siRNA molecules that regulate PCSK9 gene activity

Abstract

The present invention relates to a small interfering RNA (siRNA) for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in cells, a vector containing a nucleotide encoding the same, and a cell, and a method for treating a disease or condition associated with the expression of PCSK9 in a subject using the siRNA, the vector, or the cell.

Description

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 mainly expressed in the liver, small intestine, and kidney, and is a low-density lipoprotein regulatory substance that functions mainly by regulating the expression and secretory activity of low-density lipoprotein receptors in the liver.

[0003] Cholesterol in the blood is mainly synthesized in the liver, and the decomposition of excess cholesterol is also mainly carried out in the liver. There are low-density lipoprotein receptors (LDLR) on the surface of the liver, which can bind to cholesterol returning to the liver, decompose cholesterol into bile acids, and excrete it outside the body through the intestine. As a protease synthesized in the liver, PCSK9 can bind to LDLR and promote the penetration and degradation of LDLR into hepatocyte lysosomes, thereby reducing the number of LDLR. Conversely, inhibition of the activity of the PSCK9 enzyme can increase the number of LDLR, enhance the uptake and decomposition ability of cholesterol, and ultimately reduce the cholesterol content.

[0004] PCSK9 can bind to low-density lipoprotein receptors via extracellular and intracellular pathways and promote the degradation of LDLR. In the extracellular pathway, extracellularly secreted PCSK9 binds to LDLR on the cell membrane, causing LDLR to be endocytosed for endosome formation and ultimately entering the lysosome and being degraded. In the intracellular pathway, PCSK9 secreted from the Golgi apparatus enters the cytoplasm, directly binds to LDLR, and directly enters the lysosomal degradation pathway of LDLR intracellularly.

[0005] Therefore, PCSK9 is an effective target for the treatment of hyperlipidemia and atherosclerosis. Hypercholesterolemia is asymptomatic, but it may lead to atherosclerosis due to long-term elevation of serum cholesterol. Over decades, chronically elevated cholesterol promotes the formation of atherosclerotic plaques in arteries, which may cause progressive stenosis or even complete occlusion of arteries due to atherosclerosis. Furthermore, smaller plaques may rupture, causing thrombosis and blood flow obstruction, which may lead to, for example, myocardial infarction and / or stroke. If the formation of stenosis or occlusion is progressive, the blood supply to tissues and organs gradually decreases until the function of the organs is impaired.

[0006] In recent years, inhibitors targeting the protease PCSK9 have become novel therapeutic agents for these diseases. siRNA has great potential for huge development as a novel therapy. Compared with conventional small-molecule pharmaceuticals, siRNA can act on intracellular mRNA and directly silence target genes, thereby more effectively preventing the onset and progression of diseases fundamentally. However, due to defects such as poor stability of siRNA, easy degradation by nucleases in the body, difficulty in being absorbed by tissues, difficulty in being taken up by cells, and easy occurrence of off-target effects, its clinical application is limited. An siRNA that can effectively inhibit the expression of the PCSK9 gene in cells has been expected.

Summary of the Invention

Means for Solving the Problems

[0007] The present invention provides a novel small interfering RNA (siRNA), vector, kit, and pharmaceutical composition thereof for inhibiting the expression of proprotein convertase subtilisin 9 (PCSK9) in cells, and a novel method for inhibiting or reducing the expression of the PCSK9 gene, or treating diseases or symptoms related to PCSK9 expression using the siRNA, vector, kit, or pharmaceutical composition.

[0008] 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 cells, wherein the siRNA comprises a sense strand and an antisense strand that form a double-stranded region, and the lengths of the sense strand and the antisense strand are each independently 15 to 30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208. In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 104.

[0009] In some embodiments, the lengths of the sense strand and the antisense strand are each independently 17 to 27 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 to 23 nucleotides.

[0010] In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 17 to 21 nucleotide pairs, more preferably 19 to 21 nucleotide pairs.

[0011] In some embodiments, one or both of the sense strand and the antisense strand comprise a 3'-overhang and / or a 5'-overhang having at least 1 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 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 having 2 nucleotides.

[0012] In some embodiments, the antisense 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 the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208. Preferably, the antisense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208.

[0013] 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 represented by SEQ ID NOs: 1 to 104. Preferably, the antisense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 104.

[0014] In some embodiments, the siRNA comprises a sense strand sequence and an antisense strand sequence paired as shown in Table 3.

[0015] In some embodiments, 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, a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NO: 195, 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149 and 150, preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NO: 195, 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149 and 150.

[0016] 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, a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NO: 91, 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45 and 46, preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NO: 91, 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45 and 46.

[0017] In some embodiments, (a) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 195, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 91, (b) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 203, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 99, (c) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 204, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 100, (d) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 142, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 38, (e) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 207, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 103, (f) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 143, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 39, (g) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 197, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 93, (h) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 205, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 101, (i) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 206, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 102, (j) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 144, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 40, or (k) The antisense strand contains the nucleotide sequence represented by SEQ ID NO: 147, and the sense strand contains the nucleotide sequence represented by SEQ ID NO: 43, (l) The antisense strand contains the nucleotide sequence shown in SEQ ID NO: 148, and the sense strand contains the nucleotide sequence shown in SEQ ID NO: 44. (m) The antisense strand contains the nucleotide sequence shown in SEQ ID NO: 149, and the sense strand contains the nucleotide sequence shown in SEQ ID NO: 45, or (n) The antisense strand contains the nucleotide sequence shown in SEQ ID NO: 150, and the sense strand contains the nucleotide sequence shown in SEQ ID NO: 46.

[0018] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0019] In some specific embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorinated modified nucleotides, 2'-deoxy modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing thiophosphate esters, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural bases containing nucleotides, and cholesterol-based derivatives or terminal nucleotides conjugated to dodecanedioic acid dodecaneamide, deoxyribonucleotides.

[0020] 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'-fluorinated modified nucleotides, deprotonated nucleotides, and nucleotides containing phosphorothioate esters. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least two 2'-fluorinated modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least eight 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3'-end and / or 5'-end of the sense strand and / or the antisense strand comprise 1 to 5 phosphorothioate esters, preferably 2 to 3 phosphorothioate esters.

[0021] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any 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 of Table 6.

[0022] In some specific embodiments, (a) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm, (b) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm, (c) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmsUmsUm, and the antisense strand contains AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm. (d) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUmsUm, and the antisense strand contains AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm. (e) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (f) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (g) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (h) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (i) The sense strand contains UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand contains UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm. (j) The sense strand contains UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand contains UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm. (k) The sense strand contains UmsGmsCmUmUmUmUfGfUfAmAmCmUmUmGmAmAmsGmsAm, and the antisense strand contains UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm. (l) The sense strand contains GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGmsAmsUm, and the antisense strand contains AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm. (m) The sense strand contains CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAmsUmsAm, and the antisense strand contains UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm, or (n) The sense strand contains UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand contains AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm.

[0023] In some embodiments, the siRNA is further conjugated to a ligand moiety containing N-acetylgalactosamine by a phosphate ester or phosphorothioate ester, and preferably, the sense strand of the siRNA is conjugated to the ligand moiety by a phosphate ester or phosphorothioate ester. In some preferred embodiments, the 3'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester or phosphorothioate ester. In another preferred embodiment, the 5'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester or phosphorothioate ester.

[0024] In some embodiments, the ligand moiety comprises a linking group represented by formula (X’),

Chemical formula

Chemical formula

Chemical formula

Chem.

[0025] In some embodiments, the conjugate ligand targets asialoglycoprotein receptor (ASGPR).

[0026] In some preferred embodiments, the linking group is selected from Table 1.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0027] In some preferred embodiments, the linking group is selected from Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0028] In some embodiments, the ligand contained in the siRNA is [Chemical formula] has a structure, where [Chemical formula] represents the position where it binds to the sense strand of the siRNA via a phosphate ester or thiophosphate ester.

[0029] In some embodiments, the ligand contained in the siRNA is [Chemical formula] has a structure, where [Chemical formula] represents the position where it binds to the sense strand of the siRNA via a phosphate ester or thiophosphate ester.

[0030] In some embodiments, the ligand contained in the siRNA of the present invention is [Chemical formula] has a structure, Here, [Chemical formula] represents the position that binds to siRNA via a phosphate ester or thiophosphate ester.

[0031] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention.

[0032] In a third aspect, the present invention provides a cell comprising the siRNA or vector of the present invention.

[0033] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and any pharmaceutically acceptable carrier or excipient.

[0034] In a fifth aspect, the present invention provides a kit comprising the siRNA, vector or cell of the present invention.

[0035] In a sixth aspect, the present invention provides a method for treating a disease or condition related to PCSK9 expression in a subject, the method comprising administering to the subject the siRNA, vector, cell or pharmaceutical composition of the present invention.

[0036] In some embodiments, the disease related to PCSK9 expression is a cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from dyslipidemia, hypercholesterolemia, non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, atherosclerosis.

[0037] In some embodiments, the disease related to PCSK9 expression is an oncological disease. In some preferred embodiments, the oncological disease is selected from melanoma, hepatocellular carcinoma, and metastatic liver cancer.

[0038] In some embodiments, the disease related to 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 hepatitis, and lymphoma.

[0039] In some embodiments, the method for treating a disease or condition related to PCSK9 expression in a subject according to the present invention is to administer 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.

[0040] Description of the Invention Hereinafter, embodiments of the present invention will be described with specific examples. Those skilled in the art can easily know other advantages and effects of the present invention from the content disclosed herein. The present invention may be further applied or implemented by different specific embodiments, and various details in this specification can be variously modified and changed without departing from the technical idea of the present invention based on different viewpoints and applications.

[0041] It should be understood that the protection scope of the present invention is not limited to the following specific embodiments, and the terms used in the examples of the present invention are for explaining specific embodiments and are not intended to limit the protection scope of the present invention.

[0042] In this specification and the claims, the singular forms "one", "1", and "this" include plural forms unless the context specifically indicates otherwise.

[0043] When an embodiment represents a numerical range, unless otherwise specified, it should be understood that any of the two 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 commonly understood by those skilled in the art. In addition to the specific methods, apparatuses, and materials used in the embodiments, based on the understanding of the prior art by those skilled in the art and the description of the present invention, the present invention can also be realized using any methods, apparatuses, and materials of the prior art that are similar or equivalent to the methods, apparatuses, and materials described in the embodiments of the present invention, and they also belong to the technical scope of the present invention. The embodiments of the present invention will be described in more detail below.

[0044] Definition In this manuscript, the term "siRNA" refers to a double-stranded RNA molecule that can mediate the silencing of a target RNA complementary thereto (e.g., mRNA, e.g., the transcript of a gene encoding a protein). siRNA is usually a double-stranded molecule containing 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 the mRNA to be silenced. Such a gene is also called a target gene. Usually, the RNA to be silenced is an endogenous gene or a pathogen gene. Furthermore, RNAs other than mRNA (e.g., tRNA) and viral RNAs can also be targeted.

[0045] As used herein, the term "antisense strand" means the strand of siRNA that contains a region completely or substantially complementary to the target sequence.

[0046] As used herein, the term "complementary region" means a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. If the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal region of the molecule. Generally, the most resistant mismatches are located within 5, 4, 3, 2, or 1 nucleotide of the terminal region, e.g., the 5' and / or 3' terminus. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region". For example, in an siRNA with a 19nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0047] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, e.g., stringent conditions. For example, stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C, for 12 - 16 hours.

[0048] As used herein, a "complementary" sequence may, or may consist only of, base pairs formed from non-Watson-Crick type base pairs and / or non-natural and modified nucleotides, in that it meets the above requirements regarding the ability to hybridize. Such non-Watson-Crick type base pairs include, but are not limited to, G:U wobble base pairs or Hoogstein base pairs.

[0049] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the binding portion of the target mRNA (e.g., the mRNA encoding PCSK9). For example, if a sequence is substantially complementary to a continuous portion of the mRNA encoding PCSK9, the polynucleotide is at least partially complementary to PCSK9 mRNA.

[0050] In this manuscript, the terms "complementary", "fully complementary", and "substantially complementary" are used for base pairing between the sense and antisense strands of siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0051] As used herein, the term "sense strand" means the strand of siRNA that includes a region that is substantially complementary to the region that is the antisense strand of the term defined in this manuscript.

[0052] A "nucleoside" is a compound composed of two substances, a purine base or a pyrimidine base, and ribose or deoxyribose, and a "nucleotide" is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphoric acid. An "oligonucleotide" means a nucleic acid molecule (RNA or DNA) having a length of less than, for example, 100, 200, 300, or 400 nucleotides.

[0053] A "nucleic acid base" is the basic unit for synthesizing nucleosides, nucleotides, and nucleic acids. Its constituent elements contain nitrogen and it is also called a "nitrogenous base". In this manuscript, unless otherwise specified, the capital letters A, U, T, G, and C represent the nucleic acid base compositions of the nucleotides adenine, uracil, thymine, guanine, and cytosine, respectively.

[0054] As used herein, the term "nucleotide overhang" means at least one unpaired nucleotide that protrudes from the double-stranded structure of an siRNA (e.g., siRNA). For example, if the 3'-end of one strand of an siRNA extends beyond the 5'-end of the other strand, a nucleotide overhang is present, and vice versa. The siRNA may include an overhang having at least one nucleotide, or the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs can be located on the sense strand, the antisense strand, or any combination thereof. Further, one or more nucleotides of the overhang can be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.

[0055] By "blunt end" or "blunt terminus" is meant that there are no nucleotides at this end of the double-stranded siRNA that are not paired, i.e., there is no nucleotide overhang. A "blunt end" siRNA means an siRNA that is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule. The siRNAs of the present invention include siRNAs having a nucleotide overhang at one end (i.e., a reagent having one overhang and one blunt end) or nucleotide overhangs at both ends.

[0056] Nearly all nucleotides of the iRNA of the present invention are modified. For example, nearly all nucleotides of the sense strand are modified nucleotides, and / or nearly all nucleotides of the antisense strand are modified nucleotides, and / or nearly all nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, and / or all nucleotides of the antisense strand are modified nucleotides, and / or all nucleotides of both the sense strand and the antisense strand are modified nucleotides. Here, "nearly all nucleotides are modified" means that most but not all of the siRNAs of the present invention are modified and can contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0057] As used herein, "modified nucleotide" includes, but is not limited to, 2'-O-methyl modified nucleotide, 2'-fluorinated modified nucleotide, 2'-deoxy modified nucleotide, inosine modified ribonucleotide, deprotonated nucleotide, inverted abasic deoxyribonucleotide, nucleotide containing thiophosphate ester, vinylphosphonate modified nucleotide, locked nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, and cholesterol-based derivative or terminal nucleotide conjugated to dodecanedioic acid dodecaneamide, deoxyribonucleotide, or conventional protecting group protection, etc. For example, the 2'-fluorinated modified nucleotide refers to a nucleotide formed by substitution of the hydroxyl at the 2'-position of the ribose of the nucleotide with fluorine. The 2'-deoxy modified nucleotide refers to a nucleotide formed by substitution of the 2'-hydroxyl of the ribose with methoxy.

[0058] As used herein, "ligand moiety" means a chemical moiety attached to an siRNA that can alter the distribution, target, or lifespan of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type, compartment (e.g., a cellular or organ compartment, tissue, organ, or body region)) compared to, for example, an siRNA in the absence of such a ligand.

[0059] As used herein, the terms "inhibit" and "reduce," "silencing," "downregulation," and other similar terms are used interchangeably and include any level of inhibition.

[0060] The term "inhibiting the expression of PCSK9" is intended to inhibit the expression of any PCSK9 gene and PCSK9 gene variants or mutants. Thus, the PCSK9 gene may be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the case of genetically engineered cells, cell populations, or organisms.

[0061] "Inhibiting PCSK9 gene expression" includes any level of inhibition of the PCSK9 gene, such as at least partial inhibition of PCSK9 gene expression. Any variable level or change in level related to PCSK9 gene expression, such as PCSK9 mRNA level, PCSK9 protein level, or lipid number, can be used to evaluate PCSK9 gene expression. This level can be evaluated in individual cells or a group of cells (including samples from a subject).

[0062] Inhibition can be evaluated by comparing an absolute or relative horizontal decrease in a variable related to PCSK9 expression to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level, or a level determined from a subject, cell, or sample treated with a similar untreated or control (e.g., buffer-only control or vehicle control).

[0063] The term "hydroxyl protecting group" means a group that can protect hydroxyl from chemical reactions and can be removed under specific conditions to restore hydroxyl. It mainly includes silyl-type protecting groups, acyl-type protecting groups or ether-type protecting groups, preferably trimethylsilyl (TMS), triethylsilanol (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), acetyl chloride, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, methoxybenzoyl, 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), methoxybenzyl (PMB), allyl, trityl (Tr), 4,4-dimethoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), methoxybenzyloxymethyl (PMBM).

[0064] The term "halogenation" or "halogen" means fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

[0065] "C 1-6 alkyl halide" means the above-mentioned "C 1-6 alkyl" substituted with one or more halogens. In some embodiments, C 1-4 alkyl halide is particularly preferred, more preferably C 1-2It is an alkyl halide. Exemplary alkyl halides include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halide may be substituted with available bonding sites, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0066] “C 1-6 “alkylene” refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene and C 1-2 alkylene are preferred. Exemplary unsubstituted alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexanediyl (-CH2CH2CH2CH2CH2CH2-), etc. Examples of substituted alkylene include, for example, the alkylene substituted with one or more alkyl (methyl) groups such as 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-), etc., but are not limited thereto.

[0067] As used herein, the term "vector" means a nucleic acid molecule capable of amplifying or expressing another nucleic acid connected thereto.

[0068] I. siRNA The present invention provides a small interfering RNA (siRNA) for inhibiting the expression of proprotein convertase subtilisin / kexin type 9 (PCSK9) in cells. The siRNA includes a sense strand and an antisense strand that form a double-stranded region. The lengths of the sense strand and the antisense strand are each independently 15 to 30 nucleotides, and the antisense strand includes a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208.

[0069] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is completely complementary. In some other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may include one, two, three, four, or five non-complementary loci.

[0070] In some specific embodiments, the sense strand includes a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 104.

[0071] In some embodiments, the lengths of the sense strand and the antisense strand are each independently 17 to 27 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 to 23 nucleotides.

[0072] In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 17 to 21 nucleotide pairs, more preferably 19 to 21 nucleotide pairs.

[0073] One or both of the sense strand and the antisense strand include 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 include 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 of at least two nucleotides, preferably the antisense strand includes a 3'-overhang and / or a 5'-overhang having two nucleotides. In some embodiments, the lengths of the sense strand and the antisense strand are the same. In some embodiments, the sense strand forms a double strand complementary to the entire length of the antisense strand, that is, it has a blunt end. In another embodiment, the lengths of the sense strand and the antisense strand are the same, and a part of the sense strand is complementary to a part of the antisense strand, that is, both the sense strand and the antisense strand have a 5'-overhang. In some embodiments, the sense strand and the antisense strand have different lengths. 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.

[0074] In some embodiments, the antisense strand includes 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, a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208, preferably the antisense strand includes the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208.

[0075] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of any one of the nucleotide sequences represented by SEQ ID NOs: 1-104, and preferably the antisense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 1-104.

[0076] In some embodiments, the siRNA comprises a sense strand sequence and an antisense strand sequence of the pairing shown in Table 3.

[0077] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of any one of the nucleotide sequences represented by SEQ ID NOs: 195 / 203 / 204, 142 / 207, 143 / 197 / 205 / 206, 144, 147, 148, 149, and 150, and preferably the antisense strand comprises a nucleotide sequence represented by any one of SEQ ID NOs: 195 / 203 / 204, 142 / 207, 143 / 197 / 205 / 206, 144, 147, 148, 149, and 150.

[0078] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any of SEQ ID NO: 91 / 99 / 100, 38 / 103, 39 / 93 / 101 / 102, 40, 43, 44, 45, and 46, and preferably the antisense strand comprises a nucleotide sequence represented by any of SEQ ID NO: 91 / 99 / 100, 38 / 92, 39 / 93 / 101 / 102, 40, 43, 44, 45, and 46.

[0079] In some embodiments, (a) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 195, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 91, (b) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 203, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 99, (c) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 204, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 100, (d) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 142, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 38, (e) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 207, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 103, (f) the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 143, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 39, (g) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 197, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 93, (h) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 205, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 101, (i) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 206, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 102, (j) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 144, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 40, or (k) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 147, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 43, (l) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 148, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 44, (m) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 149, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 45, or (n) The antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 150, and the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 46.

[0080] II. Nucleotide Modifications In some embodiments, substantially all nucleotides of the sense strand and substantially all 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.

[0081] In some embodiments, all nucleotides of the sense strand and / or all nucleotides of the antisense strand are modified nucleotides.

[0082] The modification of the nucleotides of the present invention is a modification on the phosphonate, ribose group and / or basic group of the nucleotide.

[0083] In some specific embodiments, the sense strand and the antisense strand are each independently a 2'-O-methyl modified nucleotide, 2'-fluorinated modified nucleotide, 2'-deoxy-acid-modified nucleotide, inosine ribonucleotide, deprotonated nucleotide, inverted abasic deoxyribonucleotide, nucleotide containing thiophosphate ester, vinylphosphonate modified nucleotide, locked nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, and cholesterol-based derivative or terminal nucleotide conjugated to dodecanedioic acid dodecaneamide, one or more nucleotide modifications selected from the group consisting of deoxyribonucleotides.

[0084] 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'-fluorinated modified nucleotides, deoxyribonucleotides, and nucleotides containing phosphorothioate esters. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least two 2'-fluorinated modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least eight 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3'-end and / or 5'-end of the sense strand and / or the antisense strand comprise 1 to 5 phosphorothioate esters, preferably 2 to 3 phosphorothioate esters. In some preferred embodiments, the sense strand and / or antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides, and / or cytosine deoxyribonucleotides. In a more preferred embodiment, the sense strand and / or antisense strand comprise thymine deoxyribonucleotides. In the most preferred embodiment, the sense strand comprises thymine deoxyribonucleotides.

[0085] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any of Table 5 of the specification, and / or the sense strand comprises a modified nucleotide sequence shown in any 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 of Table 6 of the specification.

[0086] In some specific embodiments, (a) the sense strand comprises CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm, and the antisense strand comprises AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm, (b) The sense strand contains CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand contains AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm. (c) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmsUmsUm, and the antisense strand contains AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm. (d) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUmsUm, and the antisense strand contains AmsAfsGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm. (e) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (f) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmsUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (g) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (h) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmAm, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCmsAfsGm. (i) The sense strand contains UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand contains UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm. (j) The sense strand contains UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGmsAm, and the antisense strand contains UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm. (k) The sense strand contains UmsGmsCmUmUmUmUfGfUfAmAmCmUmUmGmAmAmsGmsAm, and the antisense strand contains UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm. (l) The sense strand contains GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGmsAmsUm, and the antisense strand contains AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm. (m) The sense strand contains CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAmsUmsAm, and the antisense strand contains UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm, or (n) The sense strand contains UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand contains AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm.

[0087] III. Ligand The siRNA of the present invention is further conjugated to a ligand moiety containing N-acetylgalactosamine by a phosphate ester or a phosphorothioate ester. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety by a phosphate ester or a phosphorothioate ester. In some preferred embodiments, the 3'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester or a phosphorothioate ester. In another preferred embodiment, the 5'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester or a phosphorothioate ester.

[0088] In some embodiments, the ligand moiety contains a linking group represented by (X'),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0089] In some embodiments, the linking group is as shown in (I’):

Chemical formula

Chemical formula

Chemical formula

[0090] In some specific embodiments, Q is independently H or [Chemical formula] and 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- with R3 being H, or R1 and R3 together form -C 1-2 alkylene- with R2 being H, where R is -OR', -CH2OR' or -CH2CH2OR', 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.

[0091] In some embodiments, the linking group is as shown by formula (I'-1), formula (I'-2) or formula (I'-3),

Chemical formula

Chemical formula

Chemical formula

[0092] In some specific embodiments, Q is independently H,

Chemical formula

[0093] In some embodiments, the linking group is as shown by formula (II’-1) or formula (II’-2),

Chemical formula

Chemical formula

[0094] In some specific embodiments Q is independently H [Chemical formula] and 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 - and 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- with R3 being H, or R1 and R3 together form -C 1-2 -alkylene- with R2 being H, where R is -OR’, -CH2OR’ or -CH2CH2OR’, and R’ is H, a hydroxyl protecting group or a solid 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.

[0095] In some embodiments, the linking group is as shown by formula (II’-2),

Chemical formula

Chemical formula

Chemical formula

[0096] In some specific embodiments, Q is independently H,

Chemical formula

Chem.

[0097] In some specific embodiments, T is -M-, -CH2-M- or -C(O)-M-, and M is

Chem.

[0098] In some specific embodiments, Q is independently H or

Chem.

[0099] In some embodiments, the linking group is as shown by formula (III’-1), formula (III’-2) or formula (III’-3),

Chemical formula

Chemical formula

[0100] In some specific embodiments, Q is independently H,

Chemical formula

[0101] In some embodiments, the linking group is as shown by formula (IV-1) or formula (IV-2),

Chemical formula

Chemical formula

[0102] In some specific embodiments, Q is independently H,

Chemical formula

Chemical formula

[0103] In some preferred embodiments, the linking group is selected from the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0104] In some preferred embodiments, the linking group is selected from the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0105] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR).

[0106] In one preferred embodiment, the ligand has the following structure,

Chemical formula

Chemical formula

[0107] In one preferred embodiment, the ligand has the following structure,

Chemical formula

Chemical formula

[0108] In one preferred embodiment, the ligand has the following structure,

Chemical formula

Chemical formula

[0109] IV. Inhibition of PCSK9 gene expression The siRNA of the present invention can inhibit 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% of PCSK9 gene expression.

[0110] Inhibition of PCSK9 gene expression can be achieved by reducing the amount of mRNA expressed by a first cell or cell population (such as a cell or these cells) in which the PCSK9 gene is transcribed and which is treated (for example, by contacting the cell or these cells with the siRNA of the present invention, or by administering the siRNA of the present invention to a subject in which these cells currently or previously existed), such that compared to a second cell or cell population (one or more control cells) that is substantially identical to the first cell or cell population but has not been treated in this way, the PCSK9 gene expression is inhibited.

[0111] In a preferred embodiment, this inhibition is evaluated by the following formula as the ratio of the mRNA level in the treated cells to the mRNA level in the control cells. In some specific embodiments, 2 -△△Ct values are calculated. △△Ct = [(Ct experimental group target gene - Ct experimental group internal standard) - (Ct control group target gene - Ct control group internal standard)].

[0112] Alternatively, inhibition of PCSK9 gene expression, such as PCSK9 protein expression, can be evaluated from the perspective of a decrease in functional parameters involved in PCSK9 gene expression (such as the number of lipids, cholesterol value, for example, LDLc level). PCSK9 gene silencing can be determined by any measurement known in the art in any cell that constitutively expresses PCSK9 or expresses it by genomic recombination. The liver is the main site of PCSK9 expression. Other important expression sites include the pancreas, kidney, and intestine.

[0113] Inhibition of PCSK9 protein expression can be achieved by a decrease in the level of PCSK9 protein expressed by a cell or cell population (e.g., the protein level expressed in a sample from a subject). As described above for the evaluation of mRNA inhibition, inhibition of the protein expression level in the treated cell or cell population can be similarly expressed as a ratio of the protein level in the control cell or cell population.

[0114] Examples of control cells or cell populations that can be used to evaluate inhibition of PCSK9 gene expression include cells or cell populations that have not yet been contacted with the siRNA of the present invention. For example, the control cells or cell populations may be derived from individual subjects (e.g., human or animal subjects) before treatment with siRNA.

[0115] V. Vectors The present invention provides vectors comprising nucleotide sequences encoding the siRNA of the present invention. The vectors of the present invention can be ligated to amplify or express the nucleotides encoding the siRNA of the present invention.

[0116] The siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be transient (from several hours to several weeks) or persistent (from several weeks to several months or more), depending on the specific construct used and the target tissue or cell type. The coding nucleotides of the siRNA targeting the PCSK9 gene can be introduced into a linear construct, circular plasmid, or 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 extrachromosomally. Generally, siRNA expression vectors are usually DNA plasmids or viral vectors.

[0117] Examples of viral vector systems containing the coding sequence of 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) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, and (j) helper virus-dependent adenoviruses or gutless adenoviruses.

[0118] VI. Cells The present invention provides a cell containing the siRNA or vector of the present invention, and the siRNA or vector of the present invention can be transcribed intracellularly.

[0119] VII. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and any pharmaceutically acceptable carrier or excipient.

[0120] As used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that, within the scope of sound medical judgment, have no excessive toxicity, irritation, allergic response, or other problems or complications, have a reasonable benefit / risk ratio, and are suitable for use in contact with the tissues of human and animal subjects.

[0121] As used herein, a pharmaceutically acceptable carrier refers to a drug carrier that contributes to the administration of a carrier or cell containing siRNA or its coding sequence to a human body and / or contributes to its absorption or action. For example, diluents, excipients such as water, fillers such as starch, sucrose, etc., binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone, wetting agents such as glycerin, disintegrants such as agar, calcium carbonate and sodium bicarbonate, absorption promoters such as quaternary ammonium compounds, surfactants such as hexadecanol, adsorbent carriers such as kaolin and soap clay, lubricants such as talc, calcium stearate / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweetening agents, etc. can be added to the composition.

[0122] For example, a pharmaceutical composition containing the siRNA, vector or cell of the present invention may contain a pharmaceutically acceptable diluent or sustained-release matrix, and the siRNA or vector of the present invention is incorporated into the sustained-release matrix.

[0123] VIII. Kit The present invention provides a kit containing the siRNA, vector or cell of the present invention.

[0124] The present invention further provides a kit for carrying out the siRNA and / or the method of the present invention. Such a kit contains one or more siRNAs, vectors or cells of the present invention and may further contain an instruction manual. The instruction manual can record instructions for inhibiting PCSK9 expression in the cell by contacting the cell with an amount of the siRNA or vector of the present invention that effectively inhibits PCSK9 expression.

[0125] When the siRNA or carrier of the present invention contacts cells in vitro, optionally, the kit of the present invention may further include a tool (e.g., an injection device) for contacting the cells with the siRNA or carrier of the present invention or a tool 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 include a device for obtaining a sample (e.g., a plasma sample, etc.) from a subject.

[0126] When the siRNA, vector, or cells into which the siRNA or vector of the present invention has been introduced in vitro are administered in vitro, the kit of the present invention may optionally include 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.

[0127] IX. Therapeutic methods, pharmaceutical uses The present invention provides a method for treating a disease or condition related to PCSK9 expression in a subject, the method comprising administering to the subject the siRNA, vector, cells, or pharmaceutical composition of the present invention.

[0128] In some embodiments, the disease related to PCSK9 expression is a cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from dyslipidemia, hypercholesterolemia, non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis, etc.

[0129] In some embodiments, the disease related to PCSK9 expression is an oncological disease. In some preferred embodiments, the oncological disease is selected from malignant melanoma, hepatocellular carcinoma, and metastatic liver cancer.

[0130] In some embodiments, the disease related to PCSK9 expression is an inflammatory immune disease mediated by T cells. In some preferred embodiments, the inflammatory immune disease mediated by T cells is selected from psoriasis or psoriatic arthritis, or eczema or atopic dermatitis, or urticaria or hormone-dependent dermatitis or rheumatoid arthritis, scleroderma or diabetes, or chronic hepatitis or lymphoma.

[0131] In some embodiments, the method for treating a disease or condition related to PCSK9 expression in a subject according to the present invention is to administer the siRNA or pharmaceutical composition to the subject, including subcutaneous administration or intravenous administration. In some embodiments, the subject is a human patient.

[0132] The present invention also relates to the siRNA, vector, cell or pharmaceutical composition of the present invention for treating a disease or condition related to PCSK9 expression in a subject.

[0133] 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 condition related to PCSK9 expression in a subject. The medicament of the present invention can be prepared into an emulsion, microemulsion, or microparticles.

[0134] Sequence The RNA sequence according to the present invention targets the human PCSK9 gene (or target gene, target mRNA sequence, target sequence).

[0135]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0136] The modified RNA sequences used in the present invention are shown in Table 4.

[0137] In this specification, the meanings of the respective abbreviations are as follows.

[0138] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0139] 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.

[0140] i represents inosine ribonucleotide.

[0141] m indicates that the nucleotide adjacent to its left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3, respectively.

[0142] f indicates that the nucleotide adjacent to its left is a nucleotide modified with 2'-F. For example, Af, Uf, Gf, and Cf represent A, U, G, and C modified with 2'-F, respectively.

[0143] “s” or “s-” indicates that the two nucleotides and / or the introduction carrier adjacent to its left and right are connected via a thiophosphate ester.

[0144] VP indicates that the nucleotide adjacent to its right is a nucleotide modified with vinylphosphonate. For publicly known information, reference can be made to, for example, PCT publication numbers WO2011139702, WO2013033230, and WO2019105419.

[0145] IB represents an inverted abasic deoxyribonucleotide and may contain the following three structures depending on its position / binding method within the siRNA. IB is known; see, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402.

Chemical formula

[0146] L96 represents a GalNAc-introducing carrier with the following known structure, among which

[0147]

Chemical formula

Chemical formula

[0148] NAG37 represents a GalNAc-introducing carrier with the following known structure, among which

Chemical formula

Chemical formula

[0149] GL6 represents a GalNAc-introducing carrier with the following structure, among which

Chemical formula

Chemical formula

[0150] GL12 represents a GalNAc-introducing carrier with the following structure, among which,

Chemical formula

Chemical formula

[0151]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

[0152]

Table 5-1

Table 5-2

Table 5-3

Table 5-4

[0153]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0154] Example Unless otherwise specified, the sources of the materials used in the examples are as follows.

[0155] The Huh7 cell line was from Nanjing Kebai with the product number CBP60202.

[0156] The Hep3B cell line was from Nanjing Kebai with the product number CBP60197.

[0157] The PHH cells were from Shanghai Xuanyi with the product number QYLF-HPMC.

[0158] The HEK293A cell line was from Nanjing Kebai with the product number CBP60436.

[0159] The Balb / c mice were purchased from Zhejiang vitalriver with the product number Balb / c.

[0160] Example 1 Preparation of Compound E7 1. Preparation of Intermediate 3-4 1.1 Preparation of Compound 2

Chemical formula

[0161] 1.2 Preparation of Compound 2A

Chemical formula

[0162] 1.3 Preparation of Compound 2B

Chemical formula

[0163] To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL), TMSOTf (137 g, 616 mmol, 111 mL) was added dropwise at 10 - 15 °C over 0.5 h. Then the mixture was stirred at 25 °C for 3 h. By TLC (dichloromethane:methanol = 20:1), compound 2A (R f = 0.54) was completely consumed and a new point (R fIt was shown that (= 0.24) was formed. Three reactions were combined. The mixture was cooled to 0 - 15 °C, and a solution of NaHCO3 (300 g dissolved in 3.00 L of water) was slowly poured in at 0 - 5 °C. The organic phase was separated, 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, the resulting yellow oily compound 2B (approx. 507 g) was used directly in the next step.

[0164] 1.4 Preparation of Compound 3

Chemical formula

[0165] 1 1H NMR: (400 MHz, DMSO). 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).

[0166] 1.5 Preparation of Intermediate 3-4 (TFA Salt)

Chem.

[0167] Under an argon atmosphere, compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) were added to a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L). The suspension was evacuated and purged with hydrogen gas three times. The mixture was stirred under H2 (50 Psi) at 30 °C for 2 h. LCMS (product: RT = 0.697 min) indicated the consumption of compound 3 and the detection of the product peak. The three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Without purification, yellow solid intermediate 3 - 4 (TFA salt) (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA) was obtained.

[0168] 1 H NMR: (400 MHz, DMSO-d6) δ = 7.92 (d, J = 9.1 Hz, 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).

[0169] 2. Preparation of Intermediate 3-3 2.1 Preparation of Compound 5

Chemical Structure

[0170] 417.0 g of Compound 3-4 was converted to Compound 5 in 9 batches.

[0171] 2.2 Preparation of Intermediate 3-3

Chem.

[0172] 1 H NMR: (400 MHz, DMSO) δ = 8.53 (t, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 3H), 8.03 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 3.6 Hz, 2H), 5.22 (d, J = 3.2 Hz, 2H), 4.96 (dd, J = 3.2 Hz, 2H), 4.55 (d, J = 8.4 Hz, 2H), 4.02 (t, J = 8.8 Hz, 6H), 3.77 - 3.59 (m, 5H), 3.58 - 3.45 (m, 21H), 3.40 - 3.20 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.17 (d, J = 8.0 Hz, 6H), 2.10 (s, 6H), 1.99 (s, 6H), 1.90 - 1.80 (m, 8H), 1.77 (s, 6H).

[0173] 3. Preparation of Compound E7 3.1 Preparation of Compound 3

Chem.

[0174] 1 H NMR (400 MHz, CD3OD) 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) + .

[0175] 3.2 Preparation of Compound 4

Chemical Structure

[0176] 1 H NMR (400 MHz, CD3OD) 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) + .

[0177] 3.3 Preparation of Compound 6

Chemical Structure

[0178] 1 H NMR (400 MHz, CD3OD) 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 - 3.97 (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) + .

[0179] 4. Preparation of Compound E7

Chemical Structure

[0180] 1 H NMR (400 MHz, CD3OD) 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) MS: m / z = 3022.36 (M+H) + .

[0181] Example 2 Preparation of Compound E13 1. Preparation of Intermediate 2-2

Chemical Structure

[0182] 1.1 Preparation of Compound 3 [Chem.] The four reactions proceeded in parallel.

[0183] 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), and the mixture was then cooled to 0 °C. Isobutyl chloroformate (243 g, 1.78 mol, 233 mL) was added to the reaction mixture over 10 minutes while maintaining the reaction temperature below 4.0 °C. After addition, the mixture was stirred for 40 minutes or more, and compound 2 (526 g, 1.78 mol) was sequentially added to the reaction mixture over 10 minutes while maintaining the reaction temperature below 4.0 °C. After addition, the ice bath was removed, and then the reaction was warmed to room temperature and continued for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, compound 1 R f = 0.43) indicated that compound 1 was completely consumed and a new spot was formed. The four reactions were combined. The reaction solution was poured into a stirred cold (0 °C) 0.50 M HCl(aq.) (12.0 L) solution and stirred for about 10 minutes. Then, EtOAc (4.00 L x 3) was added, stirred for a while, separated, and the organic phase was washed with brine (10.0 L), dried over Na2SO4, concentrated in vacuo to give a thick colorless oil. Hexane (1.20 L) was added to the stirred oil. White fumes appeared from the solution and then disappeared after further stirring. When a seed crystal (1.20 g, 0.10 wt%) was added, white crystals gradually formed. Within 20 minutes, excess hexane (6.00 L) was added to a concentration sufficient to inhibit stirring of the suspension, and the mixture was stirred for 12 hours. The suspension was filtered, washed with hexane (1.20 L), and dried to give white solid compound 3 (about 1.20 kg).

[0184] 1 H NMR: (400 MHz DMSO) 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), 4.12 - 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).

[0185] 1.2 Preparation of Compound 4

Chem.

[0186] A solution of compound 3 (240 g, 415 mmol) in HCOOH (2.40 L) was stirred at 45 °C for 2 h. LCMS (compound 4 = 0.570 min) indicated that compound 3 was completely consumed and the main peak at the required m / z appeared. The five reactions were combined. Diluted and concentrated with toluene and ACN (1.50 L each). Formic acid was removed azeotropically three times (500 mL each) with 1:1 ACN and toluene (500 mL) and ACN. Compound 4 was dried under high vacuum. Then, the residue was stirred with DCM (500 mL), the organic layer was discarded, and then azeotropically dried twice with ACN (400 mL), dried under vacuum, and then azeotropically distilled nine times with toluene (400 mL) to obtain white solid compound 4 (about 800 g).

[0187] 1 H NMR: (400 MHz DMSO) 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).

[0188] 1.3 Preparation of Compound 6

Chem.

[0189] 1 H NMR: (400 MHz DMSO) δ 7.95 - 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, 9H), 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).

[0190] 1.4 Preparation of Intermediate 2-2

Chem.

[0191] 1 1H NMR: (400 MHz DMSO) 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, 5H), 3.17 - 3.16 (m, 6H), 2.22 - 2.10 (m, 12H), 1.99 (s, 9H), 1.93 - 1.73 (m, 21H).

[0192] 2. Preparation of Compound E13

Chemical formula

[0193] 2.1 Preparation of Compound Int1 2.1.1 Preparation of Compound 1-2

Chemical formula

[0194] 1 H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz, 2 H), 7.28 - 7.39 (m, 7 H), 4.72 - 4.86 (m, 2 H), 4.42 - 4.56 (m, 2 H), 3.52 - 3.61 m, 2 H), 3.14 - 3.39 (m, 2 H), 2.43 (s, 3 H), 1.47 (s, 9 H)

[0195] 2.1.2 Preparation of Compound 1-4

Chemical Structure

[0196] 1 H NMR (400 MHz, CDCl3) δ = 7.64 - 7.77 (m, 2 H), 7.27 - 7.38 (m, 13 H), 5.05 - 5.16 (m, 1 H), 4.50 - 4.59 (m, 4 H), 4.03 - 4.11 (m, 1 H), 3.62 - 3.76 (m, 2 H), 3.44 - 3.58 (m, 3 H), 3.25 - 3.33 (m, 2 H), 3.15 - 3.20 (m, 1 H), 2.42 (s, 3 H), 1.46 (s, 9 H)

[0197] 2.1.3 Preparation of Compound 1-5

Chem.

[0198] LCMS (ESI): m / z = 700.1 (M+Na)+; 1 H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 8.4 Hz, 2 H), 7.28 - 7.39 (m, 12 H), 5.10 - 5.20 (m, 1 H), 4.93 - 5.02 (m, 1 H), 4.52 - 4.61 (m, 4 H), 3.72 - 3.88 (m, 2 H), 3.52 - 3.67 (m, 4 H), 3.27 - 3.36 (m, 1 H), 3.15 - 3.22 (m, 1 H), 3.04 (s, 3 H), 2.43 (s, 3 H), 1.43 (s, 9 H). LCMS: m / z =700.1 (M+Na) + .

[0199] 2.1.4 Preparation of Compound 1-6

Chem.

[0200] 1 H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.4 Hz, 2 H), 7.27 - 7.39 (m, 12 H), 4.48 - 4.62 (m, 4 H), 3.93 - 4.08 (m, 2 H), 3.55 - 3.69 (m, 4 H), 2.84 - 3.10 (m, 4 H), 2.44 (s, 3 H)

[0201] 1.1.5 Preparation of Compound 1-7

Chem.

[0202] 11H NMR (400 MHz, CDCl3) δ 7.27 - 7.40 (m, 10 H), 4.57 (s, 4 H), 3.90 - 4.00 (m, 2 H), 3.59 - 3.69 (m, 4 H), 2.77 - 3.04 (m, 4 H)

[0203] 1.1.6 Preparation of Compound 1-8

Chem.

[0204] 1 1H NMR (400 MHz, CDCl3) δ 4.12 - 4.22 (m, 2 H), 3.78 (d, J = 4.4 Hz, 4 H), 3.17 - 3.30 (m, 4 H)

[0205] 1.1.7 Preparation of Compound Int-1

Chem.

[0206] 1 1H NMR (400 MHz, CDCl3) δ 7.38 - 7.50 (m, 2 H), 7.25 - 7.36 (m, 6 H), 7.18 - 7.25 (m, 1 H), 6.87 (d, J = 8.8 Hz, 4 H), 4.26 - 4.40 (m, 1 H), 3.94 - 4.05 (m, 1 H), 3.85 (d, J = 4.4 Hz, 2 H) 3.73 - 3.82 (m, 6 H), 3.34 - 3.41 (m, 1 H), 3.23 - 3.30 (m, 3 H), 3.05 - 3.23 (m, 2 H)

[0207] 2.2 Preparation of Compound Int6 2.2.1 Preparation of Compound 3

Chem.

[0208] 11H NMR (400 MHz, CD3OD) δ 7.21 - 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) LCMS: m / z = 526.8 (M+H) + .

[0209] 2.2.2 Preparation of Compound 4

Chem.

[0210] 1 1H NMR (400 MHz, CD3OD) δ 3.79 - 3.89 (m, 2H), 3.73 (dd, J = 3.6, 13.2 Hz, 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 = 2.8 Hz, 11H), 1.30 - 1.37 (m, 1H). LCMS: m / z = 346.5 (M+H) + .

[0211] 2.2.3 Preparation of Compound 5

Chem.

[0212] 1 H NMR (400 MHz, CD3OD) δ 7.41-7.43 (m, 2H), 7.16-7.34 (m, 7H), 6.83-6.87 (m, 4H), 3.88-4.04 (m, 2H), 3.77-4.09 (m, 7H), 3.61-3.68 (m, 4H), 3.43-3.58 (m, 3H), 3.33-3.38 (m, 1H), 3.14-3.20 (m, 1H), 3.05 (dd, J = 8.4, 13.2 Hz, 1H), 2.20-2.42 (m, 4H), 1.51-1.60 (m, 4H), 1.25-1.39 (m, 8H). LCMS: m / z = 648.3 (M+H) + .

[0213] 2.2.4 Preparation of Compound Int-6

Chemical Structure

[0214] 2.3 Preparation of Compound E13 2.3.1 Preparation of Compound 7

Chemical Structure

[0215] 1 H NMR (400 MHz, CD3OD) δ 7.43 (d, J = 8.4 Hz, 2H), 7.18 - 7.34 (m, 7H), 6.81 - 6.91 (m, 4H), 5.34 (d, J = 2.8 Hz, 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 = 2.4 Hz, 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)

[0216] 2.3.2 Preparation of E13

Chem.

[0217] LCMS (ESI): m / z = 1149.5 (M / 2+H) + ; 1 1H NMR (400 MHz, CD3OD) δ 7.42 - 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 = 2.4 Hz, 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)

[0218] Preparation of siRNA in Example 3 The siRNA of the present invention was prepared by a known solid-phase phosphoramidite method. For specific methods, reference can be made to, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and the outline is as follows.

[0219] Preparation of siRNA not bound to a ligand 1.1 Synthesis of sense strand (SS strand) By the solid-phase phosphoramidite synthesis method, starting from a blank CPG solid support as the initial cycle, nucleoside monomers are connected one by one in the 3'-5' direction according to the sense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it involves four process reactions: deprotection, coupling, capping, and oxidation or thiolation. Regarding the synthesis scale, the synthesis conditions for 5 μmol of oligonucleic acid are as follows.

[0220] The nucleoside monomer provides a 0.05 mol / L acetonitrile solution, and the reaction conditions for each process are the same, that is, the temperature is 25 °C. For deprotection, it is deprotected three times with a 3% trichloroacetic acid-dichloromethane solution. The activator used in the coupling reaction is a 0.25 mol / L ETT-acetonitrile solution, and it is coupled twice. For capping, it is capped twice with 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). For oxidation, it is oxidized twice with a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution. For thiolation, it is thiolated twice with a 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1 / 1, v / v) solution.

[0221] 1.2 Synthesis of antisense strand (AS strand) By the solid-phase phosphoramidite synthesis method, starting from a blank CPG solid support as the initial cycle, nucleoside monomers are connected one by one in the 3'-5' direction according to the antisense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it involves four process reactions: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol of oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0222] 1.3 Purification and annealing of oligonucleotides 1.3.1 Aminolysis The synthesized solid-phase carrier (sense strand or antisense strand) is put into a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and the reaction is carried out for 16 hours (or 8 hours) under a constant temperature water bath at 35 °C (or 55 °C). After filtration, the solid-phase carrier is washed three times with ethanol / water, 1 mL each time. After the filtrate is centrifugally concentrated, the crude product is purified.

[0223] 1.3.2 Purification The methods of purification and desalting are well-known to those skilled in the art. For example, it may be eluted and washed with a strong anion packing column, a sodium chloride-sodium hydroxide system, or the product may be collected and desalted with a gel packing purification column, and the elution system is pure water.

[0224] 1.3.3 Annealing According to Table 6 above, the sense strand (SS strand) and the antisense strand (AS strand) are mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated to 70 - 95 °C in a water bath, held for 3 - 5 min, naturally cooled to room temperature, and the system is freeze-dried to obtain the product. Finally, double-stranded DR002254 - DR002343 is obtained.

[0225] 2. Preparation of siRNA with the sense strand bound to the ligand 2.1 Binding of the ligand to the CPG vendor 2.1.2 Binding of Compound E7 to the CPG vendor Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed, acetonitrile (5 mL) was added and shaken to dissolve, then DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added and shaken until clear. Blank vector Resin (550 mg, CPG pore size 1000 Å) was weighed and added to the reaction solution, the temperature was controlled at 20 °C, and the reaction was carried out overnight with a shaker. Sampling and monitoring were carried out, and thin layer chromatography TLC was performed. The reaction was complete. Here, the developing solvent was DCM / methanol = 4 / 1 and it was colored with phosphomolybdic acid. It was filtered through a sintered glass funnel, the cake was washed with anhydrous acetonitrile (20 mL * 5), the cake was taken, and vacuum extraction filtration was carried out with an oil pump for 6 hours to obtain 530 mg of a pseudo-white solid.

[0226] 530 mg of the product after the above condensation was put into a 50 mL round bottom flask, CapC (DMAP / acetonitrile), CapB (N-methylimidazole / pyridine / acetonitrile), and CapA (acetic anhydride / acetonitrile) were added sequentially, and the reaction was carried out overnight with a shaker at room temperature. It was filtered, the cake was washed with acetonitrile (20 mL * 4), the cake was collected, and after vacuum extraction filtration was carried out with an oil pump for 8 hours, 200 mg of a pseudo-white solid was obtained and used for solid phase synthesis.

[0227] 2.1.2 Binding of Compound E13 to the CPG Vector Compound E13 is connected to the CPG vector by the same method as the binding method of Compound E7 to the CPG vector.

[0228] 2.2 Synthesis of the Sense Strand (SS Strand) By the solid phase phosphoramidite synthesis method, using the above-prepared GL6 solid support as the starting cycle, nucleoside monomers are sequentially connected one by one from the 3'-5' direction according to the sense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four process reactions: deprotection, coupling, capping, and oxidation or thiolation. Regarding the synthesis scale, the synthesis conditions for 5 umol of oligonucleic acid are as follows.

[0229] The nucleoside monomer provides a 0.05 mol / L acetonitrile solution. The reaction conditions for each step are the same, that is, the temperature is 25 °C. For deprotection, it is deprotected three times with a 3% trichloroacetic acid-dichloromethane solution. The activator used in the coupling reaction is a 0.25 mol / L ETT-acetonitrile solution, and it is coupled twice. For capping, it is capped twice with 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). For oxidation, it is oxidized twice with a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution. For thiolation, it is thiolated twice with a 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1 / 1, v / v) solution.

[0230] 2.3 Synthesis of antisense strand (AS strand) Using the solid-phase phosphoramidite synthesis method, starting with a blank CPG solid support for the starting cycle, the nucleoside monomers are connected one by one from the 3'-5' direction according to the antisense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four-step reactions of deprotection, coupling, capping, oxidation or thiolation. The synthesis conditions for 5 μmol of oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0231] 2.4 Purification and annealing of oligonucleotides 2.4.1 Aminolysis The synthesized solid support (sense strand or antisense strand) is put into a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and it is reacted under a constant temperature water bath at 35 °C (or 55 °C) for 16 hours (or 8 hours), filtered, the solid support is washed three times with ethanol / water, 1 mL each time, and after the filtrate is centrifugally concentrated, the crude product is purified.

[0232] 2.4.2 Purification The methods of purification and desalination are well-known to those skilled in the art. For example, it can be eluted and washed with a strong anion-packed column and a sodium chloride-sodium hydroxide system, the product is collected, desalted with a gel-packed purification column, and the elution system is pure water.

[0233] 2.4.3 Annealing According to Table 6 below, the sense strand (SS strand) and the antisense strand (AS strand) are mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated to 70-95 °C in a water bath, held for 3-5 min, naturally cooled to room temperature, and the system is freeze-dried to obtain the product.

[0234] In the same way, siRNA conjugated with L96 was obtained.

[0235] The finally obtained siRNAs were DR002221, DR002358 to DR002364, DR005642, DR005656, DR005672, DR005674, DR005675 and DR005676.

[0236] Example 4 Cell Line Activity Screening 1. HepG2 Cell Line Activity Screening Cell Transfection On the first day, after digesting the HepG2 (Nanjing Kebai, product number CBP60199) cell line, it was resuspended, counted, plated in a 96-well plate, 100 μL / well, 1×10 4 cells / well, and transfection operation was performed after 18 hours.

[0237] On the next day, the siRNA (DR002221 or DR002254 - DR002343) prepared in Example 3 was diluted with Opti-MEM. 198 μL of Opti-MEM was taken and added to 2 μL of the compound stock solution, pipetted and mixed uniformly for use. Each time an experiment was carried out, corresponding dilution operations were performed according to different experimental needs.

[0238] The next day, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently pipetted to mix uniformly, and left at room temperature for 5 min. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA (final concentration 10 nM) were gently pipetted to mix uniformly, left at room temperature for 10 min, added to a 96-well plate, and adjusted to 10 μL / well. After culturing in a 37 °C, 5% CO₂ incubator for 24 h, RNA was extracted.

[0239] RNA Extraction According to the operation of the high-throughput cell RNA extraction kit (Fanze Medical, FG0412), the protocol was to extract cell RNA using a nucleic acid extraction device (Hangzhou Ausheng, Auto-pure96).

[0240] RNA Reverse Transcription Preparation of the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. The denaturation reaction was carried out by incubating at 65 °C for 5 min using a conventional PCR instrument. The mixture was placed on ice and rapidly cooled for 2 min.

[0241] Preparation of the reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5×Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0242] 14.5 μL of the modified reaction solution was gradually and uniformly mixed with the reverse transcription reaction solution, and incubated at 42°C for 45 minutes using a conventional PCR instrument for reverse transcription, then incubated at 95°C for 5 minutes to inactivate the enzyme, and the reverse transcription product (cDNA) was cooled at 4°C.

[0243] After the reverse transcription was completed, 30 μL of distilled water without DNA enzyme and RNA enzyme was added to the cDNA sample in each well.

[0244] Real-Time PCR TaqMan TM Referring to the operation flow of Fast Advanced Master Mix (ABI, 4444965), a real-time PCR reaction (ABI, QuantStudio3) was carried out in a 20 μL system. The reaction process was set as (50°C, 2 min) × 1 Cycle; (95°C, 20 s) × 1 Cycle; (95°C, 1 s; 60°C, 24 s) × 40 Cycles.

[0245]

Table 7

[0246] Data Statistics 2 -△△Ct Calculate the value and convert it into a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)].

[0247] The target gene was hPCSK9 and the internal standard was hACTB.

[0248] 2. Huh7 cell line activity screening Similar to the above HepG2 cell line activity screening, activity screening was carried out using the Huh7 (Nanjing Kebai, product number CBP60202) cell line. The final concentrations of siRNA were 10 nM and 0.1 nM to screen the activity of the siRNA compound.

[0249] The results of the HepG2 cell line activity screening and the Huh7 cell line activity screening are shown in Table 8.

[0250]

Table 8

[0251] Example 5 Tg mouse primary hepatocyte (PMH) activity screening (transfection) Cell Transfection Tg mouse primary hepatocytes (Tg mice were purchased from Jicaiyaokang, T053388) were isolated, counted, plated in 24-well plates, at 900 μL / well, 8×10 4 cells / well.

[0252] Transfection 10 μL of the diluted siRNA prepared in Example 3 (DR002221 or DR002254 - DR002343) (final concentration 10 nM) was added to 40 μL of Opti-MEM and mixed uniformly. 3 μL of RNAiMAX was added to 47 μL of Opti-MEM and mixed uniformly. After incubation for 5 minutes, it was mixed uniformly with the diluted siRNA, left at room temperature for 10 minutes, and then added to the corresponding wells. It was cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0253] Real-Time PCR TaqMan TM Referring to the operation flow of TaqMan Fast Advanced Master Mix (ABI, 4444965), a real-time PCR reaction (ABI, QuantStudio3) was carried out in a 20 μL system. The reaction process was (50°C, 2 min)×1 Cycle; (95°C, 20 s)×1 Cycle; (95°C, 1 s; 60°C, 24 s)×40 Cycles.

[0254] RNA Extraction A high-throughput nucleic acid extraction device was used to extract total RNA using the magnetic bead method. After reverse transcription, real-time PCR detection was performed.

[0255]

Table 9

[0256] Data Statistics 2 -△△Ct Calculate the value and convert it to a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)].

[0257] The experimental results of siRNA PMH activity screening are shown in Table 10.

[0258]

Table 10

[0259] Example 6 IC50 Activity Screening of Human Primary Hepatocytes (PHH) Using siRNAs of Multiple Concentrations A. Human Primary Hepatocyte (PHH Cell) Activity Screening Cell Transfection 1.4 mL of rat tail collagen solution (Sigma, C3867) was added to 40.6 mL of distilled water containing no DNA enzyme or RNA enzyme and mixed uniformly. 40 μL of the mixture was added to each well of a 96-well culture plate and coated overnight at 4°C. The coating solution was removed the next day.

[0260] The next day, before use, the coated cell plate was rinsed with DPBS and the DPBS was aspirated off. PHH cells were resuscitated in resuscitation medium, resuscitated at 37°C, centrifuged, resuspended, and counted. PHH cells were plated in a 96-well plate at 90 μL / well, 2×10 4 cells / well, and the complete medium was replaced 4 hours later. Transfection was performed 18 hours later.

[0261] On the 3rd day, 198 μL of Opti-MEM was added to 2 μL of 20 μM siRNA (DR002221 or DR002254 - DR002343), and pipetted to mix uniformly. As the first concentration point, it was diluted at an appropriate gradient according to the actual experimental requirements.

[0262]

[0261] On the 3rd day, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently pipetted to mix uniformly, and left at room temperature for 5 min. Then, 15 μL of the prepared RNAi-MAX mixture was mixed with 15 μL of the diluted compound, and gently pipetted to mix uniformly. The final concentrations of siRNA were 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM. It was left at room temperature for 10 min, then added to a 96-well plate at 10 μL / well. After culturing in a 37 °C, 5% CO₂ incubator for 24 h, RNA was extracted.

[0263] RNA Extraction

[0264] Similar to Example 4, according to the operation of the high-throughput cell RNA extraction kit (Fanze Medical, FG0412), the protocol was to perform cell RNA extraction using a nucleic acid extraction device (Hangzhou Ausheng, Auto-pure96).

[0264] RNA Reverse Transcription Preparation of the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. A denaturation reaction was performed by incubating at 65 °C for 5 min using a conventional PCR instrument. The mixture was placed on ice and rapidly cooled for 2 min.

[0265]

[0265] Preparation of the reverse transcription reaction solution, refer to PrimeScript TMRefer to the II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0266] 14.5 μL of the denatured reaction solution and 5.5 μL of the reverse transcription reaction solution were gradually and uniformly mixed. Incubate at 42 °C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95 °C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4 °C.

[0267] After the reverse transcription was completed, 30 μL of distilled water free of DNA enzyme and RNA enzyme was added to the cDNA sample in each well.

[0268] Real-Time PCR TaqMan TM Refer to the operation flow of TaqMan Fast Advanced Master Mix (ABI, 4444965), and perform real-time PCR reaction (ABI, QuantStudio3) in a 20 μL system. The reaction process was set as (50 °C, 2 min) × 1 Cycle; (95 °C, 20 s) × 1 Cycle; (95 °C, 1 s; 60 °C, 24 s) × 40 Cycles.

[0269]

Table 11

[0270] Data Statistics 2 -△△Ct Calculate the value and convert it to a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)].

[0271] The experimental results are shown in Table 15.

[0272]

Table 12

[0273] Example 7 Detection of psi-CHECK2 off-target activity Plasmid Preparation Based on the siRNA sequence, the corresponding antisense strand off-target plasmid was designed, and the psiCHECK2 GSSM-5Hits recombinant plasmid was manufactured by Shanghai Genechem Co., Ltd., and the recombinant plasmid was diluted to 1000 ng / μL for reserve.

[0274] Cell Transfection 100 μL of HEK293A cell resuspension was plated into each well of a 96-well plate at 8×10 3 cells / well.

[0275] The next day, first, the complete medium in the wells was aspirated and discarded, and replaced with 80 μL / well of Opti-MEM medium, and the cells were starved for about 1.5 hours.

[0276] siRNA preparation: siRNA (DR002221 or DR002254 - DR002343) was serially diluted 3-fold from a final concentration of 40 nM to a total of 11 concentration points.

[0277] Plasmid mixture: Each well of the plasmid mixture contains 0.01 μL of plasmid and 8.99 μL of Opti-MEM.

[0278] Lipo mixture: Lipo2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) was diluted with Opti-MEM and left at room temperature for 5 minutes. Each well of the Lipo mixture contains 0.2 μL of Lipo and 9.8 μL of Opti-MEM.

[0279] 22 μL of the prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of the plasmid mixture were dispensed into the corresponding identical wells, named well A, pipetted to mix uniformly, incubated at room temperature for 20 minutes, and then co-transfection was performed. Finally, 20 μL / well of the well A mixture was added to the cells in each well. The final volume of each well was 100 μL, containing the original 80 μL of Opti-MEM and 20 μL of the well A mixture. The final concentrations of siRNA were 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 culturing in a 37 °C, 5% CO2 incubator for 4 hours, 100 μL of DMEM medium containing 20% fetal bovine serum was added to each well. Detection was performed after culturing in a 37 °C, 5% CO2 incubator for 24 hours.

[0280] Result Detection Before the start of the experiment, the mixed Dual-Glo (R) Luciferase (Dual-Glo (R) Luciferase Assay System, Promega, E2940) was remelted, balanced to room temperature, and then, at the time of use, DMEM was added to each tube at a 1:1 ratio and prepared as substrate I. Dual-Glo (R) Stop&Glo (R) Buffer was remelted, balanced to room temperature, and then, at the time of use, Dual-Glo (R) Stop&Glo (R) Substrate was prepared as substrate II at a 100:1 ratio.

[0281] The culture medium in the wells of the 96-well culture plate was aspirated and removed with a vacuum pump.

[0282] 150 μL of substrate I was added to each well and incubated on a shaker at room temperature for 10 minutes.

[0283] Take 120 μL of Substrate I and transfer it to a 96-well plate. The Firefly (Photinus pyralis luciferase) chemiluminescence value is read from a microplate reader (Tecan, Infinite 200).

[0284] Furthermore, add 60 μL of Substrate II to each well, incubate at room temperature for 10 minutes on a shaker, and read the Renilla (Renilla reniformis luciferase) chemiluminescence value from the microplate reader.

[0285] Data Analysis and Processing Fluorescence activity is measured by a microplate reader, and the collected Renilla signal is normalized by the Firefly signal standard. The inhibitory effect of siRNA is obtained by comparing with the untreated result (residual inhibitory activity), and the calculation process is as follows.

[0286] Normalized Ren / Fir ratio: Ratio = Renilla / Firefly.

[0287] Residual inhibition rate = (Ratio siRNA / Ratio control ) * 100%. Take the average value of the results of two wells. Here, Ratio control is the Ratio value of the control well (without siRNA, taking the average value of the results of two wells).

[0288] Mapping: Mapped with Graphpad Prism. Half maximal inhibitory concentration (IC50): In this experiment, it was mapped at Top and Bottom, and the IC50 value was obtained by the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X)*HillSlope)), where Y = 50 and X = log(concentration).

[0289] The experimental results are shown in Table 16.

[0290]

Table 13

[0291] Example 8 IC50 Activity Screening Using Primary Hepatocytes (PMH) of Tg Mice (Free Intake) Cell Transfection Primary hepatocytes of Tg mice (the Tg mice were T053388 purchased from Jicai Pharmaceutical Co., Ltd.) were isolated, counted, plated in 24-well plates, and seeded at 900 μL / well and 8×10 4 cells / well.

[0292] 10 μL of diluted siRNA (DR002221, DR002358 - DR002364) was added to 90 μL of Opti-MEM and mixed uniformly, then added to the corresponding wells, cultured in a 37°C, 5% CO2 incubator for 24 hours, and free-intake activity screening was performed. The starting concentration of siRNA was 20 nM, and it was serially diluted 5-fold at 5 concentration points (20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM).

[0293] Real-Time PCR Total RNA was extracted using a high-throughput nucleic acid extraction device - magnetic bead method, and after reverse transcription, real-time PCR detection was performed.

[0294]

Table 14

[0295] Data Statistics 2 -△△Ct Values were calculated and converted to percentages to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)].

[0296] The experimental results are shown in Table 18.

[0297]

Table 15

[0298] Example 9 In Vivo Activity Screening Experimental animals: 40 humanized PCSK9 Tg male mice, 11 - 12 weeks old. Before administration to the animals, the baseline level of PCSK9 protein was measured by ELISA on D - 12 (i.e., 12 days before administration, the same hereinafter) and D - 5, and this baseline level was defined as 100%.

[0299] On D0, the test compound was administered subcutaneously at a single dose of 3 mg / kg. Details are shown in Table 19.

[0300] [Table 16]

[0301] On D7, D14, D21, and D28 after administration, whole blood was extracted respectively, allowed to stand at room temperature, and then centrifuged to obtain serum. The level of PCSK9 protein was measured by ELISA, and the results are shown in Table 20.

[0302] [Table 17]

Claims

**Claim 1** 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 that form a double-stranded region, the lengths of the sense strand and the antisense strand are each independently 15 to 30 nucleotides, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 105 to 208, RNA (siRNA). **Claim 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 represented by any one of SEQ ID NOs: 1 to 104. **Claim 3** The siRNA according to claim 1 or 2, wherein the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 17 to 21 nucleotide pairs, more preferably 19 to 21 nucleotide pairs. **Claim 4** The siRNA according to any one of claims 1 to 3, wherein the siRNA comprises a sense strand sequence and an antisense strand sequence of a pair as shown in Table 3. **Claim 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, a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence represented by 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 represented by any one of SEQ ID NOs: 203, 204, 142, 207, 143, 197, 205, 206, 144, 147, 148, 149 and 150. **Claim 6** The sense strand includes 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 sequences represented by any of SEQ ID NOs: 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46. Preferably, the antisense strand includes a nucleotide sequence represented by any of SEQ ID NOs: 99, 100, 38, 103, 39, 93, 101, 102, 40, 43, 44, 45, and 46. The siRNA according to any one of claims 1 to 3 and 5. **Claim 7** (a) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 203, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

99. (b) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 204, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

100. (c) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 142, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

38. (d) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 207, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

103. (e) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 143, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

39. (f) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 197, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

93. (g) The antisense strand includes the nucleotide sequence represented by SEQ ID NO: 205, and the sense strand includes the nucleotide sequence represented by SEQ ID NO:

101. (h) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 206, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 102, (i) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 144, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 40, (j) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 147, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 43, (k) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 148, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 44, (l) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 149, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 45, or (m) The antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 150, and the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 46, the siRNA according to claim 6.

8. Almost all nucleotides of the sense strand and almost all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides, the siRNA according to any one of claims 1 to 7.

9. 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'-fluorinated modified nucleotides, 2'-deoxy-acid modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing a thiophosphate ester group, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural bases containing nucleotides, and cholesterol-based derivatives or terminal nucleotides linked to dodecanedioic acid dodecanamide, deoxyribonucleotides, the siRNA according to claim 8.

10. The sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorinated modified nucleotides, deoxyribonucleotides, and nucleotides containing phosphorothioate groups, and the siRNA according to claim 8.

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

12. The siRNA contains a pair of modified sense strand sequences and modified antisense strand sequences shown in any of Table 6 in the specification, and the siRNA according to claim 8.

13. (a) The sense strand contains CmsUmsAmGmAmCmCfUmGfUm dTUmUmGmCmUmUmUmUmGmsUm, and the antisense strand contains Am sCf sAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm; (b) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCm sUmsUm, and the antisense strand contains Am sAf sGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm; (c) The sense strand contains UmsGmsUmUmUmUmGfCfUfUmUmUmGmUmAmAmCmUm sUmsUm, and the antisense strand contains Am sAf sGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmCfAmsGfsGm; (d) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUm sUmsAm, and the antisense strand contains UmsAf sAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCm sAfsGm; (e) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmsAm, and the antisense strand contains UmsAf sAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCm sAfsGm; (f) The sense strand contains GmsUmsUmUmUmGmCfUfUfUmUmGmUmAmAmCmUmUmA m, and the antisense strand contains UmsAfsAmGfUmUfAmCfAmAfAmAfGmCfAmAfAmAfCm sAfsGm. (g) The sense strand contains UmsUmsUmUmGmCmUfUfUfUmGmUmAmAmCmUmUmsGm sAm, and the antisense strand contains UmsCfsAmAfGmUfUmAfCmAfAmAfAmGfCmAfAmAfAmsCfsAm. (h) The sense strand contains UmsGmsCmUmUmUmUmGfUfAmAmCmUmUmGmAmAmsGm sAm, and the antisense strand contains UmsCfsUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmsAfsAm. (i) The sense strand contains GmsCmsUmUmUmUmGfUfAfAmCmUmUmGmAmAmGm sAmsUm, and the antisense strand contains AmsUfsCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmGfCmsAfsAm. (j) The sense strand contains CmsUmsUmUmUmGmUfAfAfCmUmUmGmAmAmGmAm sUmsAm, and the antisense strand contains UmsAfsUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmsCfsAm, or (k) The sense strand contains UmsUmsUmUmGmUmAfAfCfUmUmGmAmAmGmAmUmsAmsUm, and the antisense strand contains AmsUfsAmUfCmUfUmCfAmAfGmUfUmAfCmAfAmAfAmsGfsCm, the siRNA according to claim 8.

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

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

16. The ligand moiety contains a linking group represented by (X'), 【Chemical 1】 wherein, 【Chemical 2】 represents the connection position with the biomolecule, Q is independently H, 【Chemical Formula 3】 and, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ ), - or -NHC(O)-(CH ｄ ), and 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, -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH ２ CH ２ O) ｅ -, and where e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -C(O)-, -M-, -CH ２ -M- or -C(O)-M-, and where M is, 【Chemical Formula 4】 and, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 14 or 15.

17. The linking group is represented by formula (I'), 【Chemical Formula 5】 wherein, 【Chemical Formula 6】 represents the connection position with the biomolecule, Q is independently H, 【Chemical Formula 7】 and, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ -, -(OCH ２ CH ２ CH ２ ) ｃ -, -(OCH ２ CH ２ CH ２ CH ２ ) ｃ -, -(OCH ２ CH ２ CH ２ CH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and is 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 -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH- or -NHC(O)-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 16.

18. Q is independently H or [Chemical Formula 8] and, Here, L １ is -CH ２ O- or -NHC(O)-(CH ２ NHC(O)) ａ - and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O-, and L' is a chemical bond, R １ and R ２ are integrally CH ２ CH ２ O - or - CH ２ forms CH(R)-O-, and R ３ is H, or R １ and R ３ is integrally C １－２ forms alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 17.

19. The linking group is represented by formula (I'-1), formula (I'-2) or formula (I'-3), 【Chemical Formula 9】 wherein, 【Chemical 10】 represents the connection position with the biomolecule, Q is, 【Chemical 11】 and, Here, L １ is -CH ２ O- or -NHC(O)-, and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is - (OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O-, and R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, the siRNA according to claim 18.

20. Q is independently H, 【Chemical 12】 and, Here, L １ is -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHC(O)-(CH ２ NH C(O)) ａ - and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O- or -NHC(O)-, and L' is a chemical bond or -C(O)NH-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H, Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, The siRNA according to claim 17, wherein n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. The linking group is represented by formula (II’-1) or formula (II’-2), 【Chemical 13】 wherein, 【Chemical 14】 represents the connection position with the biomolecule, Q is independently 【Chemical Formula 15】 wherein, Here, L １ is -CH ２ O- or -CH ２ O-CH ２ CH ２ O-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, The siRNA according to claim 20.

22. Q is independently H, 【Chemical 16】 wherein, Here, L １ is -CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHC(O)-(CH ２ NH C(O)) ａ - and L ２ is a chemical bond, L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O- or -NHC(O)-, and L’ is a chemical bond or -C(O)NH-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H, Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, The siRNA according to claim 17.

23. The linking group is represented by formula (II’-2), 【Chemical 17】 wherein, 【Chemical Formula 18】 represents the connection position with the biomolecule, Q is independently 【Chemical 19】 wherein, Here, L １ is -CH ２ - or -C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - and where b = 1, 2, 3, 4 or 5, c = 1, 2, 3, 4 or 5, L is -CH ２ O- or -NHC(O)-, and R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, The siRNA according to claim 22.

24. Q is independently H, 【Chemical 20】 wherein, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ). ｃ -(OCH ２ CH ２ CH ２ ). ｃ -(OCH ２ CH ２ CH ２ CH ２ ). ｃ -(OCH ２ CH ２ CH ２ CH ２ CH ２ ). ｃ - or -NHC(O)-(CH ２ ). ｄ and 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, -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH ２ CH ２ O) ｅ -, and where e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -M-, -CH ２ -, -M- or -C(O)-M-, and where M is 【Chemical 21】 wherein, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H, Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, The siRNA according to claim 16.

25. T is -M-, -CH ２ -M- or -C(O)-M-, where M is 【Chemical 22】 The siRNA according to claim 24, wherein it is

26. Q is independently H or 【Chemical 23】 wherein, Here, L １ is -CH ２ O- or -NHC(O)-(CH ２ NHC(O)) ａ - and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O-, and L' is a chemical bond or -O(CH ２ CH ２ O) ｅ - and where e is 1, 2, 3, 4 or 5, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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. The siRNA according to claim 24 or 25, wherein T is as defined in claim 24 or 25.

27. The linking group is represented by formula (III'-1), formula (III'-2) or formula (III'-3), 【Chemical 24】 wherein, Q is 【Chemical 25】 wherein, Here, L １ is -CH ２ O- or -NHC(O)-, and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is - (OCH ２ CH ２ ) ｃ - or - NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O-, and Here, R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, and n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, The siRNA according to claim 26, wherein T is as defined in claim 24 or 25.

28. Q is independently H, 【Chemical 26】 wherein, Here, L １ is -CH ２ -, -CH ２ O- or -C(O)-, and L ２ is a chemical bond, L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and is 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, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, The siRNA according to claim 24 or 25, wherein T is as defined in claim 24 or 25.

29. The linking group is represented by formula (IV-1) or formula (IV-2), 【Chemical 27】 wherein, Q is independently 【Chemical 28】 wherein, Here, L １ is -CH ２ -, -CH ２ O- or -C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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, Here, R' is H, a hydroxyl protecting group, or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, The siRNA according to claim 24 or 25, wherein T is as defined in claim 24 or 25.

30. Q is independently H, 【Chemical 29】 wherein, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ [is] - (OCH ２ [CH ２ [CH ｃ [), - (OCH ２ [CH ２ [CH ２ [CH ｃ [), - (OCH ２ [CH ２ [CH ２ [CH ２ [CH ｃ [), - (OCH ２ [CH ２ [CH ２ [CH ２ [CH ２ [CH ｃ [), - or - NHC(O)-(CH ２ [CH ｄ [), and is​​​​​​​​​​​​​​​​​​​​​ 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, -CH ２ O- or -NHC(O)-, and L' is -O(CH ２ CH ２ O) ｅ -. wherein, e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -C(O)-, -M-, -CH ２ -M- or -C(O)-M-, and wherein, M is 【Chemical Formula 30】 wherein, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- and R ３ is H or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 16.

31. The linking group is 【Chemical 31】 【Chemical 32】 【Chemical 33】 【Chemical Formula 34】 【Chemical 35】 【Chemical 36】 selected from, the siRNA according to claim 16.

32. The linking group is 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemical 40】 【Chemical 41】 【Chemical 42】 selected from, the siRNA according to claim 16.

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

34. The ligand has the following structure, 【Chemical 43】 where, 【Chemical 44】 represents a position where it binds to the siRNA via a phosphate ester group or a thiophosphate ester group, the siRNA according to claim 14 or 15.

35. The ligand has the following structure, 【Chemical 45】 where, 【Chemical 46】 represents a position where it binds to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group, the siRNA according to claim 14 or 15.

36. The ligand has the following structure, 【Chemical 47】 where, 【Chemical 48】 represents a position where it binds to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group, the siRNA according to claim 14 or 15.

37. A cell containing the siRNA according to any one of claims 1 to 36.

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

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

40. A method for treating a disease or condition related to PCSK9 expression in a subject, comprising the step of administering to the subject the siRNA according to any one of claims 1 to 36, the cell according to claim 37, or the pharmaceutical composition according to claim 38.

41. The disease or condition related to PCSK9 expression is a cardiovascular disease, preferably the cardiovascular disease is selected from dyslipidemia, hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis, the method according to claim 40.

42. The disease or condition related to PCSK9 expression is hypercholesterolemia, preferably the hypercholesterolemia is selected from non-familial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia, and heterozygous familial hypercholesterolemia, the method according to claim 41.

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