SiRNA molecule for regulating and controlling activity of PCSK9 gene

Abstract

Provided are a small interfering RNA (siRNA) for inhibiting expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in a cell, a vector and a cell comprising a nucleotide encoding the same, and a method for treating a disease or symptom associated with PCSK9 expression in a subject using the siRNA, the vector or the cell.

Description

siRNA molecules that regulate PCSK9 gene activity Technical Field

[0001] The present invention relates to the field of RNA interference. Background Art

[0002] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a serine protease that is mainly expressed in the liver, small intestine and kidney. It is a low-density lipoprotein regulatory target substance that mainly exerts its effects by regulating the expression and secretory activity of the liver's low-density lipoprotein receptor.

[0003] Cholesterol in the blood is primarily synthesized by the liver, and the breakdown of excess cholesterol also primarily occurs in the liver. Low-density lipoprotein receptors (LDLRs) exist on the liver's surface and can bind to cholesterol circulating back into the liver, breaking it down into bile acids for excretion through the intestines. PCSK9, a protease synthesized in the liver, can bind to LDLRs, promoting their entry into the lysosomes of liver cells for degradation, thereby reducing the number of LDLRs. Conversely, inhibiting the activity of the PSCK9 enzyme can increase the number of LDLRs, thereby enhancing their ability to absorb and break down cholesterol, ultimately reducing cholesterol levels.

[0004] PCSK9 can bind to the low-density lipoprotein receptor (LDLR) through both extracellular and intracellular pathways, promoting the degradation of LDLR. In the extracellular pathway, PCSK9 secreted outside the cell binds to LDLR on the cell membrane, causing LDLR to be internalized to form endosomes and ultimately enter lysosomes for degradation. In the intracellular pathway, PCSK9 secreted from the Golgi apparatus enters the cytoplasm and directly binds to LDLR, causing LDLR to enter the lysosomal degradation pathway directly within the cell.

[0005] Therefore, PCSK9 is an effective target for treating hyperlipidemia and atherosclerosis. Although hypercholesterolemia itself is asymptomatic, long-term elevated serum cholesterol can lead to atherosclerosis. Over decades, chronically elevated cholesterol contributes to the formation of atherosclerotic plaques in arteries, which can lead to progressive stenosis or even complete occlusion of the arteries involved. In addition, smaller plaques may rupture and cause clot formation and blood flow blockage, leading to, for example, myocardial infarction and / or stroke. If the formation of stenosis or occlusion is gradual, the blood supply to tissues and organs slowly decreases until organ function becomes impaired.

[0006] In recent years, inhibitors targeting the protease PCSK9 have emerged as novel therapeutic agents for these diseases. As a new therapeutic approach, siRNA (siRNA) holds enormous potential. siRNA acts on intracellular mRNA, directly silencing target genes compared to traditional small-molecule drugs, thereby more effectively preventing the onset and progression of disease. However, siRNA's poor stability, susceptibility to nuclease degradation in the body, poor tissue absorption, difficulty in cellular uptake, and the susceptibility to off-target effects limit its clinical application. Currently, there is an urgent need for siRNA that can effectively inhibit PCSK9 gene expression in cells.

[0007] Summary of the Invention

[0008] The present invention provides novel small interfering RNA (siRNA), vectors, kits and pharmaceutical compositions for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in cells, as well as methods for inhibiting or reducing PCSK9 gene expression or treating diseases or symptoms related to PCSK9 expression using the siRNA, vectors, kits or pharmaceutical compositions.

[0009] In a first aspect, the present invention provides a small interfering RNA (siRNA) for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in a cell, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 105-208. In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-104.

[0010] In some embodiments, the sense strand and the antisense strand are each independently 17-27 nucleotides in length, preferably 19-25 nucleotides, and more preferably 19-23 nucleotides in length.

[0011] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, and more preferably 19-21 nucleotide pairs.

[0012] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide, for example, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 2 nucleotides, preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides.

[0013] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 105-208, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 105-208.

[0014] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1-104, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-104.

[0015] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0016] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 195, 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149 and 150, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 195, 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149 and 150.

[0017] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 91, 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 91, 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46.

[0018] In some embodiments, (a) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 195, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 91;

[0019] (b) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 203, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 99;

[0020] (c) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 204, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 100;

[0021] (d) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 142, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 38;

[0022] (e) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 207, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 103;

[0023] (f) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 143, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 39;

[0024] (g) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 197, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 93;

[0025] (h) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 205, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 101;

[0026] (i) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 206, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 102;

[0027] (j) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 144, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 40; or

[0028] (k) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 147, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 43;

[0029] (1) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 148, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 44;

[0030] (m) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 149, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 45; or

[0031] (n) The antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 150, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 46.

[0032] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0033] In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, and terminal nucleotides, deoxyribonucleotides attached to a cholesterol derivative or a dodecanoic acid didecamide group.

[0034] In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, abasic nucleotides, and nucleotides comprising thiophosphate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprise 1-5 thiophosphate groups, preferably 2-3 thiophosphate groups.

[0035] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4. In some preferred embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6.

[0036] In some specific embodiments, (a) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm;

[0037] (b) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm;

[0038] (c) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmsUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm;

[0039] (d) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm;

[0040] (e) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0041] (f) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0042] (g) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0043] (h) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0044] (i) the sense strand comprises UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand comprises UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm;

[0045] (j) the sense strand comprises UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand comprises UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm;

[0046] ;

[0047] (k) the sense strand comprises UmsGmsCmUmUmUmUfGfUfAmAmCmUmUmGmAmAmsGmsAm, and the antisense strand comprises UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm;

[0048] (1) the sense strand comprises GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGmsAmsUm, and the antisense strand comprises AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm;

[0049] (m) the sense strand comprises CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAmsUmsAm; and the antisense strand comprises UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm; or (n) the sense strand comprises UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand comprises AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm.

[0050] In some embodiments, the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a thiophosphate group, preferably the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group.

[0051] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0052] in,

[0053] Indicates the location of attachment to a biomolecule;

[0054] Q is independently H,

[0055] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0056] L2 is a chemical bond or -CH2CH2C(O)-;

[0057] L3 is a chemical bond, -(NHCH2CH2)b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0058] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0059] Where a = 0, 1, 2 or 3;

[0060] b = 1, 2, 3, 4, or 5;

[0061] c = 1, 2, 3, 4, or 5;

[0062] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0063] L is a chemical bond, -CH2O- or -NHC(O)-;

[0064] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0065] Where e is 1, 2, 3, 4 or 5;

[0066] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0067] Where M is

[0068] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0069] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0070] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0071] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0072] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0073] In some embodiments, the conjugated ligand targets the asialoglycoprotein receptor (ASGPR).

[0074] In some preferred embodiments, the conjugated group is selected from Table 1:

[0075] Table 1. Structure of conjugated groups

[0076] In some preferred embodiments, the conjugated group is selected from Table 2:

[0077] Table 2. Structures of conjugated groups

[0078] In some embodiments, the ligand contained in the siRNA has the following structure:

[0079] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0080] In some embodiments, the ligand contained in the siRNA has the following structure:

[0081] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0082] In some embodiments, the ligand included in the siRNA of the present invention has the following structure:

[0083] in Indicates the position of attachment to the siRNA via a phosphate group or a phosphorothioate group.

[0084] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention.

[0085] In a third aspect, the present invention provides a cell comprising the siRNA or vector of the present invention.

[0086] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0087] In a fifth aspect, the present invention provides a kit comprising the siRNA, vector or cells of the present invention.

[0088] In a sixth aspect, the present invention provides a method for treating a disease or symptom associated with PCSK9 expression in a subject, the method comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition of the present invention.

[0089] In some embodiments, the disease associated with PCSK9 expression is a cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis.

[0090] In some embodiments, the disease associated with PCSK9 expression is a tumor disease. In some preferred embodiments, the tumor disease is selected from melanoma, hepatocellular carcinoma and metastatic liver cancer.

[0091] In some embodiments, the disease associated with PCSK9 expression is a T cell-mediated inflammatory immune disease. In some preferred embodiments, the T cell-mediated inflammatory immune disease is selected from psoriasis, psoriatic arthritis, eczema or atopic dermatitis, urticaria, hormone-dependent dermatitis, rheumatoid arthritis, scleroderma or diabetes, chronic liver disease and lymphoma.

[0092] In some embodiments, the method of the present invention for treating a disease or symptom associated with PCSK9 expression in a subject comprises administering the siRNA, vector, cell or pharmaceutical composition to the subject, including subcutaneous administration, topical administration or intravenous administration. In some embodiments, the subject is a human patient.

[0093] Detailed Description of the Invention

[0094] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the spirit of the present invention.

[0095] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0096] Throughout the specification and claims of the present invention, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0097] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention, and all fall within the scope of protection of the present invention. The following describes embodiments of the present invention in more detail.

[0098] definition

[0099] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0100] As used herein, the term "antisense strand" refers to the strand of an siRNA that contains a region that is fully or substantially complementary to a target sequence.

[0101] As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, within 5, 4, 3, 2 or 1 nucleotides at 5' and / or 3' ends. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0102] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours.

[0103] As used herein, "complementary" sequences, insofar as they meet the above requirements with respect to their ability to hybridize, 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 pairing or Hoogstein base pairing.

[0104] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding PCSK9). For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.

[0105] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0106] As used herein, the term "sense strand" refers to the strand of an siRNA that includes a region that is substantially complementary to a region as that term is defined herein as the antisense strand.

[0107] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0108] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0109] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA (e.g., siRNA). For example, a nucleotide overhang is present when the 3'-end of one strand of the siRNA extends beyond the 5'-end of the other strand, or vice versa. The siRNA may comprise an overhang having at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang may be present on the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the siRNA.

[0110] "Blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded siRNA, i.e., no nucleotide overhangs. A "blunt-ended" siRNA is a siRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The siRNAs of the present invention include siRNAs that have a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or siRNAs that have nucleotide overhangs at both ends.

[0111] Substantially all of the nucleotides of the iRNA of the present invention are modified. For example, substantially all of the nucleotides of the sense strand are modified nucleotides, and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, and / or substantially all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified nucleotides. For example, all of the nucleotides of the sense strand are modified nucleotides, and / or all of the nucleotides of the antisense strand are modified nucleotides, and / or all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. Wherein "substantially all of the nucleotides are modified" means that the siRNA of the present invention is mostly but not entirely modified, and may include no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0112] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinylphosphonate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, the non-natural bases comprising nucleotides and the terminal nucleotides, deoxyribonucleotides or conventional protecting group protections on a cholesterol derivative or dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the 2 ' position of the ribose group of the nucleotide is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group of the ribose group is replaced by methoxy and formed.

[0113] As used herein, "ligand moiety" refers to a chemical moiety conjugated to an siRNA that is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a tissue, an organ, or a region of the body) compared to, for example, an siRNA in the absence of such a ligand.

[0114] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," and other similar terms and includes any level of inhibition.

[0115] The phrase "inhibiting the expression of PCSK9" is intended to refer to inhibiting the expression of any PCSK9 gene, as well as variants or mutants of the PCSK9 gene. Thus, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically manipulated cell, cell group, or organism.

[0116] "Inhibiting PCSK9 gene expression" includes inhibition of any level of the PCSK9 gene, such as at least partial suppression of PCSK9 gene expression. PCSK9 gene expression can be assessed based on the level or level change of any variable associated with PCSK9 gene expression, such as PCSK9 mRNA level, PCSK9 protein level, or lipid level. This level can be assessed in individual cells or in a group of cells (including, for example, a sample derived from a subject).

[0117] Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with PCSK9 expression compared to a control level. A control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar untreated or controlled (e.g., buffer only or inert agent only) treated subject, cell, or sample.

[0118] "Hydroxy protecting group" refers to a group that can prevent the hydroxyl group from chemical reaction and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl ( Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM).

[0119] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0120] “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0121] “C 1-6 "Alkylene" refers to the removal of C 1-6In some embodiments, C 1-4 Alkylene, C 2-4 Alkylene and C 1-2 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0122] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying or expressing another nucleic acid to which it is linked.

[0123] I.siRNA

[0124] The present invention provides a small interfering RNA (siRNA) for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in cells, wherein the siRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the lengths of the sense strand and the antisense strand are each independently 15-30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 105-208.

[0125] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is fully complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may comprise 1, 2, 3, 4 or 5 non-complementary sites.

[0126] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-104.

[0127] In some embodiments, the sense strand and the antisense strand are each independently 17-27 nucleotides in length, preferably 19-25 nucleotides, and more preferably 19-23 nucleotides in length.

[0128] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, and more preferably 19-21 nucleotide pairs.

[0129] One or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least one nucleotide, for example, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least one nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang having at least two nucleotides, preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang having two nucleotides. In some embodiments, the sense strand and the antisense strand are the same length. In some embodiments, the full length of the sense strand is complementary to the full length of the antisense strand to form a double strand, i.e., it has a blunt end. In other embodiments, the sense strand and the antisense strand are the same length, a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are different in length. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least one nucleotide, more preferably an overhang of two or three nucleotides.

[0130] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 105-208, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 105-208.

[0131] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1-104, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-104.

[0132] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0133] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 195 / 203 / 204, 142 / 207, 143 / 197 / 205 / 206, 144, 147, 148, 149, and 150, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 195 / 203 / 204, 142 / 207, 143 / 197 / 205 / 206, 144, 147, 148, 149, and 150.

[0134] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 91 / 99 / 100, 38 / 103, 39 / 93 / 101 / 102, 40, 43, 44, 45 and 46, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 91 / 99 / 100, 38 / 92, 39 / 93 / 101 / 102, 40, 43, 44, 45 and 46.

[0135] In some embodiments, (a) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 195, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 91;

[0136] (b) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 203, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 99;

[0137] (c) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 204, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 100;

[0138] (d) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 142, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 38;

[0139] (e) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 207, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 103;

[0140] (f) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 143, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 39;

[0141] (g) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 197, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 93;

[0142] (h) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 205, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 101;

[0143] (i) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 206, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 102;

[0144] (j) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 144, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 40; or

[0145] (k) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 147, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 43;

[0146] (1) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 148, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 44;

[0147] (m) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 149, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 45; or

[0148] (n) The antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 150, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 46.

[0149] II. Modification of Nucleotides

[0150] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80% of the nucleotides of the antisense strand are modified nucleotides.

[0151] In some embodiments, all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0152] The modification of the nucleotides described in the present invention may be modification of the phosphate group, ribose group and / or base group of the nucleotides.

[0153] In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, and terminal nucleotides, deoxyribonucleotides attached to a cholesterol derivative or a dodecanoic acid didecamide group.

[0154] In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, deoxyribonucleotides, and nucleotides comprising thiophosphate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprise 1-5 thiophosphate groups, preferably 2-3 thiophosphate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides, and / or cytosine deoxyribonucleotides. In a more preferred embodiment, the sense strand and / or the antisense strand comprise thymine deoxyribonucleotides. In a most preferred embodiment, the sense strand comprises thymidine deoxyribonucleotides.

[0155] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification. In some preferred embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6 of the specification.

[0156] In some specific embodiments, (a) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm;

[0157] (b) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm;

[0158] (c) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmsUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm;

[0159] (d) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm;

[0160] (e) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0161] (f) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0162] (g) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0163] (h) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm;

[0164] (i) the sense strand comprises UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand comprises UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm;

[0165] (j) the sense strand comprises UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand comprises UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm;

[0166] ;

[0167] (k) the sense strand comprises UmsGmsCmUmUmUmUfGfUfAmAmCmUmUmGmAmAmsGmsAm, and the antisense strand comprises UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm;

[0168] (1) the sense strand comprises GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGmsAmsUm, and the antisense strand comprises AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm;

[0169] (m) the sense strand comprises CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAmsUmsAm; and the antisense strand comprises UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm; or (n) the sense strand comprises UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand comprises AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm.

[0170] III. Ligand

[0171] The siRNA of the present invention is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a thiophosphate group. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group.

[0172] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0173] in,

[0174] Indicates the location of attachment to a biomolecule;

[0175] Q is independently H,

[0176] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0177] L2 is a chemical bond or -CH2CH2C(O)-;

[0178] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0179] L4 is -(OCH2CH2) c-、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0180] Where a = 0, 1, 2 or 3;

[0181] b = 1, 2, 3, 4, or 5;

[0182] c = 1, 2, 3, 4, or 5;

[0183] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0184] L is a chemical bond, -CH2O- or -NHC(O)-;

[0185] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0186] Where e is 1, 2, 3, 4 or 5;

[0187] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0188] Where M is

[0189] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0190] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0191] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0192] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0193] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0194] In some embodiments, the conjugated group is represented by formula (I'):

[0195] in,

[0196] Indicates the location of attachment to a biomolecule;

[0197] Q is independently H,

[0198] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0199] L2 is a chemical bond or -CH2CH2C(O)-;

[0200] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0201] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0202] Where a = 0, 1, 2 or 3;

[0203] b = 1, 2, 3, 4, or 5;

[0204] c = 1, 2, 3, 4, or 5;

[0205] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0206] L is -CH2O- or -NHC(O)-;

[0207] L' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0208] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0209] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0210] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0211] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0212] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0213] In some specific embodiments, wherein,

[0214] Q is independently H or

[0215] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0216] L2 is -CH2CH2C(O)-;

[0217] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0218] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0219] Where a = 0, 1, 2 or 3;

[0220] b = 1, 2, 3, 4, or 5;

[0221] c = 1, 2, 3, 4, or 5;

[0222] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0223] L is -CH2O-;

[0224] L' is a chemical bond;

[0225] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0226] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0227] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0228] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0229] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0230] In some embodiments, the conjugated group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):

[0231] in,

[0232] Indicates the location of attachment to a biomolecule;

[0233] Q is

[0234] Wherein L1 is -CH2O- or -NHC(O)-;

[0235] L2 is -CH2CH2C(O)-;

[0236] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0237] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0238] Where b = 1, 2, 3, 4 or 5;

[0239] c = 1, 2, 3, 4, or 5;

[0240] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0241] L is -CH2O-;

[0242] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0243] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0244] In some specific embodiments, wherein,

[0245] Q is independently H,

[0246] Wherein L1 is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0247] L2 is -CH2CH2C(O)-;

[0248] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0249] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0250] Where a = 0, 1, 2 or 3;

[0251] b = 1, 2, 3, 4, or 5;

[0252] c = 1, 2, 3, 4, or 5;

[0253] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0254] L is -CH2O- or -NHC(O)-;

[0255] L' is a chemical bond or -C(O)NH-;

[0256] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0257] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0258] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0259] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0260] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0261] In some embodiments, the conjugated group is as shown in Formula (II'-1) or Formula (II'-2):

[0262] in,

[0263] Indicates the location of attachment to a biomolecule;

[0264] Q is independently

[0265] Wherein L1 is -CH2O- or -CH2O-CH2CH2O-;

[0266] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0267] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0268] Where b = 1, 2, 3, 4 or 5;

[0269] c = 1, 2, 3, 4, or 5;

[0270] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0271] L is -NHC(O)-;

[0272] L' is a chemical bond or -C(O)NH-;

[0273] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0274] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0275] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0276] In some specific embodiments, wherein,

[0277] Q is independently H,

[0278] Wherein L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0279] L2 is a chemical bond;

[0280] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0281] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0282] Where a = 0, 1, 2 or 3;

[0283] b = 1, 2, 3, 4, or 5;

[0284] c = 1, 2, 3, 4, or 5;

[0285] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0286] L is -CH2O- or -NHC(O)-;

[0287] L' is a chemical bond or -C(O)NH-;

[0288] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0289] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0290] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0291] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0292] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0293] In some embodiments, the conjugated group is represented by formula (II'-2):

[0294] in,

[0295] Indicates the location of attachment to a biomolecule;

[0296] Q is independently

[0297] Wherein L1 is -CH2- or -C(O)-;

[0298] L3 is -(NHCH2CH2) b -;

[0299] L4 is -(OCH2CH2) c -;

[0300] Where b = 1, 2, 3, 4 or 5;

[0301] c = 1, 2, 3, 4, or 5;

[0302] L is -CH2O- or -NHC(O)-;

[0303] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0304] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0305] In some specific embodiments, wherein:

[0306] Q is independently H,

[0307] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0308] L2 is a chemical bond or -CH2CH2C(O)-;

[0309] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0310] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0311] Where a = 0, 1, 2 or 3;

[0312] b = 1, 2, 3, 4, or 5;

[0313] c = 1, 2, 3, 4, or 5;

[0314] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0315] L is a chemical bond, -CH2O- or -NHC(O)-;

[0316] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0317] Where e is 1, 2, 3, 4 or 5;

[0318] T is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0319] Where M is

[0320] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0321] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0322] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0323] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0324] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0325] In some specific embodiments, wherein,

[0326] T is -M-, -CH2-M- or -C(O)-M-, wherein M is

[0327] In some specific embodiments, wherein,

[0328] Q is independently H or

[0329] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0330] L2 is -CH2CH2C(O)-;

[0331] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0332] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0333] Where a = 0, 1, 2 or 3;

[0334] b = 1, 2, 3, 4, or 5;

[0335] c = 1, 2, 3, 4, or 5;

[0336] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0337] L is a chemical bond or -CH2O-;

[0338] L' is a chemical bond or -O(CH2CH2O) e -;

[0339] Where e is 1, 2, 3, 4 or 5;

[0340] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0341] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0342] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0343] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0344] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0345] wherein T is as defined in the preceding embodiments.

[0346] In some embodiments, the conjugated group is represented by Formula (III'-1), Formula (III'-2), or Formula (III'-3):

[0347] in,

[0348] Q is

[0349] Wherein L1 is -CH2O- or -NHC(O)-;

[0350] L2 is -CH2CH2C(O)-;

[0351] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0352] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0353] Where b = 1, 2, 3, 4 or 5;

[0354] c = 1, 2, 3, 4, or 5;

[0355] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0356] L is a chemical bond or -CH2O-;

[0357] Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0358] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0359] wherein T is as defined in the preceding embodiments.

[0360] In some specific embodiments, wherein,

[0361] Q is independently H,

[0362] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0363] L2 is a chemical bond;

[0364] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0365] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2)d -;

[0366] Where b = 1, 2, 3, 4 or 5;

[0367] c = 1, 2, 3, 4, or 5;

[0368] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0369] L is a chemical bond or -NHC(O)-;

[0370] L' is a chemical bond;

[0371] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0372] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0373] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0374] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0375] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0376] wherein T is as defined in the preceding embodiments.

[0377] In some embodiments, the conjugated group is represented by formula (IV-1) or formula (IV-2):

[0378] in,

[0379] Q is independently

[0380] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0381] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0382] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0383] Where b = 1, 2, 3, 4 or 5;

[0384] c = 1, 2, 3, 4, or 5;

[0385] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0386] L is a chemical bond or -NHC(O)-;

[0387] L' is a chemical bond;

[0388] Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0389] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0390] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0391] wherein T is as defined in the preceding embodiments.

[0392] In some specific embodiments, wherein:

[0393] Q is independently H,

[0394] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0395] L2 is a chemical bond or -CH2CH2C(O)-;

[0396] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0397] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0398] Where a = 0, 1, 2 or 3;

[0399] b = 1, 2, 3, 4, or 5;

[0400] c = 1, 2, 3, 4, or 5;

[0401] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0402] L is a chemical bond, -CH2O- or -NHC(O)-;

[0403] L' is -O(CH2CH2O) e -;

[0404] Where e is 1, 2, 3, 4 or 5;

[0405] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0406] Where M is

[0407] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0408] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0409] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0410] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0411] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0412] In some preferred embodiments, the conjugated group is selected from the following:

[0413] In some preferred embodiments, the conjugated group is selected from the following:

[0414] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR).

[0415] In a preferred embodiment, the ligand has the following structure:

[0416] in Indicates the position of attachment to the siRNA via a phosphate group or a phosphorothioate group.

[0417] In a preferred embodiment, the ligand has the following structure:

[0418] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0419] In a preferred embodiment, the ligand has the following structure:

[0420] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0421] IV. PCSK9 gene expression inhibition

[0422] The siRNA of the present invention is capable of inhibiting PCSK9 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, 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%.

[0423] Inhibition of PCSK9 gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject) in which the PCSK9 gene is transcribed and which cell or cells have been treated (for example, by contacting the cell or cells with an siRNA of the invention, or by administering an siRNA of the invention to a subject in which these cells are or were present) such that PCSK9 gene expression is inhibited compared to a second cell or group of cells (one or more control cells) that is substantially identical to the first cell or group of cells but has not been so treated.

[0424] In preferred embodiments, inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the formula: -△△Ct The difference between the experimental group and the control group was compared by ΔΔCt, where ΔΔCt = [(Ct target gene of experimental group - Ct internal reference of experimental group) - (Ct target gene of control group - Ct internal reference of control group)].

[0425] Alternatively, inhibition of PCSK9 gene expression, such as PCSK9 protein expression, can be assessed with respect to a reduction in parameters functionally related to PCSK9 gene expression, such as lipid levels, cholesterol levels, e.g., LDLc levels. PCSK9 gene silencing can be determined in any cell that expresses PCSK9 constitutively or through genome engineering and by any assay known in the art. The liver is the primary site of PCSK9 expression. Other important expression sites include the pancreas, kidneys, and intestines.

[0426] Inhibition of expression of PCSK9 protein can be manifested by a decrease in the level of PCSK9 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject). As explained above with respect to the assessment of mRNA suppression, inhibition of protein expression levels in treated cells or cell groups can similarly be expressed as a percentage of the level of protein in control cells or cell groups.

[0427] Control cells or cell groups that can be used to assess the inhibition of PCSK9 gene expression include cells or cell groups that have not yet been contacted with the siRNA of the present invention. For example, the control cells or cell groups can be derived from individual subjects (e.g., human or animal subjects) before treating the subject with siRNA.

[0428] V. Vector

[0429] The present invention provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0430] siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (within a few hours to a few weeks) or sustained (a few weeks to a few months or longer), depending on the specific construct and target tissue or cell type used. The coding nucleotides of the siRNA targeting the PCSK9 gene can be introduced into a linear construct, a circular plasmid or a viral vector. The nucleotides encoding the siRNA targeting the PCSK gene can be integrated into the cell genome for stable expression, or stably inherited and expressed outside the chromosome. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0431] Viral vector systems containing coding sequences for siRNA targeting the PCSK9 gene include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV40 vectors; (f) polyoma viral vectors; (g) papilloma viral vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0432] VI. Cells

[0433] The present invention provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0434] VII. Pharmaceutical Compositions

[0435] The present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0436] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0437] As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates administration of siRNA or vectors or cells containing its coding sequence into the human body and / or facilitates its absorption or function. Examples include diluents and excipients such as water; fillers such as starch and sucrose; binders such as cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; and lubricants such as talc, calcium / magnesium stearate, and polyethylene glycol. Other adjuvants such as flavoring agents and sweeteners may also be added to the composition.

[0438] For example, a pharmaceutical composition comprising the siRNA, vector or cell of the present invention may comprise a pharmaceutically acceptable diluent or a sustained-release matrix, in which the siRNA or vector of the present invention is embedded.

[0439] VIII. Kit

[0440] The present invention provides a kit comprising the siRNA, vector or cell of the present invention.

[0441] The present invention also provides kits for using the siRNAs described herein and / or performing the methods described herein. Such kits include one or more siRNAs, vectors, or cells described herein, and may further include instructions for use. The instructions may include instructions for inhibiting PCSK9 expression in cells by contacting the cells with the siRNAs or vectors described herein in an amount effective to inhibit PCSK9 expression.

[0442] In the case where the siRNA or vector of the present invention is contacted with cells in vitro, the kit of the present invention may optionally further comprise a means for contacting cells with the siRNA or vector of the present invention (e.g., an injection device) or a means for measuring the inhibitory effect of PCSK9 (e.g., a device for measuring the inhibition of PCSK9 mRNA or protein). Such a device for measuring the inhibition of PCSK9 may comprise a device for obtaining a sample (e.g., a plasma sample) from a subject.

[0443] When the siRNA, vector, or cells into which the siRNA or vector has been introduced in vitro of the present invention are administered in vivo, the kit of the present invention may optionally further comprise a device for administering the siRNA, vector, or cells of the present invention to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0444] IX. Methods of treatment and pharmaceutical uses

[0445] The present invention provides a method for treating a disease or symptom associated with PCSK9 expression in a subject, comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition of the present invention.

[0446] In some embodiments, the disease associated with PCSK9 expression is a cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis.

[0447] In some embodiments, the disease associated with PCSK9 expression is a tumor disease. In some preferred embodiments, the tumor disease is selected from melanoma, hepatocellular carcinoma and metastatic liver cancer.

[0448] In some embodiments, the disease associated with PCSK9 expression is a T cell-mediated inflammatory immune disease. In some preferred embodiments, the T cell-mediated inflammatory immune disease is selected from psoriasis or psoriatic arthritis or eczema or atopic dermatitis or urticaria or hormone-dependent dermatitis or rheumatoid arthritis or scleroderma or diabetes or chronic liver disease or lymphoma.

[0449] In some embodiments, the method of the present invention for treating a disease or symptom associated with PCSK9 expression in a subject comprises administering the siRNA or pharmaceutical composition to the subject, including subcutaneous administration or intravenous administration. In some embodiments, the subject is a human patient.

[0450] The present invention also relates to the siRNA, vector, cell or pharmaceutical composition of the present invention for treating a disease or symptom associated with PCSK9 expression in a subject.

[0451] The present invention also relates to the use of the siRNA, vector, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease or symptom associated with PCSK9 expression in a subject. The medicament of the present invention can be prepared into an emulsion, microemulsion, or microparticle.

[0452] sequence

[0453] The RNA sequence provided by the present invention targets the human PCSK9 gene (or target gene, target mRNA sequence, target sequence).

[0454] Table 3. Nucleotide sequences of the sense and antisense chains targeting the PCSK9 gene used in the present invention

[0455] Table 4 shows the modified RNA sequences used in the present invention.

[0456] In this article, the meanings of the abbreviations are as follows:

[0457] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0458] d indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0459] i represents inosine ribonucleotide.

[0460] m indicates that the nucleotide adjacent to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C.

[0461] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0462] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0463] VP indicates that the adjacent nucleotide on its right side is a vinylphosphonate-modified nucleotide, which is well known in the art, and can be found in, for example, PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0464] IB represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its position / connection mode in siRNA. IB is well known in the art, see for example F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402

[0465] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position where the siRNA is linked via a phosphate group or a phosphorothioate group can be found in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0466] NAG37 represents a GalNAc delivery vector of the following structure well known in the art, wherein Indicates the position where the siRNA is linked via a phosphate group or a phosphorothioate group, see, for example, PCT Publication No. WO2018044350

[0467] GL6 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA

[0468] GL12 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA.

[0469] Table 4 Modified RNA sequences of the positive strand of the siRNA targeting the PCSK9 gene used in the present invention

[0470] Table 5 Modified RNA sequences of the antisense strand of the siRNA targeting the PCSK9 gene used in the present invention

[0471] Table 6 Paired modified sense strands and modified antisense strands of siRNA targeting PCSK9 gene used in the present invention Example

[0472] Unless otherwise specified, the sources of materials used in the examples are as follows:

[0473] Huh7 cell line was purchased from Nanjing Kebai, catalog number CBP60202;

[0474] Hep3B cell line was purchased from Nanjing Kebai, catalog number CBP60197;

[0475] PHH cells were purchased from Shanghai Xuanyi, catalog number QYLF-HPMC;

[0476] HEK293A cell line was purchased from Nanjing Kebai, catalog number CBP60436;

[0477] Balb / c mice were obtained from Zhejiang Weitonglihua, catalog number Balb / c.

[0478] Example 1 Preparation of Compound E7

[0479] 1. Preparation of Intermediates 3-4

[0480] 1.1 Preparation of Compound 2

[0481] To compound 1 (300 g, 2.01 mol) in DCM (1.80 L) was slowly added benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) at 15°C and TEA (203 g, 2.01 mol, 280 mL) was added dropwise. After the addition, the mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol=10:1) showed that reactant 1 (R f =0.32) is retained and an important new point is detected (R f =0.52). The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2), and the organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4 and concentrated in vacuo. Without purification, compound 2 (about 385 g) was obtained as a yellow oil.

[0482] 1.2 Preparation of compound 2A

[0483] At 0-15 ℃, DMAP (19.8g, 162mmol) was added to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL) at a disposable rate and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25 ℃ for 16 hours. LCMS (product: RT=0.687min) showed that the starting reactants were completely consumed. EtOAc (1.40L) was added to the mixture at 25 ℃ and stirred for 30 minutes, then the mixture was filtered and the filter cake was washed with EtOAc (300mL). The filter cake was ground with water (1.45L) for 30 minutes at 25 ℃. The mixture was filtered and the filter cake was washed with water (175mL x 3), and the filter cake was collected to obtain compound 2A (about 580g) as a white solid.

[0484] 1.3 Preparation of compound 2B

[0485] Three reactions were performed in parallel.

[0486] To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL) was added TMSOTf (137 g, 616 mmol, 111 mL) dropwise at 10-15° C. over 0.5 h. The mixture was then stirred at 25° C. for 3 h. TLC (dichloromethane:methanol=20:1) showed that compound 2A (Rf =0.54) is completely consumed and the new point (R f =0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into a solution of NaHCO3 (300 g dissolved in 3.00 L water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00 L x 3). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo. Without purification, compound 2B (approximately 507 g) was obtained as a yellow oil and used in the next step.

[0487] 1.4 Preparation of compound 3

[0488] To a mixture of compound 2B (250 g, 759 mmol) and compound 2 (151 g, 531 mmol) in DCM (1.00 L) was added TMSOTf (84.4 g, 380 mmol, 69.0 mL) dropwise at 0-10° C. and the mixture was stirred at 20° C. for 12 hours. TLC (dichloromethane:methanol=20:1) showed that compound 2 (R f =0.33) is completely consumed and a new point is formed (R f =0.03). The combined reaction was cooled to 0-5°C and then poured into NaHCO (aqueous solution, 100 g dissolved in 1 L of water) and stirred at 5-10°C for 10 minutes. The phases were separated. The aqueous phase was extracted with DCM (500 mL x 2), the combined organic phases were dried over NaSO, filtered, and the filtrate was concentrated in vacuo. Without purification, compound 3 (approximately 360 g) was obtained as a yellow oil.

[0489] 1 H NMR: (400 MHz, DMSO).

[0490] δ=7.79-7.37(m,1H),7.35-7.26(m,5H),5.21-5.20(m,1H),5.00-4.95(m,3H),4.55-4.53(m,1H),4.03-3.86(m,3H),3.61-3.59(m,1H) ,3.59-3.57(m,1H),3.48-3.40(m,6H),3.39-3.31(m,2H),3.14-3.13(m,2H),2.09(s,3H),1.99(s,3H),1.88(s,3H),1.76-1.74(m,3H).

[0491] 1.5 Preparation of Intermediate 3-4 (TFA Salt)

[0492] Three reactions were performed in parallel.

[0493] To a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) was added compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) under an argon atmosphere. The suspension was vented and vented three times with hydrogen. The mixture was stirred at H2 (50 Psi) and 30 ° C for 2 hours. LCMS (product: RT = 0.697 min) showed that compound 3 was consumed and a product peak was detected. The three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Intermediate 3-4 (TFA salt) (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA) was obtained as a yellow solid without purification.

[0494] 1 H NMR: (400 MHz, DMSO-d6)

[0495] δ=7.92(d,J=9.1Hz,4H),5.27-5.17(m,1H),5.03-4.91(m,1H),4.60-4.50(m,1H),4.09-3.97(m,4H),3.85(s ,2H),3.65-3.46(m,10H),3.04-2.92(m,2H),2.10(s,3H),2.00(s,3H),1.94-1.86(m,3H),1.82-1.71(m,4H).

[0496] 2. Preparation of Intermediate 3-3

[0497] 2.1 Preparation of compound 5

[0498] To a solution of compound 4B (10.0 g, 35.5 mmol, 1.00 eq) and compound 3-4 (46.3 g, 78.2 mmol, 2.20 eq, TFA) prepared above in DCM (1.00 L) was added DIEA (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) in one portion at 25°C. The mixture was stirred for half an hour at 25°C. HBTU (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. LCMS (product: RT = 0.681 mins) indicated the reaction was complete, and the mixture was concentrated in vacuo. At 20 ℃, 0.50N HCl (200mL x 2) was added to the mixture, and extracted with DCM (3x 500mL), the combined organic layers were washed with saturated NaHCO (3x 800mL) until pH=8, washed with brine (3x 500mL), and dried and concentrated in vacuo to purify. The residue was purified by column chromatography (SiO , DCM:MeOH=50:1-15:1). The residue was concentrated in vacuo at 40 ℃ and purified by preparative-MPLC (column: 800g Agela C18; mobile phase: [water-ACN]; 15-45% 25min; 45% 10min). Vacuum drying was used to obtain compound 5 (approximately 180g+75.0g+87.0g+40.0g+38.0g) as a yellow solid.

[0499] 417.0 g of compound 3-4 was converted into compound 5 through 9 batches.

[0500] 2.2 Preparation of intermediate 3-3

[0501] Compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added to a solution of Pd / C (3.00 g, 10% content) in THF (300 mL) under an argon atmosphere. The suspension was degassed and purged with hydrogen three times. The mixture was stirred at 20°C under H2 (20 Psi) for 16 hours. TLC (dichloromethane:methanol = 8:1, R f =0.0) indicated that the reaction was complete. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent to obtain compound 3-3 (about 33.4 g + 129 g + 75.0 g) as a white solid.

[0502] 1 H NMR: (400 MHz, DMSO)

[0503] δ=8.53(t,J=5.2Hz,1H),8.18(d,J=2.4Hz,3H),8.03(t,J=5.2Hz,1H),7.84(d d,J=3.6Hz,2H),5.22(d,J=3.2Hz,2H),4.96(dd,J=3.2Hz,2H),4.55(d,J=8.4 Hz,2H),4.02(t,J=8.8Hz,6H),3.77-3.59(m,5H),3.58-3.45(m,21H),3.40-3.20(m,4H),2.18(t ,J=7.6Hz,2H),2.17(d,J=8.0Hz,6H),2.10(s,6H),1.99(s,6H),1.90-1.80(m,8H),1.77(s,6H).

[0504] 3. Preparation of Compound E7

[0505] 3.1 Preparation of compound 3

[0506] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method for Intermediate 3-3 above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and compound 2 (0.550 g, 0.814 mmol) were added sequentially to the solution, and the nitrogen atmosphere was replaced three times. The reaction mixture was stirred at 25°C for 16 hours. LC-MS / MS detected the product's MS response, and thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to afford compound 3 (approximately 780 mg) as a white solid.

[0507] 1 H NMR (400 MHz, CD3OD)

[0508] δ=7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67( m,4H),4.36-4.44(m,2H),4.00-4.20(m,23H),3.91-3.95(m,4H),3.69-3 .77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H),2. 14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m / z=1221.6(M / 2+H) + .

[0509] 3.2 Preparation of compound 4

[0510] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature. 10% wet Pd / C (0.050 g, 0.451 mmol) was added to the solution. The hydrogen atmosphere was replaced three times, and the reaction mixture was stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). Liquid chromatography-mass spectrometry (LC-MS / MS) detected the product. Thin-layer chromatography (dichloromethane / methanol = 10 / 1, phosphomolybdic acid) revealed complete consumption of the starting material and the formation of new spots. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to yield compound 4 (approximately 840 mg) as a white solid.

[0511] 1 H NMR (400 MHz, CD3OD)

[0512] δ=5.32-5.34(m 4H),5.06-5.10(m,4H),4.63-4.65(m,4H),4.38-4.40(m,2H), 3.99-4.20(m,20H),3.90-3.97(m,4H),3.69-3.76(m,6H),3.50-3.68(m,36H),3.35-3.44(m,11H ),2.28-2.38(m,4H),2.15(s,12H),2.03(s,12H),1.90-1.94(m,24H).LCMS:m / z=1154.7(M / 2+H) + .

[0513] 3.3 Preparation of compound 6

[0514] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and compound 4 (840 mg, 0.364 mmol) were added sequentially to the solution, and the atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. LC / MS analysis revealed the disappearance of the starting material, while thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1 to 5 / 1) to afford compound 6 (approximately 620 mg) as a white solid.

[0515] 1 H NMR (400 MHz, CD3OD)

[0516] δ=7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m ,4H),4.63-4.65(m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.9 7(m,6H),3.58-3.81(m,45H),3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s ,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H),1.28-1.38(m,8H).LCMS:m / z=1460.0(M / 2+H) + .

[0517] 4. Preparation of Compound E7

[0518] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were added sequentially to the solution, and the atmosphere was replaced with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. LC / MS / MS analysis confirmed the disappearance of the starting material. The reaction solution was concentrated under reduced pressure, and the resulting crude product was separated by preparative HPLC (Preparative-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to afford compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid.

[0519] 1 H NMR (400 MHz, CD3OD)

[0520] δ=7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63- 4.67(m,4H),4.38-4.47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m ,7H),3.49-3.76(m,45H),3.33-3.47(m,10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H), 2.14(s,12H),2.05-2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)

[0521] MS: m / z = 3022.36 (M+H) + .

[0522] Example 2 Preparation of Compound E13

[0523] 1. Preparation of Intermediate 2-2

[0524] 1.1 Preparation of compound 3

[0525] Four reactions were performed in parallel.

[0526] To a solution of compound 1 (250 g, 741 mmol) in THF (1.75 L) was added 4-methylmorpholine (434 g, 4.30 mol, 472 mL), followed by cooling to 0°C. Isobutyl chloroformate (243 g, 1.78 mol, 233 mL) was added to the reaction mixture over 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the mixture was stirred for 40 minutes or longer, and compound 2 (526 g, 1.78 mol) was added to the reaction mixture over 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the ice bath was removed, and the reactants were then allowed to warm to room temperature for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, compound 1R f=0.43) shows that compound 1 is completely consumed and new spots are formed. 4 reactions were combined. The reaction solution was poured into a stirred cold (0 ℃) 0.50M HCl (aq.) (12.0L) solution and stirred for about 10 minutes. EtOAc (4.00L x 3) was subsequently added, stirred for a period of time, separated, and the organic phase was washed with brine (10.0L), dried with Na2SO4, and concentrated in vacuo to produce a thick colorless oil. Hexane (1.20L) was added to the stirred oil. White smoke appeared in the solution and then disappeared after further stirring. Seed crystals (1.20g, 0.10wt%) were added, and white crystals slowly formed. Within 20 minutes, the suspension was thick enough to hinder stirring, and additional hexane (6.00L) was added and stirred for 12 hours. The suspension was filtered, washed with hexane (1.20L) and dried to obtain compound 3 (about 1.20kg) as a white solid.

[0527] 1 H NMR: (400MHz DMSO)

[0528] δ=8.10-8.08(m,1H),7.62-7.60(m,1H),7.38-7.31(m,5H),5.11-4.94(m,2H),4.1 2-4.09(m,1H),3.92-3.87(m,1H),2.25-2.19(m,4H),1.90-1.88(m,2H),1.73-1.67 (m,2H),1.40-1.39(m,27H).

[0529] 1.2 Preparation of compound 4

[0530] Five reactions were performed in parallel.

[0531] A solution of compound 3 (240 g, 415 mmol) in HCOOH (2.40 L) was stirred at 45 ° C for 2 hours. LCMS (compound 4 = 0.570 min) showed that compound 3 was completely consumed and the main peak of the desired m / z appeared. Five reactions were combined. Diluted with toluene and ACN (1.50 L each) and concentrated. Formic acid was removed by azeotropy using 1: 1 ACN and toluene (500 mL) and ACN three times (500 mL each). Compound 4 was dried under high vacuum. The residue was then stirred with DCM (500 mL) and the organic layer was poured off, followed by azeotropic drying with ACN (400 mL) twice, vacuum drying, and then azeotroped with toluene (400 mL) 9 times to obtain compound 4 (about 800 g) as a white solid.

[0532] 1 H NMR: (400MHz DMSO)

[0533] δ=12.4-12.2(m,2H),8.12-8.10(m,1H),7.62-7.60(m,1H),7.38-7.34(m,5H),5.07-5.02(m,2H) ,4.22-4.17(m,1H),3.99-3.96(m,1H),2.31-2.21(m,4H),2.00-1.93(m,2H),1.76-1.75(m,2H).

[0534] 1.3 Preparation of compound 6

[0535] To a stirred solution of compound 4 (80.0 g, 194 mmol) and (384 g, 700 mmol, TFA) in DMF (2.00 L) were added HOBT (103 g, 760 mmol), EDCI (146 g, 760 mmol), and DIEA (113 g, 877 mmol, 153 mL) in sequence. The reaction was stirred at 20°C for 2 hours. TLC (dichloromethane / methanol = 5 / 1, compound 6R f =0.43) showed that compound 4 was completely consumed and a new spot was formed. The reaction mixture was slowly poured into a stirred cold 0.5M HCl aqueous solution (230 mL), stirred for 10 minutes, a white solid was formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L). The combined organic phase was washed with 5% NaHCO3 (aq.) (200 mL), then dried (Na2SO4), and then concentrated by pressure evaporation. The product was purified by column chromatography (SiO2, dichloromethane / methanol=5 / 1, compound 6R f =0.43) to give compound 6 (about 180 g) as a yellow solid.

[0536] 1 H NMR: (400MHz DMSO)

[0537] δ7.95-7.91(m,3H),7.82-7.80(m,4H),7.39-7.31(m,6H),5.21-5.01 (m,3H),5.00-4.96(m,5H),4.56-4.53(m,3H),4.02(s,1H),3.88(s,9 H),3.85(s,4H),3.76(s,3H),3.50-349(m,9H),3.39-3.36(m,6H),3. 19-3.15(m,6H),2.15-2.05(m,12H),1.99(s,9H),1.89-1.77(m,21H).

[0538] 1.4 Preparation of intermediate 2-2

[0539] In a dry hydrogenation bottle under argon atmosphere, Pd(OH)2 / C (4.94 g, 3.52 mmol, 10% content) was added, MeOH (350 mL) was added, and compound 6 (47.4 g, 28.5 mmol) and TFA (3.26 g, 28.5 mmol, 2.11 mL) were added in sequence, and stirred at H2 (50 psi) and 20°C for 3 hours. By TLC (dichloromethane / methanol = 5 / 1, intermediate 2-2R f =0.25) and the reaction was monitored, showing the consumption of compound 6. The reaction mixture was filtered and the filtrate was concentrated in vacuo to afford intermediate 2-2 (TFA salt) as a white solid (46.3 g, 94.6% yield, 95.7% purity, TFA).

[0540] 1 H NMR: (400MHz DMSO)

[0541] δ=8.56-8.50(m,1H),8.14-8.05(m,4H),7.99(s,1H),7.88-7.82(m,4H),5. 22-5.21(m,3H),4.99-4.96(m,3H),4.55-4.53(m,3H),4.09(s,1H),4.03(s, 9H),3.87-3.79(m,6H),3.59-3.52(m,1H),3.51-347(m,9H),3.39-3.37(m,5 H),3.17-3.16(m,6H),2.22-2.10(m,12H),1.99(s,9H),1.93-1.73(m,21H).

[0542] 2. Preparation of Compound E13

[0543] 2.1 Preparation of compound Int1

[0544] 2.1.1 Preparation of Compound 1-2

[0545] At room temperature, compound 1-1 (38.0 g, 231 mmol), TsNHBoc (75.4 g, 278 mmol), K2CO3 (6.40 g, 46.3 mmol), and TEBA (5.27 g, 23.1 mmol) were added to a three-necked flask. The reaction was allowed to proceed at 95°C for 2 hours, then cooled to 25°C. TLC (PE / EA = 2 / 1, UV 254 nm) indicated the reaction was complete. The reaction solution was diluted with water (500 mL) and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE / EA = 10 / 1 to 3 / 1) to afford compound 1-2 (40.0 g) as a colorless oil.

[0546] 1 H NMR (400 MHz, CDCl3)

[0547] δ7.72(d,J=8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.5 6(m,2H),3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)

[0548] 2.1.2 Preparation of Compounds 1-4

[0549] Compound 1-2 (79.0 g, 181 mmol), compound 1-3 (4.21 g, 30.5 mmol), and TEBA (3.47 g, 15.2 mmol) were reacted at 95° C. for 3 hours. TLC (PE / EA=2 / 1, UV 254 nm) showed that the reaction was complete. The reaction solution was diluted with water (500 mL) and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was separated and purified by column chromatography (PE / EA=10 / 1-3 / 1) to obtain compound 1-4 (about 40.0 g) as a colorless oil.

[0550] 1 H NMR (400 MHz, CDCl3)

[0551] δ=7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m,1H),3 .62-3.76(m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)

[0552] 2.1.3 Preparation of Compounds 1-5

[0553] Compound 1-4 (77.0 g, 128 mmol) and Et3N (28.6 mL, 205 mmol) were added sequentially to dichloromethane (800 mL) under an ice bath, and methanesulfonyl chloride (24.2 g, 212 mmol) was slowly added dropwise. The reaction was allowed to proceed at 25°C for 2 hours, and LCMS indicated the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to afford compound 1-5 (approximately 90.0 g) as a colorless oil.

[0554] LCMS (ESI): m / z = 700.1 (M+Na) + ;

[0555] 1 H NMR (400MHz, CDCl3) δ7.68 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 12H), 5.10-5.20 (m, 1H), 4.93-5.02 (m, 1H), 4.52-4.61 (m, 4H), 3.72-3. 88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H),2.43(s,3H),1.43(s,9H).LCMS:m / z=700.1(M+Na) + .

[0556] 2.1.4 Preparation of Compounds 1-6

[0557] Compound 1-5 (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to MeOH (900 mL) at room temperature. The reaction was allowed to proceed at 66°C for 2 hours. TLC (PE / EA = 2 / 1, UV 254 nm) indicated the formation of new spots. The organic phase was concentrated under reduced pressure, the reaction mixture was diluted with water (200 mL), and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE / EA = 30 / 1-2 / 1) to obtain compound 1-6 (approximately 64.0 g) as a white solid.

[0558] 1 H NMR (400MHz, CDCl3) δ7.62 (d, J = 8.4Hz, 2H), 7.27-7.39 (m, 12H), 4.48-4.62 ( m,4H),3.93-4.08(m,2H),3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H)

[0559] 1.1.5 Preparation of Compounds 1-7

[0560] Compound 1-6 (69.0 g, 143 mmol) and magnesium turnings (54.7 g, 2.28 mol) were added to MeOH (400 mL) at room temperature and reacted at 66°C for 1 hour. TLC (DCM / MeOH = 10 / 1, UV 254 nm) showed complete reaction of the starting materials with the formation of new spots. The reaction mixture was diluted with water (3000 mL) and saturated aqueous ammonium chloride (3000 mL), extracted with dichloromethane (1000 mL x 3), and the organic phase was washed with saturated sodium bicarbonate (300 mL x 3). The organic phase was concentrated under reduced pressure to afford compound 1-7 (approximately 32.0 g) as a colorless oil.

[0561] 1 H NMR (400MHz, CDCl3) δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m,4H)

[0562] 1.1.6 Preparation of Compound 1-8

[0563] At room temperature, compound 1-7 (8.00 g, 24.4 mmol) was added to HCl (100 mL, 12 M) and reacted at 50°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a colorless oily compound 1-8 (about 3.60 g, HCl salt).

[0564] 1 H NMR (400MHz, CDCl3) δ4.12-4.22(m,2H),3.78(d,J=4.4Hz,4H),3.17-3.30(m,4H)

[0565] 1.1.7 Preparation of Compound Int-1

[0566] Compound 1-8 (8.29 g, 24.5 mmol) was added to pyridine (50 mL, 618 mmol) at room temperature, followed by the addition of DMTrCl (4.77 g, 12.3 mmol). The reaction system was allowed to react at 25°C for 18 hours. TLC (DCM / MeOH = 10 / 1, UV 254 nm) indicated the formation of the product. The reaction solution was concentrated under reduced pressure, diluted with saturated ammonium chloride (200 mL), extracted with DCM (100 mL x 3), and the organic phase was concentrated under reduced pressure. The crude product was isolated by column chromatography (DCM / MeOH = 99 / 1-10 / 1) to afford Int-1 (approximately 2.6 g, 5.78 mmol, 23.6%) as a yellow solid.

[0567] 1 H NMR (400MHz, CDCl3) δ7.38-7.50(m,2H),7.25-7.36(m,6H),7.18-7.25(m,1H),6.87(d,J=8.8Hz,4H),4.26-4.40(m,1H ),3.94-4.05(m,1H),3.85(d,J=4.4Hz,2H)3.73-3.82(m,6H),3.34-3.41(m,1H),3.23-3.30(m,3H),3.05-3.23(m,2H)

[0568] 2.2 Preparation of compound Int6

[0569] 2.2.1 Preparation of compound 3

[0570] Compound 1 (1.00 g, 3.05 mmol) was dissolved in anhydrous N,N-dimethylformamide (10.0 mL) at 25°C. HOBt (0.54 g, 3.97 mmol), EDCI (0.76 g, 3.97 mmol), and DIEA (1.51 mL, 9.16 mmol) were added sequentially, followed by compound 2 (0.79 g, 3.66 mmol). The mixture was stirred at 25°C for 2 hours. LC / MS analysis showed complete reaction of the starting material and formation of the product. Thin-layer chromatography (dichloromethane:methanol = 10:1) also showed complete reaction of the starting material and formation of the product. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated citric acid solution (20 mL x 3), saturated sodium bicarbonate solution (20 mL x 3), and saturated brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol=10:1) to give compound 3 (about 1.00 g) as a yellow oil.

[0571] 1H NMR (400MHz, CD3OD) δ7.21-7.41(m,10H),4.47-4.60(m,4H),3.88-4.01(m,2H),3.75(dd,J=3.2,13.2 Hz,1H),3.63-3.65(m,3H),3.40-3.63(m,7H),2.27-2.34(m,4H),1.50-1.60(m,4H),1.28-1.34(m,8H)

[0572] LCMS: m / z=526.8 (M+H) + .

[0573] 2.2.2 Preparation of compound 4

[0574] Compound 3 (380 mg, 0.20 mmol) was dissolved in anhydrous methanol (10.0 mL) at 25°C, and 10% wet Pd / C (500 mg, 4.70 mmol) was added. The reaction was stirred at 50°C for 12 hours. LC / MS analysis indicated the formation of the product. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to yield compound 4 (approximately 600 mg) as a yellow oil.

[0575] 1 H NMR (400MHz, CD3OD) δ3.79-3.89(m,2H),3.73(dd,J=3.6,13.2Hz,1H),3.64-3.68(m,5H),3.57-3.62(m,2H),3.48-3 .56(m,2H),3.38-3.47(m,1H),1.58-1.62(m,5H),1.34(d,J=2.8Hz,11H),1.30-1.37(m,1H).LCMS:m / z=346.5(M+H) + .

[0576] 2.2.3 Preparation of compound 5

[0577] Compound 4 (600 mg, 1.74 mmol) was dissolved in pyridine (3.00 mL) at 25°C, and DMTrCl (883 mg, 2.61 mmol) was added. The reaction mixture was stirred at 25°C for 1 hour. LC-MS / MS indicated the formation of the product. Thin-layer chromatography (dichloromethane:methanol = 10:1) showed complete reaction of the starting materials and the formation of the product. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (20 mL x 3) and then with brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound 5 (approximately 475 mg) as a colorless liquid.

[0578] 1 H NMR(400MHz,CD3OD)δ7.41-7.43(m,2H),7.16-7.34(m,7H),6.83-6.87(m,4 H),3.88-4.04(m,2H),3.77-4.09(m,7H),3.61-3.68(m,4H),3.43-3.58(m,3 H),3.33-3.38(m,1H),3.14-3.20(m,1H),3.05(dd,J=8.4,13.2Hz,1H),2.2 0-2.42(m,4H),1.51-1.60(m,4H),1.25-1.39(m,8H).LCMS:m / z=648.3(M+H) + .

[0579] 2.2.4 Preparation of Compound Int-6

[0580] Compound 5 (475 mg, 0.73 mmol) was dissolved in tetrahydrofuran (8.00 mL) and H₂O (2.00 mL) at 25°C, and lithium hydroxide monohydrate (36.9 mg, 0.89 mmol) was added. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) indicated complete reaction of the starting materials and formation of the product. The reaction mixture was concentrated under reduced pressure and lyophilized directly to yield compound 6 (approximately 430 mg) as a white solid.

[0581] 2.3 Preparation of compound E13

[0582] 2.3.1 Preparation of compound 7

[0583] Compound Int-6 (100 mg, 0.156 mmol) was dissolved in anhydrous N,N-dimethylformamide (3.00 mL) at 25°C. HBTU reagent (89.0 mg, 0.234 mmol), DIEA (0.078 mL, 0.47 mmol), and Intermediate C (254 mg, 0.156 mmol, synthesized according to the method for Intermediate 2-2 in Example 2) were added sequentially. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) showed the formation of a new spot. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (20 mL x 3) and three times with brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain Compound 7 (approximately 140 mg) as a yellow oil.

[0584] 1 H NMR (400MHz, CD3OD) δ7.43(d,J=8.4Hz,2H),7.18-7.34(m,7H),6.81-6.91(m,4H),5.34(d,J=2.8Hz,3H),5.09-5 .11(m,3H),4.62-4.66(m,3H),4.27-4.35(m,2H),4.07-4.17(m,9H),4.00-4.07(m,4H),3.89-3.98(m,5H),3.79 (d,J=2.4Hz,6H),3.69-3.75(m,5H),3.60-3.64(m,9H),3.52-3.56(m,10H),3.43-3.50(m,4H),3.39-3.40(m,1H ),2.25-2.40(m,8H),2.13-2.15(m,9H),2.02(s,9H),1.94-1.96(m,18H),1.48-1.66(m,4H),1.21-1.36(m,11H)

[0585] 2.3.2 Preparation of E13

[0586] Compound 7 (400 mg, 0.187 mmol) was dissolved in anhydrous dichloromethane (5.00 mL). DIEA (0.18 mL, 1.12 mmol) and DMAP (5.71 mg, 0.047 mmol) were added sequentially, followed by compound 8 (112 mg, 1.12 mmol). The mixture was stirred at 25°C for 3 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) revealed the formation of a new spot. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated brine (15.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product. The crude product was separated by HPLC (column: Welch Xtimate C18 150*25mm*5um; mobile phase: water-ACN; gradient: 27%-57% / 11min; flow rate: 25ml / min) to give white solid compound E13 (74.8mg, 0.033mmol, yield 17.9%).

[0587] LCMS (ESI): m / z = 1149.5 (M / 2+H) + ; 1 H NMR (400MHz, CD3OD) δ7.42-7.44(m,2H),7.18-7.34(m,7H),6.83-6.90(m,4H),5.33-5.34(m,3H),5.08-5.11(m,3H),4.61-4.68(m,3H),4.27-4 .36(m,2H),4.19-4.27(m,1H),4.10-4.19(m,7H),4.08(s,2H),4.00-4. 07(m,4H),3.90-3.94(m,4H),3.83-3.89(m,1H),3.79(d,J=2.4Hz,6H), 3.70-3.72(m,4H),3.53-3.54(m,15H),3.44-3.48(m,3H),3.35-3.40(m,4H),3.10-3.23(m,2H),2.53-2.63(m,4H),2.22- 2.40(m,8H),2.11-2.16(m,9H),2.05-2.10(m,2H),2.02(s,9H),1.90-1.97(m,19H),1.48-1.65(m,4H),1.29-1.32(m,8H)

[0588] Example 3 Preparation of siRNA

[0589] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

[0590] 1. Preparation of siRNA without Ligand

[0591] 1.1 Synthesis of the positive sense chain (SS chain)

[0592] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle, and nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each linking step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for oligonucleotide synthesis at a 5 μmol scale are as follows:

[0593] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25 degrees Celsius. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0594] 1.2 Synthesis of antisense strand (AS strand)

[0595] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0596] 1.3 Oligonucleotide purification and annealing

[0597] 1.3.1 Ammonolysis

[0598] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35 degrees (or 55 degrees) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, each time 1 mL, and centrifuge the filtrate to purify the crude product.

[0599] 1.3.2 Purification

[0600] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0601] 1.3.3 Annealing

[0602] According to Table 6, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio of SS strand / AS strand = 1 / 1.05. The mixture was heated in a water bath to 70-95°C for 3-5 minutes, cooled naturally to room temperature, and lyophilized to obtain the product. Double-stranded proteins DR002254 to DR002343 were obtained.

[0603] 2. Preparation of siRNA with Sense Strand Linked to Ligand

[0604] 2.1 Ligand-CPG Carrier Connection

[0605] 2.1.2 Ligation of compound E7 to CPG carrier

[0606] Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and added to acetonitrile (5 mL) and shaken to dissolve. DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were then added and shaken to dissolve until the solution became clear. ) was added to the reaction solution, and the reaction was shaken overnight at 20°C. Samples were taken and monitored for TLC, which indicated the reaction was complete. The developing solvent was DCM / methanol = 4:1, and the color was developed with phosphomolybdic acid. Filtered using a fritted funnel, the filter cake was washed with anhydrous acetonitrile (20 mL x 5), and the filter cake was filtered under reduced pressure using an oil pump for 6 h to obtain 530 mg of an off-white solid.

[0607] Place 530 mg of the condensed product in a 50 mL round-bottom flask. Add Cap C (DMAP / acetonitrile), Cap B (N-methylimidazole / pyridine / acetonitrile), and Cap A (acetic anhydride / acetonitrile) in that order. Shake overnight at room temperature. Filter, wash the filter cake with acetonitrile (20 mL x 4). Remove the filter cake and filter under reduced pressure with an oil pump for 8 hours to obtain 200 mg of an off-white solid, which was used for solid-phase synthesis.

[0608] 2.1.2 Ligation of compound E13 to CPG carrier

[0609] Compound E13 was linked to the CPG carrier in a manner similar to that of compound E7.

[0610] 2.2 Synthesis of the positive sense chain (SS chain)

[0611] Using the solid-phase phosphoramidite synthesis method, using the GL6 solid support prepared above as the starting cycle, nucleoside monomers were attached one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each ligation step involved deprotection, coupling, capping, and oxidation or thiolation. The conditions for oligonucleotide synthesis at a 5 μmol scale were as follows:

[0612] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25 degrees Celsius. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0613] 2.3 Synthesis of antisense strand (AS strand)

[0614] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0615] 2.4 Oligonucleotide Purification and Annealing

[0616] 2.4.1 Ammonolysis

[0617] Add the synthesized solid phase carrier (sense chain or antisense chain) to a 5 mL centrifuge tube, add 3% diethylamine / ammonia water (v / v), react in a constant temperature water bath at 35 degrees (or 55 degrees) for 16 hours (or 8 hours), filter, wash the solid phase carrier three times with ethanol / water, each time 1 mL, and centrifuge the filtrate to purify the crude product.

[0618] 2.4.2 Purification

[0619] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification. The product can be collected and pipetted, and desalination can be performed using a gel-filled purification column, and the elution system can be pure water.

[0620] 2.4.3 Annealing

[0621] According to Table 6, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95 degrees, maintained for 3-5 minutes, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0622] siRNA conjugated with L96 was obtained in a similar manner.

[0623] Finally, the following siRNAs were obtained: DR002221, DR002358 to DR002364, DR005642, DR005656, DR005672, DR005674, DR005675, and DR005676.

[0624] Example 4 Cell line activity screening

[0625] 1. HepG2 cell line activity screening

[0626] Cell transfection

[0627] On the first day, HepG2 cells (Nanjing Kebai, catalog number CBP60199) were digested and resuspended, counted, and plated in 96-well plates at 100 μL / well, with 1×10 4 cells / well, and transfection was performed 18 h later.

[0628] On the second day, dilute the siRNAs prepared in Example 3 (DR002221 or DR002254 to DR002343) with Opti-MEM. Add 2 μL of the compound stock solution to 198 μL of Opti-MEM, pipette to mix, and set aside. For each experiment, adjust the dilution accordingly based on your experimental needs.

[0629] The next day, dilute 0.9 μL of RNAiMAX (Thermo, 13778150) with 14.1 μL of Opti-MEM, gently pipette to mix, and let stand at room temperature for 5 minutes. Next, gently pipette to mix 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA (final concentration 10 nM). Let stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before RNA extraction.

[0630] RNA extraction

[0631] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the protocol of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0632] RNA reverse transcription

[0633] For preparation of denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Incubate at 65°C for 5 minutes in a conventional PCR instrument to perform the denaturation reaction. Rapidly cool the mixture on ice for 2 minutes.

[0634] Preparation of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0635] 14.5 μL of the denatured reaction solution was slowly mixed with the reverse transcription reaction solution, incubated at 42° C. for 45 min in a conventional PCR instrument for reverse transcription, incubated at 95° C. for 5 min to inactivate the enzyme, and the reverse transcription product (cDNA) was cooled at 4° C.

[0636] After reverse transcription, 30 μL of distilled water free of DNase and RNase was added to the cDNA sample in each well.

[0637] Fluorescence quantitative PCR

[0638] ReferenceTaqMan TM Fast Advanced Master Mix (ABI, 4444965) was used to perform fluorescent quantitative PCR reactions (ABI, QuantStudio3) in 20 μL of the system. The reaction program was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0639] Table 7 Primer information

[0640] Statistics

[0641] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0642] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0643] The target gene was hPCSK9, and the internal reference was hACTB.

[0644] 2. Huh7 cell line activity screening

[0645] Similar to the HepG2 cell line activity screening described above, activity screening was performed using the Huh7 (Nanjing Kebai, Cat. No. CBP60202) cell line. siRNA compound activity screening was performed at final concentrations of 10 nM and 0.1 nM.

[0646] The results of HepG2 cell line activity screening and Huh7 cell line activity screening are shown in Table 8.

[0647] Table 8 Results of high-throughput screening experiments on HepG2 and Huh7 cell lines

[0648] Example 5 Activity screening of Tg mouse primary hepatocytes (PMH) (transfection)

[0649] Cell transfection

[0650] Isolate Tg mouse liver primary cells (Tg mice were obtained from Jicui Yaokang, T053388), count, and plate in 24-well plates at 900 μL / well, 8 × 10 4 cells / well.

[0651] Transfection

[0652] Add 10 μL of diluted siRNA (DR002221 or DR002254 to DR002343) prepared in Example 3 (final concentration 10 nM) to 40 μL of Opti-MEM and mix thoroughly. Add 3 μL of RNAiMAX to 47 μL of Opti-MEM and mix thoroughly. Incubate for 5 minutes, then mix with the diluted siRNA. Let stand at room temperature for 10 minutes before adding to the corresponding wells. Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0653] Fluorescence quantitative PCR

[0654] ReferenceTaqMan TM Fast Advanced Master Mix (ABI, 4444965) was used to perform fluorescent quantitative PCR reactions (ABI, QuantStudio3) in 20 μL of the system. The reaction program was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0655] RNA extraction

[0656] Total RNA was extracted using a high-throughput nucleic acid extraction instrument-magnetic bead method, and then reverse transcribed and detected by fluorescence quantitative PCR.

[0657] Table 9 Primer information

[0658] Statistics

[0659] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0660] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0661] siRNA PMH activity screening, experimental results are shown in Table 10.

[0662] Table 10 Results of the activity screening experiment of primary hepatocytes from Tg mice (transfection)

[0663] Example 6 IC50 Activity Screening in Primary Human Hepatocytes (PHH) Using Various Concentrations of siRNA

[0664] A. Activity screening of primary human hepatocytes (PHH cells)

[0665] Cell transfection

[0666] Add 1.4 mL of rat tail collagen solution (Sigma, C3867) to 40.6 mL of distilled water free of DNase and RNase and mix thoroughly. Add 40 μL of the mixture to each well of a 96-well culture plate and coat overnight at 4°C. Remove the coating solution the next day.

[0667] The next day, before use, the coated cell plate was rinsed with DPBS and then the DPBS was aspirated. PHH cells were added to the recovery medium, recovered at 37°C, centrifuged, resuspended, and counted. PHH cells were plated into a 96-well plate at 90 μL / well, 2×10 4 cells / well; complete culture medium was replaced after 4 hours, and transfection was performed after 18 hours.

[0668] On the third day, 2 μL of 20 μM siRNA (DR002221 or DR002254 to DR002343) was added to 198 μL of Opti-MEM and mixed by pipetting. This was used as the first concentration point and the corresponding gradient dilution was performed according to the actual experimental needs.

[0669] On the third day, dilute 0.9 μL of RNAiMAX (Thermo, 13778150) in 14.1 μL of Opti-MEM. Gently pipette to mix thoroughly, and let stand at room temperature for 5 minutes. Then, mix 15 μL of the prepared RNAi-MAX mixture with 15 μL of the diluted compound and gently pipette to mix thoroughly. The final siRNA concentrations were 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM. Let stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before extracting RNA.

[0670] RNA extraction

[0671] Similar to Example 4, cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating protocol of a high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0672] RNA reverse transcription

[0673] Refer to PrimeScript for the preparation of denaturation reaction mixture TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Incubate at 65°C for 5 minutes in a conventional PCR instrument to perform the denaturation reaction. Rapidly cool the mixture on ice for 2 minutes.

[0674] For the configuration of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0675] Slowly mix 14.5 μL of the denatured reaction solution with 5.5 μL of the reverse transcription reaction solution. Incubate the reaction at 42°C for 45 minutes in a conventional PCR instrument for reverse transcription. Incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0676] After inversion, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0677] Fluorescence quantitative PCR

[0678] ReferenceTaqMan TM Fast Advanced Master Mix (ABI, 4444965) was used to perform fluorescent quantitative PCR reactions (ABI, QuantStudio3) in 20 μL of the system. The reaction program was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0679] Table 11 Primer information

[0680] Statistics

[0681] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0682] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0683] The experimental results are shown in Table 15.

[0684] Table 12 IC50 activity screening results using PHH cell line

[0685] Example 7 Detection of off-target activity of psi-CHECK2

[0686] Plasmid preparation

[0687] The corresponding antisense off-target plasmid was designed according to the siRNA sequence, and the psiCHECK2GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for use.

[0688] Cell transfection

[0689] 100 μL of HEK293A cell resuspension was plated into each well of a 96-well plate, 8 × 10 3 cells / well.

[0690] On the second day, the complete medium in the wells was first aspirated and discarded, and replaced with 80 μL / well of Opti-MEM medium, and the cells were starved for about 1.5 h.

[0691] siRNA preparation: siRNA (DR002221 or DR002254 to DR002343) was diluted 3-fold starting from a final concentration of 40 nM, for a total of 11 concentration points.

[0692] Plasmid mixture: Each well contains 0.01 μL of plasmid and 8.99 μL of Opti-MEM.

[0693] Lipo mix: dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019), and incubated at room temperature for 5 min. The LIpo mixture in each well contained 0.2 μL of Lipo and 9.8 μL of Opti-MEM.

[0694] Aliquot 22 μL of the prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of the plasmid mixture into corresponding wells, designated Well A. Mix thoroughly by pipetting and incubate at room temperature for 20 minutes before co-transfection. Finally, add 20 μL / well of the Well A mixture to each well of cells. The final volume per well is 100 μL, containing 80 μL of Opti-MEM and 20 μL of the Well A mixture. The final siRNA concentrations are 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.041 nM, 0.013 nM, 0.0045 nM, 0.0015 nM, 0.0005 nM, and 0.00016 nM. After incubation at 37°C in a 5% CO2 incubator for 4 hours, add 100 μL of DMEM medium supplemented with 20% fetal bovine serum to each well. The cells were cultured in a 37°C 5% CO2 incubator for 24 hours and then tested.

[0695] Result detection

[0696] Before starting the experiment, mix Luciferase( Luciferase Assay System, Promega, E2940) was reconstituted and equilibrated to room temperature. DMEM was added to each tube at a ratio of 1:1 to prepare substrate I, which was used immediately after preparation. Buffer was reconstituted and equilibrated to room temperature. Substrate was prepared into substrate II at a ratio of 100:1 and used immediately.

[0697] Use a vacuum pump to remove the original culture medium in the 96-well culture plate;

[0698] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min;

[0699] 120 μL of substrate I was transferred to a 96-well microplate, and the chemiluminescence value of Firefly (firefly luciferase) was read on a microplate reader (Tecan, Infinite 200);

[0700] Then, 60 μL of substrate II was added to each well, and the mixture was incubated on a shaker at room temperature for 10 min. The chemiluminescence value of Renilla luciferase was read on a microplate reader.

[0701] Data analysis and processing

[0702] Fluorescence activity was measured using a microplate reader, and the collected Renilla signal was normalized using a Firefly signal standard. The inhibitory effect of the siRNA was determined by comparing it with the untreated results (residual inhibitory activity), and the calculation process is as follows:

[0703] Normalized Ren / Fir ratio: Ratio = Renilla / Firefly.

[0704] Residual inhibition rate = (Ratio siRNA / Ratio control )*100%, take the average of the results of the two holes: control Ratio value of the control well (without siRNA) (the average value of the results of two wells) is taken.

[0705] Graphing: Graphpad Prism

[0706] Half maximal inhibitory concentration (IC50): In this experiment, Top and Bottom plots were used. The IC50 value was calculated using the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0707] The experimental results are shown in Table 16.

[0708] Table 13 psi-CHECK2 off-target activity detection experimental results

[0709] Example 8 IC50 Activity Screening Using Tg Mouse Primary Hepatocytes (PMH) (Free Uptake)

[0710] Cell transfection

[0711] Isolate primary liver cells from Tg mice (Tg mice were obtained from Jicui Yaokang, T053388).

[0712] Count and plate in 24-well plates, 900 μL / well, 8×10 4 cells / well.

[0713] Add 10 μL of diluted siRNA (DR002221, DR002358 to DR002364) to 90 μL of Opti-MEM, mix thoroughly, and add to the corresponding wells. Incubate in a 37°C, 5% CO2 incubator for 24 hours to screen for free uptake activity. The starting siRNA concentration was 20 nM, and five 5-fold dilutions were used to determine the concentrations (20 nM, 4 nM, 0.8 nM, 0.16 nM, and 0.032 nM).

[0714] Fluorescence quantitative PCR

[0715] Total RNA was extracted using a high-throughput nucleic acid extraction instrument-magnetic bead method, and then reverse transcribed and detected by fluorescence quantitative PCR.

[0716] Table 14 Primer information

[0717] Statistics

[0718] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0719] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0720] The experimental results are shown in Table 18.

[0721] Table 15 IC50 activity screening results of Tg mouse primary hepatocytes using 5 concentrations of siRNA (free uptake)

[0722] Example 9 In vivo activity screening

[0723] Experimental animals: 40 humanized PCSK9Tg mice, male, 11-12 weeks old.

[0724] The PCSK9 protein baseline levels of the animals were measured by ELISA on D-12 (ie, 12 days before administration, the same below) and D-5 before administration, and the baseline level was defined as 100%.

[0725] The test compound was administered subcutaneously on D0; 3 mg / kg, single dose, see Table 19 for details.

[0726] Table 16 Dosing details for in vivo activity screening

[0727] Whole blood was collected on D7, D14, D21, and D28 after administration, respectively. After standing at room temperature, serum was obtained by centrifugation. The level of PCSK9 protein was determined by ELISA. The results are shown in Table 20.

[0728] Table 17 Results of in vivo activity screening

Claims

1. A small interfering RNA (siRNA) for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in a cell, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 105-208.

2. The siRNA according to claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-104.

3. The siRNA according to claim 1 or 2, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, more preferably 19-21 nucleotide pairs.

4. The siRNA according to any one of claims 1 to 3, wherein the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

5. The siRNA according to any one of claims 1 to 3, wherein the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149, and 150. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149, and 150.

6. The siRNA according to any one of claims 1 to 3 and 5, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46.

7. The siRNA of claim 6, wherein: (a) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 203, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 99; (b) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 204, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 100; (c) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 142, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 38; (d) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 207, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 103; (e) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 143, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 39; (f) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 197, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 93; (g) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 205, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 101; (h) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 206, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 102; (i) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 144, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 40; (j) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 147, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 43; (k) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 148, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 44; (1) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 149, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 45; or (m) the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 150, and the sense strand comprises the nucleotide sequence shown in SEQ ID NO:

46.

8. The siRNA of any one of claims 1 to 7, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

9. The siRNA of claim 8, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, and terminal nucleotides, deoxyribonucleotides attached to a cholesterol derivative or a dodecanoic acid didecylamide group.

10. The siRNA of claim 8, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, deoxyribonucleotides, and nucleotides comprising phosphorothioate groups.

11. The siRNA according to claim 8, wherein the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification.

12. The siRNA of claim 8, wherein the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6 of the specification.

13. The siRNA of claim 8, wherein: (a) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm; (b) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmsUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm; (c) the sense strand comprises UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUmsUm, and the antisense strand comprises AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm; (d) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm; (e) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm; (f) the sense strand comprises GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmAm, and the antisense strand comprises UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm; (g) the sense strand comprises UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand comprises UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm; (h) the sense strand comprises UmsGmsCmUmUmUmUfGfUfAmAmCmUmUmGmAmAmsGmsAm, and the antisense strand comprises UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm; (i) the sense strand comprises GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGmsAmsUm, and the antisense strand comprises AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm; (j) the sense strand comprises CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAmsUmsAm, and the antisense strand comprises UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm; or (k) the sense strand comprises UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand comprises AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm.

14. The siRNA according to any one of claims 1 to 13, wherein the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a phosphorothioate group, preferably the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

15. The siRNA of claim 14, wherein the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

16. The siRNA of claim 14 or 15, wherein the ligand moiety comprises a conjugated group represented by formula (X'): in, Indicates the location of attachment to a biomolecule; Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c - or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

17. The siRNA of claim 16, wherein the conjugated group is represented by formula (I'): in, Indicates the location of attachment to a biomolecule; Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c - or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH- or -NHC(O)-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

18. The siRNA according to claim 17, wherein Q is independently H or Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; L' is a chemical bond; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The siRNA of claim 18, wherein the conjugated group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3): in, Indicates the location of attachment to a biomolecule; Q is Wherein L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

20. The siRNA of claim 17, wherein Q is independently H, Wherein L1 is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond or -C(O)NH-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. The siRNA of claim 20, wherein the conjugated group is represented by formula (II'-1) or formula (II'-2): in, Indicates the location of attachment to a biomolecule; Q is independently Wherein L1 is -CH2O- or -CH2O-CH2CH2O-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -NHC(O)-; L' is a chemical bond or -C(O)NH-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

22. The siRNA of claim 17, wherein Q is independently H, Wherein L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond or -C(O)NH-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. The siRNA of claim 22, wherein the conjugated group is represented by formula (II'-2): in, Indicates the location of attachment to a biomolecule; Q is independently Wherein L1 is -CH2- or -C(O)-; L3 is -(NHCH2CH2) b -; L4 is -(OCH2CH2) c -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; L is -CH2O- or -NHC(O)-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

24. The siRNA of claim 16, wherein: Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

25. The siRNA of claim 24, wherein T is -M-, -CH2-M- or -C(O)-M-, wherein M is 26. The siRNA according to claim 24 or 25, wherein Q is independently H or Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -CH2O-; L' is a chemical bond or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

27. The siRNA of claim 26, wherein the conjugated group is represented by Formula (III'-1), Formula (III'-2), or Formula (III'-3): in, Q is Wherein L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -CH2O-; Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

28. The siRNA according to claim 24 or 25, wherein Q is independently H, Wherein L1 is -CH2-, -CH2O- or -C(O)-; L2 is a chemical bond; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -NHC(O)-; L' is a chemical bond; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

29. The siRNA of claim 24 or 25, wherein the conjugated group is represented by formula (IV-1) or formula (IV-2): in, Q is independently Wherein L1 is -CH2-, -CH2O- or -C(O)-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -NHC(O)-; L' is a chemical bond; Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

30. The siRNA of claim 16, wherein: Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. The siRNA of claim 16, wherein the conjugated group is selected from the group consisting of:

32. The siRNA of claim 16, wherein the conjugated group is selected from the group consisting of:

33. The siRNA of any one of claims 1-32, wherein the ligand targets the asialoglycoprotein receptor (ASGPR).

34. The siRNA of claim 14 or 15, wherein the ligand has the structure: in Indicates the position of attachment to the siRNA via a phosphate group or a phosphorothioate group.

35. The siRNA of claim 14 or 15, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

36. The siRNA of claim 14 or 15, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

37. A cell comprising the siRNA of any one of claims 1-36.

38. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 36 or the cell of claim 37, and optionally a pharmaceutically acceptable carrier or excipient.

39. A kit comprising the siRNA of any one of claims 1 to 36 or the cell of claim 37.

40. A method of treating a disease or symptom associated with PCSK9 expression in a subject, the method comprising the step of administering to the subject the siRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

41. The method of claim 40, wherein the disease or symptom associated with PCSK9 expression is a cardiovascular disease, preferably, the cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, coronary heart disease, myocardial infarction, stroke and atherosclerosis.

42. The method of claim 41, wherein the disease or symptom associated with PCSK9 expression is hypercholesterolemia, preferably, the hypercholesterolemia is selected from non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia and heterozygous familial hypercholesterolemia.