.

[0035] In another preferred embodiment, the siRNA is selected from the siRNA sequence code L3-02 in Table B.

[0036] In another preferred embodiment, the siRNA is selected from the siRNA sequence code L45-01 in Table B.

[0037] In another preferred embodiment, the siRNA is selected from the siRNA sequence code L5N-03 in Table B.

[0038] In another preferred embodiment, the sense strand is selected from the group consisting of sequences shown in SEQ ID NO: 104, SEQ ID NO: 151 or SEQ ID NO: 138.

[0039] In another preferred embodiment, the antisense strand is selected from the group consisting of sequences shown in SEQ ID NO: 105, SEQ ID NO: 152 or SEQ ID NO: 139.

[0040] In another preferred embodiment, the sense strand and antisense strand are shown as SEQ ID NO: 104 and SEQ ID NO: 105, respectively.

[0041] In another preferred embodiment, the sense strand and antisense strand are shown as SEQ ID NO: 151 and SEQ ID NO: 152, respectively.

[0042] In another preferred embodiment, the sense strand and antisense strand are shown as SEQ ID NO: 138 and SEQ ID NO: 139, respectively.

[0043] In the second aspect of the present invention, a siRNA conjugate is provided, wherein the siRNA conjugate comprises the siRNA described in the first aspect of the present invention and a targeting group.

[0044] In another preferred embodiment, the conjugate further comprises a linker, and the siRNA, the linker and the targeting group are covalently or non-covalently linked once.

[0045] In another preferred embodiment, the targeting group contains a group derived from carbohydrates, and the carbohydrates include monosaccharides, disaccharides, trisaccharides or polysaccharides.

[0046] In another preferred embodiment, the targeting group is selected from the following monosaccharides: allose, maltose, arabinose, cladinose, brown sugar, erythrose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fucose, galactosamine, D-galactosamine alcohol, N-acetyllactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose, glucose-6-phosphate, glucose glyceraldehyde, L-glycero-D-mannose-heptose, glycerol, glycerol, glucose, iodose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnose, ribose, ribulose, heptose, sorbose, tagatose, talose, tartaric acid, threose, xylose and xylose.

[0047] In another preferred embodiment, the targeting group contains a group derived from N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0048] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0049] (a) the siRNA according to the first aspect of the present invention, and / or the conjugate according to the second aspect of the present invention; and

[0050] (b) a pharmaceutically acceptable carrier.

[0051] In another preferred embodiment, the pharmaceutical composition is used to inhibit the expression of ANGPTL3 gene.

[0052] In another preferred embodiment, the inhibition rate of expression can be calculated by the following formula:

[0053]

[0054] In another preferred embodiment, the value of Y is 50% to 90%.

[0055] In the fourth aspect of the present invention, provided is the use of the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a medicament for preventing and / or treating ANGPTL3-mediated diseases or conditions, for inhibiting the expression of the ANGPTL3 gene.

[0056] In another preferred embodiment, the disease or condition mediated by ANGPTL3 is a disease or condition in which ANGPTL3 is overexpressed.

[0057] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0058] In another preferred embodiment, the diseases or conditions include: atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0059] In another preferred embodiment, the preparation comprises a laboratory preparation.

[0060] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0061] In another preferred embodiment, the inhibition rate of expression can be calculated by the following formula:

[0062]

[0063] In another preferred embodiment, the value of Y is 50% to 90%.

[0064] In a fifth aspect of the present invention, a method for inhibiting ANGPTL3 expression in cells in vitro is provided, the method comprising the following steps:

[0065] (z1) Co-culturing cells with an effective amount of the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention.

[0066] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0067] In another preferred embodiment, the siRNA described in the first aspect of the present invention, the conjugate described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention enters the cell through endocytosis.

[0068] In the sixth aspect of the present invention, a method for treating an ANGPTL3-mediated disease or condition is provided, wherein a therapeutically effective amount of the siRNA of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention is administered to a subject.

[0069] In another preferred embodiment, the ANGPTL3-mediated disease or condition is a disease or condition in which ANGPTL3 is overexpressed.

[0070] In another preferred embodiment, the administration dose is 1 to 20 mg / kg.

[0071] In another preferred embodiment, the disease or condition includes lipid metabolism disorder.

[0072] In another preferred embodiment, the diseases or conditions include: atherosclerosis, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, and cardiovascular disease.

[0073] In another preferred embodiment, the subject is a human or a non-human mammal.

[0074] In another preferred embodiment, the subject is a human.

[0075] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The statistical results of the inhibition rate of the modified siRNA on the serum ANGPTL3 protein content of ANGPTL3 transgenic mice in one embodiment of the present invention are shown.

[0077] Figure 2 The results of the detection of blood lipids (TG, TC, HDL-C, LDL-C) of ANGPTL3 transgenic mice by the modified siRNA in one embodiment of the present invention are shown.

[0078] Figure 3 The results of Hep3B cell viability detection using modified siRNA in one embodiment of the present invention are shown.

[0079] Figure 4 The results of gene expression detection after the modified siRNA (L3-02) was detected by qPCR in one embodiment of the present invention are shown, wherein TIPIN, PLPPR5, WDR66 and UBASH3B represent TIMELESS interacting protein gene, phospholipid phosphatase-related 5 gene, WD repeat domain 66 gene, ubiquitin-related and SH3 domain B gene, respectively.

[0080] Figure 5The results of gene expression detection after the modified siRNA (L45-01) was detected by qPCR in one embodiment of the present invention are shown, wherein B3GNT5, CDYL, COX10, KIF2A, and MAP9 represent beta-1,3-N-acetylglucosamine transferase gene, crotonyl-CoA hydratase gene, cytochrome c oxidase assembly factor heme A gene, kinesin family member 2A gene, and microtubule-associated protein 9 gene, respectively. DETAILED DESCRIPTION

[0081] After extensive and in-depth research, the inventors have developed for the first time a class of siRNA drugs that inhibit the expression of ANGPTL3 protein, which can achieve strong reduction of TG and LDL-C at the same time, and can also achieve extended medication cycle. The siRNA of the present invention can effectively inhibit the expression of the ANGPTL3 gene, thereby effectively treating diseases or conditions with overexpression of ANGPTL3; the siRNA of the present invention is a class of siRNA drugs with a long-acting dosing interval, which provides a more convenient treatment method for the treatment of lipid metabolism disorders including preventive treatment of atherosclerotic cardiovascular diseases; the 3' end of the siRNA sense chain of the present invention is coupled with GalNAc, so that liver-specific ANGPTL3 silencing can be achieved without affecting the intestinal ANGPTL3 expression. The present invention was completed on this basis.

[0082] The siRNA provided by the present invention comprises a sense chain and an antisense chain, wherein the antisense chain induces the formation of an RNA-induced silencing complex (RISC), and binds to a human ANGPTL3 transcript (NM_014495.4) through complementary base pairing, thereby cleaving the transcript of the ANGPTL3 gene and reducing the expression of ANGPTL3.

[0083] the term

[0084] As used herein, the term "ANGPTL3" refers to angiopoietin-like protein 3 from humans, rodents and primates, including the full-length unprocessed precursor form, the mature form (signal peptide cleavage) and the ANGPTL3 protein after protease processing. The human ANGPTL3 mRNA transcript is numbered as NM_014495.4, and the cynomolgus monkey ANGPTL3 mRNA predicted sequence is numbered as (XM_005543185.3).

[0085] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA), which can reduce or inhibit the expression of the target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating degradation and inhibiting the translation of mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA complementary to the target mRNA sequence or a double-stranded RNA formed by two complementary strands or by a single self-complementary strand.

[0086] Interfering RNA includes "small interfering RNA" or "siRNA", each strand of the siRNA contains about 15 to about 60 nucleotides (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, 17-25, 19-25, 17-23, 17-21, 19-23 or 19-21 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present invention. In a preferred embodiment, the siRNA is chemically synthesized. The siRNA of the present invention is capable of silencing the expression of a target sequence in vitro and / or in vivo. In other embodiments, the siRNA contains at least one modified nucleotide, for example, the siRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides in the double-stranded region.

[0087] As used herein, the terms "dsRNA," "double-stranded ribonucleic acid," or "precursor RNAi molecule" are intended to include any precursor molecule that is processed by an endonuclease in vivo to generate an active siRNA.

[0088] As used herein, the "dsRNA" in the present invention refers to a double helix structure formed by two antiparallel and base-complementary nucleic acid chains, which form two directions of nucleic acid chains, namely "sense chain" and "antisense chain", relative to the target gene mRNA, depending on whether they are complementary.

[0089] The term "siRNA" refers to a molecule that contains an siRNA as that term is defined herein and that mediates targeted cleavage of an RNA transcript through an RNA-induced silencing complex (RISC) pathway. The iRNA directs sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). The iRNA modulates (e.g., inhibits) expression of ANGPTL3 in a cell (e.g., a cell in a subject such as a mammalian subject).

[0090] Typically, most of the nucleotides of each strand of the siRNA are ribonucleotides, but as described in detail herein, each or both of the two strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. In addition, as used in this description, "siRNA" may include ribonucleotides with chemical modifications; siRNA may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Therefore, the term modified nucleotide encompasses substitution, addition, or removal of, for example, a functional group or atom of an internucleoside linkage, a sugar moiety, or a nucleobase. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art.

[0091] As used herein, "G", "C", "A" and "U" are nucleotides containing guanine, cytosine, adenine and uracil as bases, respectively. "Ribonucleotide" or "nucleotide" may also indicate a modified nucleotide. "dT" is 2'-deoxythymine.

[0092] As used herein, mG is 2'-O-methylguanylate, mA is 2'-O-methyladenylate, mU is 2'-O-methyluridylate, and mC is 2'-O-methylcytidine; 2FG is 2'-fluoroguanylate, 2FA is 2'-fluoroadenylate, 2FU is 2'-fluorouridylate, and 2FC is 2'-fluorocytidine; the letter S indicates that the two nucleotides adjacent to the letter S are connected by a thiophosphate group; dA is 2'-deoxyadenylate, dC is 2'-deoxycytidine, dG is 2'-deoxyguanylate, dT is 2'-deoxythymidylate, and GalNac is L96.

[0093] The siRNA of the present invention includes a single-stranded RNA that interacts with the ANGPTL3 mRNA sequence, thereby guiding the cleavage of the mRNA. After the double-stranded siRNA enters the cell and binds to AGO2, the double helix is ​​opened and then enters RISC, further guiding the antisense strand to recognize and complementarily pair with ANGPTL3 mRNA, and finally the nuclease such as AGO2 inside RISC cleaves ANGPTL3 mRNA, thereby reducing the level of ANGPTL3 mRNA.

[0094] As used herein, the term "nucleotide overhang" refers to unpaired nucleotides that protrude from the double helix structure of siRNA. For example, when the 3' end of the antisense strand of siRNA extends beyond the 5' end of the sense strand, the nucleotides in the excess portion are referred to as nucleotide overhangs.

[0095] As used herein, the term "antisense strand" or "guide strand" refers to a nucleotide sequence in a double-stranded siRNA that is complementary to the target sequence (ANGPTL3 mRNA). The nucleotide sequence with this complementary feature is defined as the antisense strand. The antisense strand may not completely match the target sequence mRNA, and mismatches may exist in the terminal region.

[0096] As used herein, the term "complementarity" refers to the ability of a nucleotide sequence to hybridize with another nucleotide sequence under certain conditions and form a double-stranded structure. The bases of the two chains of siRNA are completely paired throughout the entire length, and this feature is called "complete complementarity". If there are 1-3 base mispairings, it can be called "substantially complementary". The terms "complementarity", "completely complementary" and "substantially complementary" can be used inside double-stranded siRNA; they can also be used for base pairing between the antisense strand of siRNA and the target gene mRNA.

[0097] As used herein, the term "inhibit" is used interchangeably with "reduce", "silence", "downregulate" and similar descriptive language to indicate any level of inhibition. "Inhibiting the expression of ANGPTL3" includes inhibiting the expression of any mRNA of ANGPTL3 gene and its variants, and ANGPTL3 and its variant proteins.

[0098] As used herein, "delivering siRNA into cells" means that siRNA can be delivered into target cells by incubating liposome-encapsulated siRNA with cells by lipofection in vitro. In in vivo experiments, siRNA needs to be coupled to the ligand GalNAc, which binds to the asialoglycoprotein receptor on the surface of liver cells, thereby specifically delivering siRNA into liver cells.

[0099] As used herein, "lipid metabolism disorder" refers to a disorder or disease or condition related to lipid metabolism such as hyperlipidemia caused by the disturbance of lipid metabolism and metabolic imbalance, which is mainly manifested as an abnormal increase in the levels of some lipid metabolites and lipoproteins in the blood. "Lipid metabolism disorder" includes (1) hereditary diseases: familial hypertriglyceridemia; (2) acquired disorders, dietary or drug-related abnormalities. "Lipid metabolism disorder" in a general sense is believed to include various types of diseases such as atherosclerosis, dyslipidemia, hypertriglyceridemia, acute pancreatitis related to hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, hyperlipidemia (including familial combined hyperlipidemia), and hypercholesterolemia. Among cardiovascular diseases, coronary artery disease (ischemic heart disease) and inflammation related to coronary artery disease are also considered to be related to "lipid metabolism disorder".

[0100] The term "conjugate" (sometimes also referred to as conjugate, conjugate, coupling, coupling, and sometimes referred to as conjugate in literature) used in the present invention corresponds to "conjugate" or "conjugates" in English. A conjugate refers to a new compound generated by covalently connecting (coupling) two or more compound molecules through a bivalent or multivalent compound molecule with a connecting function. A conjugate can also be generated by directly coupling or condensing two molecules. A common antibody-drug conjugate (ADC) is a conjugate, also known as an antibody-drug conjugate. In some embodiments, the product produced by coupling an siRNA molecule to a targeting group via a linker (or linker) is also a conjugate or siRNA conjugate. In some embodiments, the product produced by coupling siRNA to a targeting group is a siRNA conjugate.

[0101] The term "coupling" used in the present invention refers to a chemical process in which two or more compound molecules undergo a certain reaction to generate new chemical bonds and new molecules. In certain contexts, "coupling" can be used interchangeably with "connection" or can replace each other.

[0102] As used herein, "inhibiting ANGPTL3 gene expression" includes any level of ANGPTL3 gene inhibition, such as at least partial inhibition of ANGPTL3 gene expression, such as at least about 20% inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0103] ANGPTL3 mRNA levels or ANGPTL3 protein levels may be assessed based on the level of any variable associated with ANGPTL3 gene expression. ANGPTL3 gene expression may also be assessed indirectly based on the level of serum lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol) or free fatty acids. Inhibition may be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level may be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., a buffer-only control or an inert agent control) treated subject, cell, or sample.

[0104] As used herein, "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to the amount of the siRNA of the present invention, when administered to a cell, tissue or subject alone or in combination with other therapeutic agents, that is effective to prevent or improve the symptoms of one or more diseases or conditions or the development of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to cause symptomatic improvement, such as an amount to treat, cure, prevent or improve a related medical condition or to increase the rate of treatment, cure, prevention or improvement of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0105] siRNA of the present invention

[0106] Provided herein are siRNAs for inhibiting ANGPTL3 gene expression. Each siRNA comprises a sense strand and an antisense strand. The antisense strand is substantially complementary to the ANGPTL3 gene mRNA sequence, and the length of the complementary region is 19-23 nucleotides. The lengths of the sense strand and the antisense strand may each be 19-23 nucleotides. The sense strand and the antisense strand may be the same length, or they may be different lengths. In some embodiments, the lengths of the sense and antisense strands are each independently 19-23 nucleotides. In some embodiments, the length of the sense strand is 19-21 nucleotides, and the length of the antisense strand is 19-23 nucleotides. In some embodiments, the length of the sense strand is about 19 nucleotides, and the length of the antisense strand is about 20 nucleotides. In some embodiments, the length of the sense strand is about 19 nucleotides, and the length of the antisense strand is about 21 nucleotides.

[0107] After the siRNA of the present invention is delivered into cells, the siRNA inhibits the expression of the ANGPTL3 gene by at least about 50%. The expression level of the ANGPTL3 gene was determined using PCR and Western blotting techniques.

[0108] siRNA can be synthesized by an automated Oligo synthesizer. The siRNA of the present invention is prepared by the following steps: (1) preparing the sense strand and the antisense strand of the double-stranded RNA molecule respectively; (2) annealing the sense strand and the antisense strand at a molar ratio of 1:1 to assemble a double-stranded siRNA composition. The sense strand and the antisense strand of the double-stranded RNA molecule are prepared by solid phase organic synthesis. The siRNA of the present invention includes two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the sequences provided in Table A and Table B, and the antisense strand corresponding to the sense strand is a sequence selected from Table A and Table B, and the two sequences are complementary, wherein the antisense strand in the sequence is substantially complementary to the ANGPTL3 mRNA sequence.

[0109] The siRNA provided by the present invention may contain one or more mismatches relative to the target gene mRNA sequence, generally containing no more than 3 mismatches, and for a 21-nucleotide siRNA, the mismatch is generally not located in the central 2-14 nucleotide region. If the antisense strand of the siRNA contains a mismatch in the target gene mRNA sequence, it is preferred that the mismatch region is generally limited to the last 5 nucleotides from the 5' or 3' end of the complementary region. The methods described herein or methods known in the art can be used to determine whether the siRNA containing a mismatch in the target sequence is effective in inhibiting ANGPTL3 gene expression.

[0110] In a preferred embodiment, the sense strand and antisense strand of the siRNA of the present invention are selected from the sense strand and antisense strand shown in the siRNA sequence code in Table A:

[0111] Table A

[0112]

[0113]

[0114] Modified siRNA of the present invention

[0115] The present invention also provides siRNA chemically modified with RNA, thereby enhancing the stability of siRNA. The nucleic acid characterized by the present invention can be synthesized and modified by methods established in the art. Modifications include: (1) terminal modification, 5' terminal modification (phosphorylation) or 3' terminal modification (covalent binding of conjugates, etc.); morpholino modification at the 5' end of the sense chain, etc.; (2) base modification, replacing the base at a specific site, generally can be replaced with a stable base, a stable base, or a removed base (non-basic nucleotide); (3) ribose modification: introducing a methoxy group at the 2' position of ribose, or introducing fluorine after deoxygenation; (4) modification of the main chain, including 'terminal modification or the introduction of thiophosphate at the 3' end. In one embodiment, the 3' end of the sense chain of the siRNA composition is covalently bound to a GalNac ligand, thereby enhancing the distribution of the siRNA in hepatocytes and the ability to target liver tissue.

[0116] In another preferred embodiment, the modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

[0117] The GalNAc covalently bound siRNA conjugate provided by the present invention can be conventionally prepared by solid phase synthesis technology, and can also be commissioned to a commercial company with such synthesis technology for preparation.

[0118] In a preferred embodiment, the sense strand and antisense strand of the modified siRNA of the present invention are selected from the sense strand and antisense strand shown in the siRNA sequence code in Table B:

[0119] Table B

[0120]

[0121]

[0122]

[0123] Pharmaceutical composition

[0124] The present invention also includes pharmaceutical compositions and formulations. They include the siRNA, siRNA conjugates of the present invention and clinically applicable formulations to form pharmaceutical compositions. The siRNA of the present invention is prepared using a variety of buffer solutions. The buffer solution may include acetate, citrate, carbonate or phosphate. In one embodiment, the buffer solution is a phosphate buffer solution (PBS). The pharmaceutical composition of the siRNA can be used to treat diseases or disorders associated with ANGPTL3 gene expression or activity, including but not limited to lipid metabolism disorders (e.g., hypertriglyceridemia) and other diseases. The pharmaceutical composition provided by the present invention can be administered intravenously (IV) or subcutaneously, and the effect of a single dose of the pharmaceutical composition of the present invention on ANGPTL3 levels can be maintained for a long time, so the pharmaceutical composition can be administered once a day. Or it can be administered at intervals of 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks.

[0125] The pharmaceutical composition of the present invention comprises an active ingredient within a safe and effective amount and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of active ingredient / dose, more preferably, 10-200 mg of active ingredient / dose. Preferably, the "one dose" is an injection.

[0126] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, etc.), gelatin, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, olive oil, etc.), polyols (such as propylene glycol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0127] The administration method of the pharmaceutical composition described in the present invention is consistent with the application method (or administration method) described in the present invention.

[0128] The dosage form of the pharmaceutical composition of the present invention is consistent with the mode of administration, for example (but not limited to): tablets, capsules, powders, pills, granules, syrups, solutions, suspensions, emulsions, suspensions, sprays, aerosols, powder sprays, volatile liquids, injections, powder injections, external solutions, lotions, pour-ons, liniments, papules, plasters, rubber plasters, ointments, plasters, pastes, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, suppositories, etc.

[0129] Method of the present invention

[0130] The present invention also provides methods for inhibiting ANGPTL3 expression in cells using siRNA, siRNA conjugates or pharmaceutical compositions containing the same. These methods include: delivering the siRNA of the present invention into cells and maintaining the cells for a period of time, thereby effectively degrading ANGPTL3 mRNA and inhibiting ANGPTL3 gene expression in cells. The reduction of gene expression can be evaluated by any method known in the art, including qRT-PCR to determine the expression level of ANGPTL3 mRNA; using Western blotting to determine the protein level of ANGPTL3.

[0131] The present invention also provides methods for inhibiting ANGPTL3 gene expression in animals. These methods include: targeting and delivering a GalNAc-conjugated siRNA conjugate to liver cells by intravenous or subcutaneous injection, and continuing to feed the animal for a period of time, thereby effectively reducing the expression level of ANGPTL3. The reduction of gene expression can be assessed by any method known in the art, including qRT-PCR to determine the expression level of ANGPTL3 mRNA; using Western blotting to determine the protein level of ANGPTL3.

[0132] The present invention further provides methods for treating a subject in need thereof using siRNA or a pharmaceutical composition thereof. These treatment methods of the present invention include administering the siRNA, siRNA conjugate or pharmaceutical composition of the present invention to a subject suffering from a disease including but not limited to atherosclerotic cardiovascular disease. The above methods can be used in combination with other drugs and / or other treatment methods. For example, cholesterol synthesis inhibitors (statins), bile acid sequestrants; cholesterol acyltransferase inhibitors; farnesoid X receptor antagonists. Other therapeutic drugs also include those that increase high-density lipoprotein, such as cholesterol ester transfer protein inhibitors.

[0133] The main advantages of the present invention are:

[0134] 1. The siRNA of the present invention can effectively inhibit the expression of the ANGPTL3 gene, thereby effectively treating diseases or conditions caused by overexpression of ANGPTL3.

[0135] 2. The siRNA of the present invention is a type of siRNA drug with a long-acting dosing interval, providing a more convenient treatment method for the treatment of lipid metabolism disorders including preventive treatment of atherosclerotic cardiovascular diseases.

[0136] 3. The 3' end of the positive strand of the siRNA of the present invention is coupled to GalNAc, so that liver-specific ANGPTL3 silencing can be achieved without affecting intestinal ANGPTL3 expression, and in theory, the intestinal side effects of ANGPTL3 small molecule inhibitors, such as abdominal pain and diarrhea, can be avoided.

[0137] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0138] Example 1 Design and synthesis of siRNA:

[0139] Based on the analysis of ANGPTL3 gene NM_014495.4, 47 pairs of siRNAs were designed as shown in Table 1, and the sequences were sent to Beijing Qingke Biotechnology Co., Ltd. (Qingke for short), and 0.2-1 μmol of oligonucleotides were synthesized using a solid phase oligonucleotide synthesis protocol. Among them, G is guanylic acid, A is adenylic acid, U is uridylic acid, C is cytidylic acid, and dT is 2'-deoxythymine.

[0140] Table 1. Sequences of the sense and antisense strands of unmodified siRNA

[0141]

[0142]

[0143] Example 2. In vitro activity assay of unmodified siRNA

[0144] Hep3B cells (purchased from Pronocell Biotechnology Co., Ltd.) were cultured in DMEM high-glucose medium (Adamas) supplemented with 10% fetal bovine serum (FBS, ExCell) and 1% NeAA (Gibco) at 5% CO2 and 37°C. When the cells grew to almost completely cover the culture flask, they were digested with trypsin (Gibco) and resuspended. The cell density after resuspension was adjusted and the number of cells was 3×10 5cells / well were inoculated into 6-well plates and cultured until the cell confluence reached 80%-90%. The 6-well plate culture medium was replaced with 2 ml DMEM / F-12 (Adamas) supplemented with 10% FBS culture medium for standby use. siRNA, transfection reagent (Polyplus) and OptiPRO SFM (Gibco) were mixed and suspended and added to the 6-well plate for transfection and cultured overnight in a 37°C, 5% CO2 incubator. The final concentration of siRNA used for preliminary screening was 5 nM, and IC 50 During the measurement, siRNA was diluted 3-fold to 0.07 nM with a starting and ending concentration of 5 nM. In addition to the experimental group, a control group (CK) without siRNA and a positive control group (PC, JS401) were set up.

[0145] About 24 hours after transfection, total RNA was extracted using an RNA kit (Qingke). cDNA was synthesized using a reverse transcription kit (Titan), and 1ug RNA, 2ul gDNA Remover, and 5ul 5×RT

[0146] Mix and add Nuclease free ddH2O to make up to 20ul, centrifuge briefly to the bottom of the tube. Use a PCR instrument (ThermoFisher) to synthesize cDNA, react at 42℃ for 5 minutes, 95℃ for 30 seconds, and then continue to store at -20℃.

[0147] Quantitative PCR detection was performed using the SYBR green method (Qingke). The human HMBS gene was used as the internal reference gene. The upstream primer sequence of HMBS was 5'-TACTGGCACACTGCAGCCTC-3' (SEQ ID NO.163), and the downstream primer sequence was 5'-CACGATCCCGAGACTCTGCT-3' (SEQ ID NO.164); the upstream primer sequence of the human ANGPTL3 gene was 5'-CCTGAAACTCCAGAACACCCAG-3' (SEQ ID NO.165), and the downstream primer sequence was 5'-TTCCACGGTCTGGAGAAGGTCT-3' (SEQ ID NO.166). The relative mRNA level of ANGPTL3 was detected using 2-△△Ct.

[0148] Quantitative PCR reaction was performed using the instrument QuantStudio3 (ABI). The reaction conditions were: (1) pre-denaturation at 95°C for 30 seconds; (2) denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds. Step (2) lasted for 40 cycles. (3) Melting curve: 95°C for 15 seconds; 60°C for 60 seconds; 95°C for 15 seconds. The results were normalized with the control to obtain the relative mRNA expression level and knockdown efficiency.

[0149] Excel software was used for plotting during screening. If IC50 was required, Graphpad Prism was used for four-parameter fitting. The results of the initial screening of unmodified siRNA are shown in Table 2; the IC50 results of unmodified siRNA are shown in Table 3.

[0150] Table 2. In vitro activity assay of unmodified siRNA (5 nM concentration)

[0151]

[0152] Table 3. Unmodified siRNA IC50 determination

[0153] Group <![CDATA[IC 50 (nM)]]> Group <![CDATA[IC 50 (nM)]]> Group <![CDATA[IC 50 (nM)]]> L2 0.048 L12 0.064 L29 0.041 L3 0.038 L18 0.215 L33 0.034 L5 0.052 L23 0.043 L45 0.036 L10 0.255 L24 0.11 L46 0.1

[0154] Example 3 Design and preparation of modified siRNA

[0155] The experimental results obtained from Tables 2 and 3 above showed that multiple pairs of siRNA inhibited the expression of ANGPTL3 in Hep3B cells to varying degrees, among which L2, L3, L5, L12, L23, L29, L33 and L45 had relatively high inhibitory activity against ANGPTL3, with an inhibition rate of greater than 83%, which was similar to the positive reference (PK) activity.

[0156] In order to improve the stability and activity of siRNA in vivo, the sequences of L2, L3, L5, L12, L29, L33 and L45 were modified, and each sequence had multiple modifications, wherein mG is 2'-O-methylguanylic acid, mA is 2'-O-methyladenylic acid, mU is 2'-O-methyluridylic acid, and mC is 2'-O-methylcytidylic acid; 2FG is 2'-fluoroguanylic acid, 2FA is 2'-fluoroadenylic acid, 2FU is 2'-fluorouridylic acid, and 2FC is 2'-fluorocytidylic acid; the letter S indicates that the two nucleotides adjacent to the letter S are connected by a thiophosphate group; dA is 2'-deoxyadenylic acid, dC is 2'-deoxycytidylic acid, dG is 2'-deoxyguanylic acid, dT is 2'-deoxythymidylic acid, and GalNac is GalNac-L96. For specific sequence modifications, see Table 4. The sequences were sent to Beijing Qingke for oligonucleotide synthesis of 0.2-1 μmol using a solid phase oligonucleotide synthesis protocol.

[0157] Table 4. Sequences of the sense and antisense strands of modified siRNA

[0158]

[0159]

[0160]

[0161] Example 4. In vitro activity assay of modified siRNA

[0162] For the specific method, see Example 2. The results of in vitro activity detection (5 nM concentration) of the modified siRNA are shown in Table 5; the IC50 results of the modified siRNA are shown in Table 6.

[0163] Table 5. In vitro activity detection of modified siRNA (5 nM concentration)

[0164]

[0165] Table 6. Modified siRNA IC50 determination

[0166] Group <![CDATA[IC 50 (nM)]]> Group <![CDATA[IC 50 (nM)]]> Group <![CDATA[IC 50 (nM)]]> JS401 0.072 L23-01 0.097 L33-01 0.141 L3-02 0.046 L23-02 0.098 L33-04 0.171 L5-04 5.2 L29-02 0.097 L33-05 0.067 L5N-03 0.083 L29-03 0.08 L45-01 0.06

[0167] Example 5 Evaluation of the activity of modified siRNA in ANGPTL3 transgenic mice

[0168] After adaptive feeding of ANGPTL3 transgenic mice (purchased from Shanghai Model Organisms Technology Co., Ltd., 6 weeks old), the serum was collected after fasting for 4 to 6 hours two days before administration (i.e., Day-2) to detect the content of ANGPTL3 protein and blood lipid levels. The mice were randomly divided into 10 groups according to the content of ANGPTL3 in serum (see Table 7 below for details). A single (once) subcutaneous injection (sc) was administered, and the administration volume was 5 mL / kg. Weekly testing. Among them, the positive control drug was JS401.

[0169] Table 7

[0170]

[0171] The following measurements were performed:

[0172] (1) Detection of ANGPTL3 protein expression in serum: detection time: before administration (Day-2), Day 1, Day 3, Day 7, Day 14, Day 21, Day 28, Day 35, Day 42, Day 49 after administration. ELISA kit was used for detection; serum samples were prevented from repeated freezing and thawing.

[0173] The experimental data were expressed as mean ± standard deviation (Mean ± SD), and the data were analyzed using GraphPad Prism 8.3 analysis software. The LSD test was used to test for homogeneity of variance, and the Dunnett T3 test was used for heterogeneity of variance. P < 0.05 was considered statistically significant. The experimental results are shown in Table 8 and Figure 1 As shown:

[0174] Table 8. Inhibition rate of serum ANGPTL3 protein in each group of mice (n=5 or 7, Mean±SD)

[0175]

[0176] Note: The low-dose group refers to the 3mg / kg dose group, and the high-dose group refers to the 9mg / kg dose group; compared with the control group: # P<0.05, ## P<0.01, ### P<0.001; compared with G10 positive drug group: & P<0.05, && P<0.01, &&& P<0.001.

[0177] The results showed that compared with the G1 control group, the ANGPTL3 protein content in the serum of mice in the 9 mg / kg dose group (G9 group) of L45-01 was significantly reduced on Day 3-49 after administration (P < 0.001 or 0.01 or 0.05); the ANGPTL3 protein content in the serum of mice in the 3 mg / kg dose group (G2 group) of L3-02 was significantly reduced on Day 1, 3, and 42 after administration (P < 0.001 or 0.05); the ANGPTL3 protein content in the serum of mice in the 9 mg / kg dose group (G5 group) of L5N-03 was significantly reduced on Day 3 and 7 after administration (P < 0.001 or 0.05); the ANGPTL3 protein content in the serum of mice in the 3 mg / kg dose group (G6 group) of L29-03 was significantly reduced on Day 3, 7, 14, and 28 after administration (P < 0.001 or 0.01 or 0.05); the ANGPTL3 protein content in the serum of mice in the positive drug group (G10) was significantly reduced on Day 3, 7, and 14 after administration (P < 0.001 or 0.01 or 0.05). At the same time, compared with the G10 positive drug group, the ANGPTL3 protein content in the serum of mice in the 9 mg / kg dose group (G9 group) of L45-01 was significantly reduced on Day 14, 21, 28, 35, 42, and 49 after administration (P < 0.001 or 0.01 or 0.05), and its effect was better than that of the positive drug group ( Figure 1 ).

[0178] In summary, the four modified siRNAs, L3-02, L45-01, L5N-03 and L29-03, were able to effectively knock down the expression level of ANGPTL3 protein at 21 days, among which L45-01 could still maintain a knockdown rate of about 50% at 49 days.

[0179] (2) Blood lipid determination. The detection time is: before administration (Day-2), Day 1, Day 3, Day 7, Day 14, Day 21, Day 28, Day 35, Day 42, and Day 49 after administration. About 200 μL of blood is collected from the inner canthus of the mouse after fasting for 6 hours. The whole blood sample is temporarily stored in an ice box before centrifugation, centrifuged at 2-8°C and 3500rpm for 10 minutes, and divided into 3 tubes (one of which is about 70 μL for blood lipid detection (TG, TC, HDL-C, LDL-C), and the remaining serum is divided into 2 tubes for protein ANGPTL3 content detection, and stored at -70 to -86°C. The results of blood lipid detection (TG, TC, HDL-C, LDL-C) of each group of mice are shown in Tables 9-12 and Figure 2 As shown:

[0180] Table 9

[0181]

[0182] Note: The low-dose group refers to the 3mg / kg dose group, and the high-dose group refers to the 9mg / kg dose group; compared with the control group: # P<0.05, ## P<0.01, ### P<0.001.

[0183] Table 10 Inhibition rate of serum TG content in each group of mice (n=5 or 7, Mean±SD)

[0184]

[0185] Note: The low-dose group refers to the 3mg / kg dose group, and the high-dose group refers to the 9mg / kg dose group; compared with the control group: # P<0.05, ## P<0.01, ### P<0.001.

[0186] Table 11 Inhibition rate of serum HDL-C content in each group of mice (n=5 or 7, Mean±SD)

[0187]

[0188]

[0189] Note: The low-dose group refers to the 3mg / kg dose group, and the high-dose group refers to the 9mg / kg dose group; compared with the control group: # P<0.05, ## P<0.01, ### P<0.001.

[0190] Table 12 Inhibition rate of serum LDL-C content in each group of mice (n=5 or 7, Mean±SD)

[0191]

[0192] Note: The low-dose group refers to the 3mg / kg dose group, and the high-dose group refers to the 9mg / kg dose group; compared with the control group: # P<0.05, ## P<0.01, ### P<0.001.

[0193] Table 9-12 and Figure 2 The results showed that compared with the control group (i.e., SC control group), the TC, TG, and LDL-C levels in the serum of mice in the L45-01 3 mg / kg and L45-01 9 mg / kg groups were significantly reduced after administration (P < 0.05), and there were no significant differences among the other groups.

[0194] Example 6. Detection of cytotoxicity of modified siRNA

[0195] Hep3B cells were cultured at 2×10 4 cells / well were plated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator.

[0196] Transfection was performed 24 hours after plating. siRNA, transfection reagent and OptiPRO SFM were mixed and added to 96-well plates for transfection. The initial concentration of siRNA was 50nM, and 10 concentration gradients were diluted 3 times to 0.003nM. The experimental groups included L45-01 group; L3-02 group; L29-03 group; L5N-03 group; CK was the Hep3B cell control and was not transfected.

[0197] 48 hours after transfection, add 100ul GMTiterTM Luminescent Cell Viability Assay (Jiman) to each well of the 96-well plate. Place in a 37°C incubator for 30 minutes and shake well; set the wavelength of the microplate reader (Molecular Device) to 570nm and measure the OD value. The calculation formula is Cell Viability (%) = (Exp / CK) × 100%.

[0198] like Figure 3As shown, each modified siRNA (numbered L5N-03, L3-02, L29-03 and L45-01) did not cause cytotoxicity after transfection into cells.

[0199] Example 7. RNA-SEQ analysis of off-target effects

[0200] 7.1 Cell culture and transfection:

[0201] Hep3B cells were cultured at 3×10 5 cells / well were plated in a 6-well plate (Corning). The cells were cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the 6-well plate culture medium was replaced with 2 ml DMEM / F-12 (Adamas) supplemented with 10% FBS (ExCell) culture medium for standby use. The siRNA, transfection reagent (Polyplus) and OptiPROSFM (Gibco) were mixed and suspended and added to the 6-well plate for transfection. The cells were cultured in a 37°C, 5% CO2 incubator overnight. The final concentration of siRNA was 50 nM. The experimental groups included L45-01 group; L3-02 group; L29-03 group; L5N-03 group. A control group (CK) without siRNA and a positive control group (PC) were also set up. Every two wells were a replicate, and 3 biological replicates were set up for each group.

[0202] 7.2 Cracking and testing:

[0203] 24 h after transfection, cells were lysed with TRIzol lysis buffer (Invitrogen), placed in 1.5 ml EP tubes (Axygen), and stored at −80° C. The samples were transported on dry ice to Qingke Biotechnology Co., Ltd. and Kidio Biotechnology Co., Ltd. for transcriptome sequencing.

[0204] 7.3 Transcriptome sequencing process:

[0205] 1) Preparation and library inspection of cDNA library: First, the target mRNA is enriched using Oligo (dT) magnetic beads, and then the fragmentation buffer is added to break the mRNA into fragments. The first chain of cDNA is then synthesized using the broken mRNA as a template, followed by RNaseH to degrade the RNA chain and synthesize the second chain of cDNA. Next, AMPure XP beads are used to screen cDNA of about 200 bp, and the purified cDNA is A-tailed, end-repaired, and connected to sequencing adapters. PCR amplification is then performed, and the product is purified using AMPure XP beads to finally obtain the desired library. To ensure the quality of sequencing, Qubit 2.0 and Agilent 2100 are used to strictly control the quality of library construction. After ensuring that the library inspection quality is qualified, Illumina HiSeq 2500 is used for sequencing.

[0206] 2) Data quality control: In order to ensure data quality, the raw data should be filtered before information analysis to reduce the analysis interference caused by invalid data. First, the raw reads are quality controlled using fastp to filter low-quality data and obtain clean reads. The steps of read filtering are as follows:

[0207] (1) Remove reads containing adapters.

[0208] (2) Remove reads containing N ratio greater than 10%.

[0209] (3) Remove reads that are all A bases.

[0210] (4) Remove low-quality reads (the number of bases with a quality value Q≤20 accounts for more than 50% of the entire read).

[0211] 3) Sequence alignment: The transcriptome sequence is aligned with the human genome sequence as a reference using HISAT2 software. Based on the alignment results of all reads that can be located on the genome (Total Mapped reads), the distribution position of reads in the reference genome is calculated.

[0212] 7.4 qRT-PCR analysis of down-regulated genes

[0213] First, the nucleotide sequences of L29-03, L3-02, L45-01, and L5N-03 were BLASTed with the human genome on NCBI. After BLAST, genes with higher sequence matching were selected and the intersection with RNA-Seq down-regulated genes was taken as candidate genes, which were subsequently used for q-PCR verification.

[0214] The primer sequences of the relevant genes are shown in Table 13 below:

[0215] Table 13

[0216]

[0217]

[0218] 7.5 Results:

[0219] As shown in Table 14, the two companies used different analytical methods, resulting in different numbers of differentially expressed genes. As can be seen from Table 15, the comparison of the target genes obtained by the two companies with the control showed consistent results, which was the same as the qPCR detection effect.

[0220] Table 14. Statistics of differentially expressed genes in RNA-seq

[0221]

[0222] Table 15. Differential expression of ANGPTL3 in different comparison groups

[0223]

[0224] (The values ​​in the table are log2FC, FC = mRNA level of negative control CK / mRNA level of test sample, i.e., the difference fold of mRNA level)

[0225] After BLAST, we selected genes with high sequence matching and RNA-Seq down-regulated genes and took the intersection as candidate genes. Through q-PCR verification, we found that L3-02 and L45-01 did not significantly down-regulate these selected genes with high homology, and there should be no obvious off-target effect; in the CK vs L45-01 grouping, the relative expression levels of the selected differentially expressed genes (B3GNT5, CDYL, COX10, KIF2A or MAP9) in L45-01 and CK were not significantly different. ( Figure 4 and Figure 5 )

[0226] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A siRNA for inhibiting the expression of angiopoietin-like protein 3 (ANGPTL3) gene, characterized in that: The siRNA comprises a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide. wherein the antisense strand comprises at least 19 consecutive nucleotides that differ from any antisense strand sequence shown in Table A or Table B by 0, 1, 2 or 3 nucleotides, The sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides of complementarity with the antisense strand.

2. The siRNA according to claim 1, wherein The sense strand comprises at least 19 consecutive nucleotides that differ from any of the sense strand sequences shown in Table A or Table B by 0, 1, 2 or 3 nucleotides.

3. The siRNA according to claim 1, wherein The sense strand and antisense strand are selected from the sense strand and antisense strand shown in the following siRNA sequence codes:

4. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide, and the modified nucleotide is selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides and locked nucleotides.

5. The siRNA according to claim 4, characterized in that The sense strand and antisense strand are selected from the sense strand and antisense strand shown in the following siRNA sequence codes: Among them, the letters mG are 2'-O-methylguanylic acid, mA is 2'-O-methyladenylic acid, mU is 2'-O-methyluridylic acid, and mC is 2'-O-methylcytidylic acid; 2FG is 2'-fluoroguanylic acid, 2FA is 2'-fluoroadenylic acid, 2FU is 2'-fluorouridylic acid, and 2FC is 2'-fluorocytidylic acid; the letter S indicates that the two nucleotides adjacent to the letter S are connected by thiophosphate groups; dA is 2'-deoxyadenylic acid, dC is 2'-deoxycytidylic acid, dG is 2'-deoxyguanylic acid, and dT is 2'-deoxythymidylic acid.

6. A siRNA conjugate, characterized in that: The siRNA conjugate comprises the siRNA according to claim 1 and a targeting group.

7. The siRNA conjugate according to claim 6, characterized in that The targeting group contains a group derived from N-acetylgalactosamine (GalNAc) or a derivative thereof.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the siRNA according to claim 1, and / or the conjugate according to claim 6; and (b) a pharmaceutically acceptable carrier.

9. Use of the siRNA according to claim 1, the conjugate according to claim 6, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating an ANGPTL3-mediated disease or condition, for inhibiting the expression of the ANGPTL3 gene.

10. A method for inhibiting ANGPTL3 expression in cells in vitro, the method comprising the following steps: (z1) Co-culturing an effective amount of the siRNA of claim 1, the conjugate of claim 6, or the pharmaceutical composition of claim 8 with cells, wherein the method is non-diagnostic and non-therapeutic.

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