CN115572726A

CN115572726A - siRNA for inhibiting PCSK9 gene expression and application thereof

Info

Publication number: CN115572726A
Application number: CN202210707102.1A
Authority: CN
Inventors: 潘隽; 马辛辛; 宋培明; 吴纯; 冯辉; 姚盛; 吕佳声; 司杨海啸; 殷毅杰; 郭万涛; 李海明; 陈大为; 顾家敏; 孔宪起
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2021-06-21
Filing date: 2022-06-21
Publication date: 2023-01-06

Abstract

The invention relates to siRNA for inhibiting PCSK9 gene expression and application thereof. The siRNA for inhibiting the expression of the PCSK9 gene comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 continuous nucleotides which are different from any one sequence shown in a table 1 or a table 2 by 0, 1,2 or 3 nucleotides, the sense strand and the antisense strand have at least 15, 16, 17, 18, 19, 20 or 21 nucleotides in complementarity, and at least one nucleotide in the sense strand and/or the antisense strand is a modified nucleotide. The invention also provides the siRNA conjugate and a pharmaceutical composition thereof, and a method for reducing the expression of the PCSK9 gene by using the siRNA, the siRNA conjugate and the pharmaceutical composition thereof. The siRNA, the siRNA conjugate and the pharmaceutical composition thereof can be used for treating and/or preventing PCSK9 gene-mediated diseases or symptoms.

Description

siRNA for inhibiting PCSK9 gene expression and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to siRNA for inhibiting human protein convertase subtilisin/kexin9 (PCSK 9) gene expression and application thereof in preparation of drugs for treating PCSK 9-mediated related diseases.

Background

Proprotein convertase subtilisin/kexin9 (PCSK 9) is a glycoprotein consisting of 692 amino acids, belongs to the ninth member of the Proprotein Convertase (PCs) family, is a secreted serine protease, is mainly expressed in tissues such as liver and intestinal tract, and is then secreted into blood. After entering blood circulation, PCSK9 can specifically bind to the epidermal growth factor-like domain of low density lipoprotein receptor (LDL-R) on the surface of hepatocytes, guide the low density lipoprotein receptor (LDL-R) to enter hepatocytes and reach lysosomes, so that the LDL-R is degraded in the lysosomes, and the LDL-R on the surface of hepatocytes is reduced, thereby reducing the ability of the liver to bind and remove LDL-C, and finally increasing the level of LDL-C in blood. Thus, hypercholesterolemia can be treated by inhibition of PCSK 9. In addition, recent studies have shown that PCSK9 elevation is closely related to obesity and type 2 diabetes, and also to chronic kidney disease such as nephrotic syndrome and proteinuria, and therefore, inhibition of PCSK9 can be an important means for preventing and treating these diseases related thereto.

RNA interference (RNAi) technology was first discovered by Fire et al in 1998 and was rapidly becoming widely used. The double-stranded RNA that causes gene silencing in RNA interference is siRNA, and generally consists of a length of double-stranded RNA of about 21-23 nucleotides, including a sense strand and an antisense strand that pair with a target mRNA, thereby inducing a host cell degradation response against these mRNAs. Although siRNA can specifically inhibit gene expression, intracellular siRNA is easily degraded and stable gene silencing is difficult to achieve. The slow virus vector has high infection efficiency in cells and low immune prototype, can interfere cells in a division stage and infect cells in a non-division stage, and the mediated RNAi can stably express siRNA in various animal cells for a long term and inhibit the expression of target genes, so the slow virus vector has the characteristics of high efficiency, stability, strong specificity and wide application range. Small interfering RNAs (sirnas) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference, thereby achieving the purpose of treating diseases.

There is also a need for agents that inhibit PCSK9 gene expression and that can treat diseases associated with PCSK9 gene expression, such as hyperlipidemia.

Inhibitors targeting PCSK9 and their use for the treatment of blood lipid disorders have been reported, but there is still a need to develop other RNAi agents with better therapeutic efficacy, specificity, stability, targeting or tolerance, etc.

Disclosure of Invention

The invention provides an siRNA for inhibiting PCSK9 gene expression, which comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 continuous nucleotides which are different from any one sequence shown in Table 1 or Table 2 by 0, 1,2 or 3 nucleotides, the sense strand and the antisense strand have at least 15, 16, 17, 18, 19, 20 or 21 nucleotides which are complementary, and at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

In some embodiments, the sense strand of the invention comprises at least 17 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from any one of the sequences set forth in table 1 or table 2.

In some embodiments, the modified nucleotide of the invention is selected from the group consisting of: 2 '-O-methyl modified nucleotide, 2' -fluoro modified nucleotide, 2 '-deoxynucleotide, 2' -fluoro, 2 '-deoxy modified nucleotide, 2' -methoxyethyl modified nucleotide, 2 '-amino modified nucleotide, 2' -alkyl modified nucleotide, 2 '-alkoxy modified nucleotide, 2' -F-arabinose nucleotide, phosphorothioate modified nucleotide, abasic nucleotide, morpholino nucleotide and locked nucleotide.

In some embodiments, the modified nucleotide of the invention is selected from the group consisting of: 2' -O-methyl modified nucleotide, 2' -fluoro modified nucleotide, 2' -deoxynucleotide and phosphorothioate modified nucleotide.

In some embodiments, the modified nucleotides of the invention include:

(1) According to the direction from the 5 'end to the 3' end, the nucleotides at the 7 th and the 9 th positions of the sense strand are 2 '-fluorine modified nucleotides, and the nucleotides at the rest positions are 2' -O-methyl modified nucleotides; or the nucleotides at the 3 rd, 5 th, 8 th, 9 th and 12 th positions of the sense strand are 2 '-fluorine modified nucleotides, and the nucleotides at the rest positions are 2' -O-methyl modified nucleotides; and/or

(2) According to the direction from the 5 'end to the 3' end, the nucleotides at the 2 nd, 4 th, 6 th, 11 th, 12 th, 14 th, 16 th and 18 th positions of the antisense strand are 2 '-fluorine modified nucleotides, and the nucleotides at the rest positions are 2' -O-methyl modified nucleotides; or the nucleotides at the 1 st, 2 nd, 3 rd, 5 th, 7 th, 11 th, 13 th, 14 th, 15 th, 17 th, 19 th and 21 st positions of the antisense strand are 2 '-fluorine modified nucleotides, and the nucleotides at the rest positions are 2' -O-methyl modified nucleotides; and/or

(3) In the direction from the 5' end to the 3' end, the 11 th nucleotide of the sense strand is a 2' -deoxynucleotide; and/or

(4) The 5' end of the sense strand comprises 1 or 2 phosphorothioate modified nucleotides in the direction from the 5' end to the 3' end; and/or the 5 'end and the 3' end of the antisense strand each independently comprise 1 or 2 phosphorothioate modified nucleotides.

In some embodiments, the antisense strand of the invention comprises any one of the antisense strand nucleotide sequences set forth in table 2 and the sense strand comprises any one of the sense strand nucleotide sequences set forth in table 2.

In some embodiments, the sense and antisense strands of the invention comprise or consist of a nucleotide sequence (5 '→ 3') selected from:

XR-1

sense strand mCMUGfUGfmCMUAfGfmCAfmCAfmCAmCMAMmA (SEQ ID NO: 11)

Antisense strand mU Uf mGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmCAfmCAfmCAfmG Cf mC (SEQ ID NO: 29)

XR-2

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 12)

Antisense chain mU Uf mGGfmGUmGmUmUmGCfUfmAGfmCAfmCAfmG Cf mC (SEQ ID NO: 30)

XR-3

Sense strand mC mU GfmUGfmCUafGfmcAafmCMmmA (SEQ ID NO: 13)

Antisense strand mU Uf mGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmCAfmCAfmCAfmG Cf mC (SEQ ID NO: 31)

XR-4

Sense strand mC mUGfUGfmCUFAfGfmCDAAfmCMMCmCMmCMmmA (SEQ ID NO: 14)

Antisense strand mU Uf mGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmCAfmCAfmCAfmG Cf mC (SEQ ID NO: 32)

XR-5

Sense strand mCMU GfmUGfmCUAFGfmCGaAfmCMmCMmMamA (SEQ ID NO: 15)

Antisense strand mU Uf mGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmCAfmCAfmCAfmG Cf mC (SEQ ID NO: 33)

XR-6

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 16)

Antisense chain mUufmGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmGCfmC (SEQ ID NO: 34)

XR-7

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 17)

Antisense strand mUdTMGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmGCfmC (SEQ ID NO: 35)

XR-8

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 18)

Antisense strand mUufmGGfmGUfmGmUmGCfUfmAoGmCAfmGCfmC (SEQ ID NO: 36)

XR-9

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 19)

Antisense strand mU Uf mGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmCAfmCAfmCAfmG Cf mC (SEQ ID NO: 37)

XR-10

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 20)

Antisense chain mU UfmGGfmGUfmGmUmUmGCfUfmaGfmCAfmG Cf mC (SEQ ID NO: 38)

XR-11

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmMAmA (SEQ ID NO: 21)

Antisense chain mU ufmggfmgufmgmumumgumgfugufmgcfumagmcafmgcf mC (SEQ ID NO: 39)

XR-12

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 40)

XR-13

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain mUUUUUUfGfmGGfmUGfUmUmGCfUAfGfCfmACfmAGfmCCf (SEQ ID NO: 41)

XR-14

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfmUGfmGGfmUGfmUmUmGCfmUAfGfCfmACfmAGfmCCf (SEQ ID NO: 42)

XR-15

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfmGmGGfmUGfUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 43)

XR-16

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfGfmGfmUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 44)

XR-17

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUmGmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 45)

XR-18

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmUmGmCmUAfGfCfmACfmAGfmcCf (SEQ ID NO: 46)

XR-19

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfmUmAGfCfmACmAGfmcCf (SEQ ID NO: 47)

XR-20

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmUmGCfUAfGCfmACfmAGfmCCf (SEQ ID NO: 48)

XR-21

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfUAfGfmCMACmAGfmCCf (SEQ ID NO: 49)

XR-22

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfmUAfGfCfmmAGfmcCf (SEQ ID NO: 50)

XR-23

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmGCfmUAfGfCfmmmmmmmGmCCf (SEQ ID NO: 51)

XR-24

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfmUAfGfCfmACfmAGfmCMC (SEQ ID NO: 52)

SR-1

Sense strand mC mUmmGmCCfUGfUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 23)

Antisense strand mA x Cf x mAAfAfmGCfmAAfmAAfmCAfmGGfUCfUmAMmmA (SEQ ID NO: 53)

SR-2

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 24)

Antisense strand mA mcmAAfmAAfmGCfmAAfmAAfmCMmmGMUmAMOMA mA (SEQ ID NO: 54)

SR-3

Sense strand mC mU mAMmCCfUGfmUdTmUmUmGmGmUmUmGmU (SEQ ID NO: 25)

Antisense strand mA x Cf x mAAfmAAfmGCfmAAfmAAfmCMmmGmUmAMAMmG x mA (SEQ ID NO: 55)

SR-4

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 26)

Antisense strand mA mmCAfmAAfmGCfmAAfmAAfmCAfmGmUmAMOMG mA (SEQ ID NO: 56)

SR-5

Sense strand mC mU mAMmCCfUGfmUdTmUmUmGmGmUmUmGmU (SEQ ID NO: 27)

Antisense strand mA mmC mAAfmAAfmGCfmAAfmAAfmCMmMAGmGGfUmCUmmA (SEQ ID NO: 57)

SR-6

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 28)

The antisense strand mA, mAAfmAAfmGCfmAAfmAAfmmCMmGmUCfUmAMmG, mA (SEQ ID NO: 58),

wherein C, G, U, A represents cytidine-3 '-phosphate, guanosine-3' -phosphate, uridine-3 '-phosphate, adenosine-3' -phosphate, respectively; m represents that one nucleotide adjacent to the right side of the letter m is a 2' -O-methyl modified nucleotide; f denotes that one nucleotide adjacent to the left side of the letter f is a 2' -fluoro modified nucleotide; * Indicates that the left adjacent nucleotide is a phosphorothioate modified nucleotide; f denotes that the left adjacent nucleotide of f is a nucleotide modified by phosphorothioate and 2' -fluorine simultaneously; d indicates that one nucleotide adjacent to the right side of the letter d is a 2' -deoxyribonucleotide; o indicates that one nucleotide adjacent to the right side of the letter d is a 2' -methoxyethyl modified nucleotide.

In some embodiments, the sense and antisense strands of the invention are each independently 17-25 nucleotides in length; preferably, the sense and antisense strands are each independently 19-23 nucleotides in length.

In yet another aspect, the invention provides an siRNA conjugate comprising an siRNA described herein and a targeting group.

In some embodiments, the targeting group of the present invention is a ligand that has affinity for asialoglycoprotein receptors.

In some embodiments, the invention discloses siRNA conjugates comprising a targeting group comprising a group derived from a lipophile selected from the group consisting of cholesteryl, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3- (oleoyl) lithocholic acid, O-3- (oleoyl) cholic acid, dimethoxytribenzyl, and phenoxazine. It is understood that the term "group derived from a lipophile" means a monovalent group formed after removal of an atom or a group from the lipophile, which monovalent group retains the original biological activity and function of the lipophile. One skilled in the art can readily determine the atoms or groups on the lipophile that can be removed so that the sites at which the resulting lipophile groups are used to attach to the remainder of the siRNA conjugate do not interfere with its biological function as a lipophile. For example, in the case of a liquid, can be covalently linked with the rest part of the siRNA conjugate in an ester group (-COO-), ether (-O-), amide (-CO-NH-) way and the like through a hydroxyl group in the carboxyl group or a hydroxyl group on a compound and the like.

In some embodiments, the targeting moiety of the present invention comprises a moiety from a carbohydrate selected from the group consisting of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fucoidan, galactosamine, D-galactosamine, N-acetyl-galactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucitol, glucose-6-phosphate, gulose glyceraldehyde, L-glycerol-D-mannose-heptose, glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, tartaric acid, threose, xylose, and xylulose. Specifically, the targeting group is a ligand group containing a ligand derived from N-acetyl-galactosamine (GalNAc). It is understood that the "group derived from a carbohydrate" refers to a monovalent group formed after removal of an atom or a group, which monovalent group retains the original biological activity and function of the carbohydrate. One skilled in the art can readily determine the atoms or groups on each carbohydrate that can be removed so that the site at which the resulting carbohydrate group is attached to the rest of the siRNA conjugate does not affect its biological function as a carbohydrate. For example, the moiety can be covalently attached to the remainder of the siRNA conjugate via O in the hydroxyl group in the form of an ether. The hydroxyl group attached to a ring carbon atom adjacent to the epoxy atom is generally selected for attachment. Exemplary ligand groups from N-acetyl-galactosamine (GalNAc) are shown below:

wherein denotes the position at which the ligand group is attached to the rest of the siRNA conjugate.

In other embodiments, the siRNA conjugates disclosed herein specifically bind to specific receptors of a particular tissue, thereby achieving tissue-specific targeting. In some embodiments, the conjugates of the invention specifically target hepatocyte surface receptors and thus specifically target liver tissue. In some embodiments, the conjugates of the invention specifically target asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. In some embodiments, the targeting group is a ligand group containing a ligand from N-acetyl-galactosamine (GalNAc).

In some embodiments, the targeting group of the present invention comprises the following structure:

wherein the wavy line indicates the position of connection with the joint. Typically, the targeting group is attached to the linker in the form of an amide bond.

In some embodiments, the siRNA conjugate of the invention further comprises a linker, said siRNA, said linker and said targeting group being, in turn, covalently or non-covalently linked.

In some embodiments, the linker of the invention is selected from the group consisting of:

wherein the wavy line indicates the attachment position of the linker to the rest of the siRNA conjugate, wherein the targeting group is covalently linked to the carbonyl group of the linker and the siRNA is covalently linked to the O at the other end of the linker via a phosphate bond.

In some embodiments, the siRNA is linked through the 3' end of its sense strand to a targeting group or linker via a phosphono group. In some embodiments, the targeting group is amide-bonded to the linker.

The invention discloses a siRNA conjugate for delivering siRNA or active molecule groups. In some embodiments, the siRNA conjugates disclosed herein facilitate tissue-specific targeting. In some embodiments, the targeting groups disclosed herein bind to a cell surface receptor. Thus, any cell surface receptor or biomarker, or targeting group corresponding to a portion thereof, is contemplated as suitable for use in the present invention.

Specifically, the aforementioned conjugates for targeted delivery of siRNA to liver include, but are not limited to, the structural examples of the compounds in table a, wherein R is ² Is an siRNA as defined herein.

Table a: examples of siRNA conjugates

The compounds of the present invention include, but are not limited to, optical isomers, racemic compounds and other mixtures thereof. In these cases, single enantiomers or diastereomers, i.e., optically active configurations, can be obtained by asymmetric synthesis or chiral resolution. Resolution of the racemates can be accomplished, for example, by conventional methods, such as recrystallization in the presence of a resolving agent, or using, for example, chiral High Pressure Liquid Chromatography (HPLC) column chromatography. In addition, some compounds containing carbon-carbon double bonds have Z-and E-configurations (or cis-and trans-configurations). When tautomerism exists in the compounds of the present invention, the term "compound" (including conjugates) includes all tautomeric forms of the compounds. Such compounds also include crystals and chelates. Similarly, the term "salt" includes all tautomeric forms of the compound and crystal forms of the compound. The structures shown in Table 1, the amino acid residues being derived from L-amino acids, D-amino acids, dl-amino acids, and any combination thereof, the specific structures disclosed above do not limit the configuration of the specific amino acids.

In some embodiments, R ² Comprising any one of the antisense strand nucleotide sequences shown in Table 1 or 2, and any one of the sense strand nucleotide sequences shown in Table 1 or 2.

In some embodiments, R ² Comprising the sense strand and antisense strand nucleotide sequences of any one of the siRNAs shown in Table 2. Preferably, the siRNA is selected from the group consisting of the siRNAs as shown in any of the siRNA numbers in Table 2.

In some embodiments, R ² Comprises the amino acid sequence of SEQ ID NO:59 and the sense strand sequence shown in SEQ ID NO:60, or a pharmaceutically acceptable salt thereof:

S1

sense strand mC mU mAMMcCfUGfmUTmUmGmCmUmUmUmGmU (SEQ ID NO: 59); the antisense strand mA x Cf x mAAfAfmGCfmAAfmAAfmCAfmGGfUCfUmAMmG x mA (SEQ ID NO: 60).

In some embodiments, the phosphono group in the table a structural formula is attached to the 3' end of the siRNA sense strand.

The invention also provides a pharmaceutical composition comprising an siRNA or siRNA conjugate described herein, and a pharmaceutically acceptable carrier thereof.

In yet another aspect, the present invention provides a pharmaceutical combination for inhibiting PCSK9 gene expression, comprising an siRNA, an siRNA conjugate or a pharmaceutical composition thereof described herein, and one or more additional therapeutic agents for inhibiting PCSK9 gene expression.

In yet another aspect, the invention provides the use of an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein for the manufacture of a medicament for the treatment and/or prevention of a PCSK 9-mediated disease or disorder.

In yet another aspect, the present invention provides a method of treating and/or preventing a PCSK 9-mediated disease or disorder, wherein the method comprises administering to a subject in need thereof an siRNA, an siRNA conjugate, a pharmaceutical composition, or a pharmaceutical combination as described herein.

In yet another aspect, the invention provides an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein for use in the treatment and/or prevention of a PCSK 9-mediated disease or disorder.

In yet another aspect, the invention provides a method of inhibiting PCSK9 gene expression in a subject, the method comprising administering to a subject in need thereof an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein.

In some embodiments, the diseases or conditions described herein include atherosclerosis, hypercholesterolemia, hypertriglyceridemia, acute coronary syndrome, dyslipidemia, myocardial infarction, coronary artery disease, stroke, coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type ii diabetes, and kidney disease.

Drawings

FIG. 1: effect of

siRNA conjugates

1 and 5 on PCSK9 protein levels in plasma in mice.

FIG. 2: structural formula of positive control InclissiRNA.

Detailed Description

Definition of

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined as follows. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, including the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In addition, it should be noted that whenever a value or range of values of a parameter is recited, it is intended that the value or range of values recited is also part of the invention.

The term "about," when used in conjunction with numerical values, is intended to encompass numerical values within the range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value, including, but not limited to, ± 5%, ± 2%, ± 1% and ± 0.1%, as such variations are suitable for carrying out the disclosed methods.

The term "and/or" should be understood to mean any one of the options or a combination of any two or more of the options.

The term "or" is to be understood as having the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" and/or "should be interpreted as being inclusive, i.e., including at least one of a quantity or a list of elements, but also including more than one, and optionally, additional unlisted items. Only if the contrary terms are explicitly indicated, such as "only one" or "exactly one" or "consisting of … …" are used in the claims, it will refer to only one number listed or one element of a list.

As used herein, the terms "a" and "an" should be understood to mean "at least one" unless explicitly indicated to the contrary.

The term "including" or "comprising" is intended to be used interchangeably with the phrase "including, but not limited to".

The term "at least" preceding a number or series of numbers is to be understood to include the numbers adjacent to the term "at least" as well as all subsequent numbers or integers that may be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified properties. When at least one series of numbers or range precedes, it is understood that at least each number in the series or range can be modified.

The term "PCSK9" refers to proprotein convertase subtilisin Kexin9 gene or protein. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARCl. The term PCSK9 includes human PCSK9, the amino acid and nucleotide sequence of which can be found, for example, in GenBank accession number GI:299523249 (SEQ ID NO: 1); mouse PCSK9, the amino acid and nucleotide sequence of which can be found, for example, in GenBank accession No. GI: 163644257; rat PCSK9, the amino acid and nucleotide sequence of which can be found, for example, in GenBank accession No. GI: 77020249; additional examples of PCSK9mRNA sequences are conveniently obtained using publicly available databases, e.g., genBank, uniProt, and OMIM.

The term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that is capable of reducing or inhibiting expression of a target gene or sequence when the interfering RNA is in the same cell as the target gene or sequence (e.g., by mediating degradation and inhibition of translation of mRNA complementary to the interfering RNA sequence). Interfering RNA thus refers to single-stranded RNA complementary to a target mRNA sequence or double-stranded RNA formed from two complementary strands or from a single self-complementary strand.

Interfering RNAs include "small interfering RNAs" or "siRNAs" in which each strand of the siRNA comprises from about 15 to about 60 nucleotides (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, 17-25, 19-25, 17-23, 17-21, 19-23, or 19-21 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). Ranges and length intermediate values to the ranges and lengths recited above are also contemplated as part of the invention. In a specific embodiment, the siRNA is chemically synthesized. The sirnas of the invention are capable of silencing expression of a target sequence in vitro and/or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region.

As used herein, the term "dsRNA" or "precursor RNAi molecule" is intended to include any precursor molecule that is processed in vivo by an endonuclease to produce an active siRNA.

The term "siRNA" refers to a molecule that contains siRNA as that term is defined herein and which mediates targeted cleavage of RNA transcripts by an RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). The iRNA modulates (e.g., inhibits) expression of PCSK9 in a cell (e.g., a cell within a subject, such as a mammalian subject).

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

The term "antisense strand" refers to a strand of an iRNA (e.g., an siRNA) that includes a region that is substantially complementary to a target sequence (e.g., a PCSK9 mRNA). As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence. Where the complementary region is not fully complementary to the target sequence, there may be mismatches within or at terminal regions of the molecule. Typically, the most tolerated mismatches are present in the terminal regions, e.g., within 5, 4,3, or 2 nucleotides of the 5 '-and/or 3' -terminus of the siRNA.

The term "sense strand" or "sense strand" means a strand of an iRNA that comprises a region that is substantially complementary to a region of an antisense strand as defined herein.

The antisense and sense strands of the siRNA can be of the same or different lengths, as described herein and as known in the art.

As used herein, and unless otherwise specified, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, the term "complementary" refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence. Such conditions may for example be stringent conditions, wherein stringent conditions may comprise: 400mM NaCl,40mMPIPES, pH 6.4,1mM EDTA,50 ℃ or 70 ℃ for 12-16 hours.

The term "conjugate" (also sometimes referred to as conjugate, coupling, conjugate, also sometimes referred to in the literature as conjugate) as used herein corresponds to "conjugate" or "conjugates" in the english language. The conjugate is a new compound generated after two or more molecules of the compound are covalently linked (coupled) through bivalent or multivalent compound molecules with linking function. Conjugates can also be formed from two molecules directly via coupling or condensation. The common antibody-drug conjugate (ADC) is a conjugate, also known as an antibody drug conjugate. In some embodiments, the product resulting from coupling the siRNA molecule with the targeting group via a linker is also a conjugate or an siRNA conjugate. In some embodiments, the product produced by coupling the siRNA and targeting group together is an siRNA conjugate.

The term "coupled" as used herein refers to a chemical process in which two or more molecules of a compound undergo some reaction to form a new chemical bond and a new molecule. In certain contexts, "coupled" may be used interchangeably with "connected" or in place of one another.

The term "carbohydrate" refers to a monosaccharide, disaccharide, trisaccharide or polysaccharide.

The term "monosaccharide" includes allose, maltose, arabinose, cladinose, brown sugar, erythrose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, galactosamine, D-galactosamine, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucamine alcohol, glucose-6-phosphate, glucose glyceraldehyde, L-glycerol-D-mannose-heptose, glycerol, glucose, iodine, lyxose, mannosamine, mannose-6-phosphate, psicose, isorhamnese (quinovose), quinovosamine (quinovosamine), rhamnose, ribose, ribulose, glycoheptose, sorbose, talose, tartaric acid, threose, xylose and xylose. The monosaccharide may be in the D-or L-configuration. The monosaccharide may also be a deoxy sugar (alcoholic hydroxyl group substituted with hydrogen), an amino sugar (alcoholic hydroxyl group substituted with amino), a thio sugar (alcoholic hydroxyl group substituted with thiol), or CO substituted with CS, or a cyclic epoxy substituted with sulfur), a seleno sugar, a tellurose, an aza sugar (ring carbon substituted with nitrogen), an iminosugar (epoxy substituted with nitrogen), a phosphoro sugar (epoxy substituted with phosphorus), a phosphoro sugar (ring carbon substituted with phosphorus), a C-substituted monosaccharide (hydrogen on a non-terminal carbon atom substituted with carbon), an unsaturated monosaccharide, a sugar alcohol (carbonyl substituted with a CHOH group), an aldonic acid (aldehyde group substituted with carboxyl), a ketoaldonic acid, an uronic acid, an aldonic acid, or the like. Amino sugars include amino monosaccharides, preferably galactosamine, glucosamine, mannosamine, fucosamine, quinovosamine, neuraminic acid, muramic acid, lactosamine, acosamine, bacillosamine, daunomamine, desosamine, fu Luo Shaming, aminocarboxamide, carnosamine, mannosamine, trehalose, mycophenolide, peroxidase, pneumococcal amine, purinoceramine, rhodamine. It is understood that monosaccharides and the like may be further substituted.

The terms "disaccharide", "trisaccharide" and "polysaccharide" include Abbe quinone sugar, aclacinose, glucosamine, amylopectin, amylose, apiose, glucosamine, ascose, ascorbic acid, flavone sugar, cellobiose, cellotriose, cellulose, chalcone trisaccharide, thioether, chitin, collagen, cyclodextrin, melamine, dextrin, 2-deoxyribose, 2-deoxyglucose, diglucose, maltose, digital ketose, EVALOSE, evodia, fructo-oligosaccharide, galacto-oligosaccharide, gentiobiose, dextran, glycogen, hamamelose, heparin, inulin, isoevodiamine, isomaltose, isomaltotriose, isopentyl sugar, curdlan, lactose, lactosamine, lactodiamine, layered arabinose, levoglucosan ketone, -maltose, mannooligosaccharide, mannotriose, maltose, melibiose, muramic acid, trehalose, neuraminic acid, black glucose, nogeniclin, sophorose, stachyose, streptococcal sugar, sucrose, trehalose, and, furthermore, it is understood that "disaccharide", "trisaccharide" and "polysaccharide" and the like may be further substituted. Disaccharides also include amino sugars and derivatives thereof, particularly the mycoamine sugars derivatized at the C-4 'position or the 4-deoxy-3-amino-glucose derivatized at the C-6' position.

The phrase "inhibiting PCSK9 gene expression" as used herein comprises inhibiting the expression of any PCSK9 gene (such as, e.g., a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene) as well as variants or mutants of the PCSK9 gene encoding a PCSK9 protein.

"inhibiting PCSK9 gene expression" encompasses any level of PCSK9 gene inhibition, e.g., at least partial inhibition of PCSK9 gene expression, such as at least about 20% inhibition. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

PCSK9 gene expression can be assessed based on the level of any variable associated with PCSK9 gene expression, e.g., PCSK9mRNA levels or PCSK9 protein levels. PCSK9 gene expression can also be assessed indirectly based on the levels of serum lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer only control or inert agent control) treated subject, cell, or sample.

"subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

"therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an siRNA of the invention that is effective to prevent or ameliorate one or more symptoms of a disease or condition or the development of the disease or condition when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When an individual is administered a single administration of an active ingredient, a therapeutically effective dose refers to that ingredient alone. When administered in combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of the therapeutic agent will result in an increase in a diagnostic standard or parameter of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

The term "treating" or "treatment" means that a favorable or desired result includes, such as reducing the level of triglycerides in the subject. The term "treating" or "treatment" also includes, but is not limited to, alleviation or amelioration of one or more symptoms of a lipid metabolism disorder, such as, for example, a reduction in the size of eruptive xanthomas. "treatment" also refers to an extended survival as compared to the expected survival without treatment.

The term "preventing" or "prevention," when used in reference to a disease, disorder, or condition thereof that would benefit from a reduction in PCSK9 gene expression, means reducing the likelihood that the subject will develop symptoms associated with such disease, disorder, or condition, e.g., high triglyceride levels or xanthoma. A reduced likelihood of developing high triglyceride levels or xanthoma is, for example, when an individual has one or more risk factors for high triglyceride levels or xanthoma either fails to develop high triglyceride levels or xanthoma or develops high triglyceride levels or xanthoma of lower severity relative to a population having the same risk factors but not receiving treatment as described herein. Failure to develop a disease, disorder, or condition, or reduced development of symptoms associated with the disease, disorder, or condition (e.g., at least about 10% reduction in clinically acceptable proportions for the disease or condition), or exhibiting a delay in symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prophylaxis.

The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (directed to carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body).

SiRNA and modified nucleotide

Provided herein are sirnas for inhibiting PCSK9 gene expression. Each siRNA comprises a sense strand and an antisense strand. The sense and antisense strands can each be 17-25 nucleotides in length. The sense and antisense strands may be the same length, or they may be different lengths. In some embodiments, the sense and antisense strands are each independently 17-25 nucleotides in length. In some embodiments, the sense and antisense strands are each independently 19-23 nucleotides in length. In some embodiments, the sense strand is 19-21 nucleotides in length and the antisense strand is 19-23 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length and the antisense strand is about 20 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length and the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 20 nucleotides in length and the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 20 nucleotides in length and the antisense strand is about 22 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 22 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. In some embodiments, the siRNA sense and antisense strands are each independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the duplex of the siRNA has about 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, the sense and antisense strands have a length of at least 15, 16, 17, 18, 19, 20, or 21 nucleotides complementary.

Examples of nucleotide sequences used to form sirnas are provided in tables 1,2, respectively.

In some embodiments, the antisense strand of the sirnas disclosed herein differs from any of the antisense strand sequences shown in table 1 or 2 by 0, 1,2, or 3 nucleotides. In some embodiments, the sense strand of the PCSK9 sirnas disclosed herein differs from any of the sense strand sequences set forth in table 1 or 2 by 0, 1,2, or 3 nucleotides.

In some embodiments, the siRNA antisense strand comprises or consists of any of the antisense strand nucleotide sequences shown in table 1 or 2. In some embodiments, the siRNA antisense strand comprises the sequence at positions 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24 or 2-24 of the nucleotide sequence of any of the antisense strands in Table 1 or 2 (from the 5 'end → the 3' end). In certain embodiments, the siRNA antisense strand comprises or consists of a modified sequence of any one of the sequences in table 1 or 2.

In some embodiments, the siRNA sense strand comprises or consists of any one of the sense strand nucleotide sequences shown in table 1 or 2. In some embodiments, the siRNA sense strand comprises a sequence from position 1-17, position 2-15, position 2-17, position 1-18, position 2-18, position 1-19, position 2-19, position 1-20, position 2-20, position 1-21, position 2-21, position 1-22, position 2-22, position 1-23, position 2-23, position 1-24 or position 2-24 of the nucleotide sequence of any of the sense strands in Table 1 or 2 (from the 5 'end → the 3' end). In certain embodiments, the siRNA sense strand comprises or consists of a modified sequence of any one of the sequences in tables 1, 2.

In some embodiments, the siRNA is prepared or provided as a salt, a mixed salt, or a free acid. In some embodiments, the siRNA is prepared as a sodium salt. Such forms, which are well known in the art, are within the scope of the invention disclosed herein.

In some embodiments, the siRNA contains one or more modified nucleotides. In some embodiments, the modified nucleotides include, but are not limited to: 2' -O-methyl modified nucleotides, 2' -fluoro, 2' -deoxy modified nucleotides, 2' -deoxy nucleotides, 2' -methoxyethyl modified nucleotides, 2' -amino modified nucleotides, 2' -alkyl modified nucleotides, 2' -alkoxy modified nucleotides, 2' -F-arabinonucleotides, abasic nucleotides, morpholino nucleotides and locked nucleotides. It is not necessary that all positions in a given compound be uniformly modified. Rather, more than one modification can be added to a single siRNA or even to a single nucleotide thereof. The modification of one nucleotide is independent of the modification of another nucleotide.

In some embodiments, all or substantially all of the nucleotides of the siRNA are modified nucleotides. In some embodiments, the siRNA is an unmodified nucleotide having 4 or fewer (i.e., 0, 1,2, 3, or 4) nucleotides in both the sense and antisense strands. In some embodiments, the sense strand of the siRNA comprises two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified nucleotides. In some embodiments, the antisense strand of the siRNA comprises two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified nucleotides. In some embodiments, one or more nucleotides of the siRNA are unmodified ribonucleotides.

In some embodiments, one or more nucleotides of the siRNA are linked by a non-standard linkage or backbone (i.e., a modified internucleoside linkage or a modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, chiral phosphonates, phosphinates, phosphoramidates, thioalkyl phosphonates, thioalkyl phosphotriesters, morpholino linkages wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

In some embodiments, the sense strand of the siRNA may contain 1,2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides) and the antisense strand of the siRNA may contain 1,2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides). In some embodiments, the sense strand of the siRNA may contain 1 or 2 phosphorothioate linkages and the antisense strand of the siRNA may contain 1,2, 3, or 4 phosphorothioate linkages.

In some embodiments, the sense strand of the siRNA contains 2 phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 1-3 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 1-3 from the 3' terminus of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is located at the 5 'end of the sense strand and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand of the siRNA contains 1 phosphorothioate internucleoside linkage. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 1-2 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 2-3 from the 5' end of the sense strand. In some embodiments, the targeting ligand is linked to the sense strand by a phosphorothioate linkage.

In some embodiments, the siRNA antisense strand contains 4 phosphorothioate internucleoside linkages. In some embodiments, the 4 phosphorothioate internucleoside linkages are between the nucleotides 1 to 3 from the 5 'terminus and between the nucleotides 1 to 3 from the 5' terminus of the antisense strand. In some embodiments, the siRNA antisense strand contains 3 phosphorothioate internucleoside linkages. In some embodiments, 3 phosphorothioate internucleoside linkages are located between the nucleotides 1-2 from the 5 'terminus and between the nucleotides 1-3 from the 3' terminus of the antisense strand, respectively. In some embodiments, 3 phosphorothioate internucleoside linkages are located between the nucleotides 1-3 from the 5 'terminus and between the nucleotides 1-2 from the 3' terminus of the antisense strand, respectively. In some embodiments, the siRNA antisense strand contains 2 phosphorothioate internucleoside linkages. In some embodiments, the 2 phosphorothioate internucleoside linkages are between the nucleotides 1-2 from the 5 'terminus and between the nucleotides 1-2 from the 5' terminus of the antisense strand.

In some embodiments, the siRNA antisense strand comprises the nucleotide (from 5 'end → 3' end) sequence of any one of the antisense strand sequences in table 1 or 2. In some embodiments, the siRNA sense strand comprises the nucleotide (from 5 'end → 3' end) sequence of any one of the sense strands in table 1 or 2. In some embodiments, the antisense strand of the siRNA comprises the nucleotide (from 5 'terminus → 3' terminus) sequence of any of the antisense strands in table 1 or 2, and the sense strand comprises the nucleotide (from 5 'terminus → 3' terminus) sequence of any of the sense strands in table 1 or 2.

The nucleotide abbreviations herein are as follows:

a = adenosine-3' -phosphate ester

C = cytidine-3' -phosphate

G = guanosine-3' -phosphate

U = uridine-3' -phosphate

mA =2 '-O-methyladenosine-3' -phosphate

mA =2 '-O-methyladenosine-3' -phosphorothioate

mC =2 '-O-methylcytidine-3' -phosphate

mC =2 '-O-methylcytidine-3' -phosphorothioate

mG =2 '-O-methylguanosine-3' -phosphate

mG =2 '-O-methylguanosine-3' -phosphorothioate

mU =2 '-O-methyluridine-3' -phosphate

mU =2 '-O-methyluridine-3' -phosphorothioate

Nf = any 2' -fluoro modified ribonucleotide

Af =2 '-fluoroadenosine-3' -phosphate ester

Af =2 '-fluoroadenosine-3' -phosphorothioate

Cf =2 '-fluorocytidine-3' -phosphate ester

Cf =2 '-fluorocytidine-3' -phosphorothioate

Gf =2 '-fluoroguanosine-3' -phosphate

Gf =2 '-fluoroguanosine-3' -phosphorothioate

Tf =2' -fluoro-5 ' -methyluridine-3 ' -phosphate

Tf =2' -fluoro-5 ' -methyluridine-3 ' -phosphorothioate

Uf =2 '-fluorouridine-3' -phosphate

Uf =2 '-fluorouridine-3' -phosphorothioate

dN = any 2' -deoxyribonucleotide

dA =2 '-deoxyadenosine-3' -phosphate ester

dA =2 '-deoxyadenosine-3' -phosphorothioate

dC =2 '-deoxycytidine-3' -phosphate ester

dC × =2 '-deoxycytidine-3' -phosphorothioate

dG =2 '-deoxyguanosine-3' -phosphate

dG =2 '-deoxyguanosine-3' -phosphorothioate

dT =2 '-deoxythymidine-3' -phosphate

dT =2 '-deoxythymidine-3' -phosphorothioate

dU =2 '-deoxyuridine-3' -phosphate ester

dU =2 '-deoxyuridine-3' -phosphorothioate

N = any ribonucleotide

Note: the sequences in the electronic sequence listing show only the base sequence, and their modifications are not shown. The modifications in the siRNA sequences with modified bases and the sequences are shown in Table 2 in their entirety.

Table 1: nucleotide sequences of sense strand and antisense strand of siRNA

Note: NC is negative control.

The sense and antisense strands of the siRNA comprising or consisting of the sequences in table 1 may be modified nucleotides or unmodified nucleotides. In some embodiments, all or substantially all of the sirnas having sense and antisense strand sequences comprising or consisting of the sequences in table 1 are modified nucleotides.

In some embodiments, the antisense strand of the sirnas disclosed herein differs from any of the antisense strand sequences in table 1 by 0, 1,2, or 3 nucleotides. In some embodiments, the sense strand of the sirnas disclosed herein differs from any of the sense strand sequences in table 1 by 0, 1,2, or 3 nucleotides.

In some embodiments, the sense strand of the siRNA of the invention comprises the sequence of any one of the sense strands of table 1 and the antisense strand comprises the sequence of any one of the antisense strands of table 1; preferably, the siRNA of the present invention is selected from the group consisting of the siRNAs as shown in any one of the siRNA numbers in Table 1.

In some embodiments, the sense strand of the siRNA comprises or consists of a modified sequence of any one of the sequences in table 1. In some embodiments, the siRNA antisense strand comprises or consists of a modified sequence of any one of the sequences in table 1.

The antisense and sense strand nucleotide sequences of certain modified sirnas are provided in table 2. In forming the siRNA, each of the nucleotides listed in table 2 above as well as table 1 can be a modified nucleotide.

Table 2 provides examples of sense and antisense strands containing modified nucleotides.

Table 2: siRNA modified sense strand and antisense strand nucleotide sequences

In some embodiments, the antisense strand of the sirnas disclosed herein differs from any one of the antisense strand sequences in table 2 by 0, 1,2, or 3 nucleotides. In some embodiments, the sense strand of the sirnas disclosed herein differs from any one of the sense strand sequences in table 2 by 0, 1,2, or 3 nucleotides.

In some embodiments, the siRNA antisense strand comprises any one of the antisense strand nucleotide sequences in table 2. In some embodiments, the siRNA antisense strand comprises the sequence of nucleotides (from 5 'terminus → 3' terminus) from positions 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24 or 2-24 of any of the sequences in table 2. In certain embodiments, the siRNA antisense strand comprises or consists of a modified sequence of any one of the antisense strand sequences in table 2.

In some embodiments, the siRNA sense strand comprises any one of the sense strand nucleotide sequences in table 2. In some embodiments, the sense strand of the siRNA comprises the sequence at positions 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 of the nucleotides of any of the sequences in table 2 (from the 5 'end → the 3' end). In certain embodiments, the siRNA sense strand comprises or consists of a modified sequence of any one of the sense strand sequences in table 2.

In some embodiments, the siRNA antisense strand comprises any one of the antisense strand nucleotide sequences in table 2 and the siRNA sense strand comprises any one of the sense strand nucleotide sequences in table 2.

In some embodiments, the siRNA comprises, consists of, or consists essentially of a duplex represented by any one of the siRNA numbers presented herein. In some embodiments, the siRNA comprises the sense strand and antisense strand nucleotide sequences of any duplex represented by any siRNA number presented herein. In some embodiments, the siRNA comprises the sense strand and antisense strand nucleotide sequences of any duplex represented by any siRNA number presented herein and a targeting group and/or linking group, wherein the targeting group and/or linking group is covalently linked (i.e., conjugated) to the sense strand or antisense strand. In some embodiments, the siRNA comprises sense and antisense strand modified nucleotide sequences of any of the siRNA numbers presented herein. In some embodiments, the siRNA comprises sense and antisense strand modified nucleotide sequences and targeting groups and/or linking groups of any of the siRNA numbering presented herein, wherein the targeting groups and/or linking groups are covalently linked to the sense or antisense strand.

In some embodiments, the siRNA comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense/sense strand duplexes of table 1 or table 2, and further comprises a targeting group. In some embodiments, the siRNA comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense/sense strand duplexes of table 1 or table 2, and further comprises a biological ligand group targeted for recognition.

siRNA conjugates

The invention also provides an siRNA conjugate which comprises the siRNA and a targeting group for targeting identification. In some embodiments, the siRNA conjugate comprises a targeting group for targeted recognition and a linker for coupling the two directly or indirectly. The siRNA conjugate recognizes a receptor on the cell surface through a targeting group, thereby delivering the siRNA to a target.

In some embodiments, the siRNA conjugate comprises a pharmaceutically acceptable targeting group and optionally a linker (linker), and the selected siRNA, the linker and the targeting group are linked sequentially. The siRNA molecule may be non-covalently or covalently bound to the linker, e.g., may be covalently linked to the linker.

In some embodiments, the siRNA is attached to the targeting group without the use of a linker. In some embodiments, the targeting group itself is designed with a linker or other site to facilitate facile coupling.

In some embodiments, the linker is attached to the end of the sense or antisense strand of the siRNA, which refers to the first 4 nucleotides from one end of the sense or antisense strand. In some embodiments, the linker is attached to the 3 'end or the 5' end of the sense strand of the siRNA. In some embodiments, the linker is attached to the 3 'end or the 5' end of the antisense strand of the siRNA. In some embodiments, the linker is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, the pharmaceutically acceptable targeting group of the present invention can comprise ligands conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607, the entire disclosure of which is incorporated herein by reference.

In some embodiments, examples of linkers can include (but are not limited to): reactive groups (such as primary amines and alkynes), alkyl groups, abasic nucleotides, ribitol (abasic ribose) and/or PEG groups. In some embodiments, the linkers of the present invention can comprise linkers known in the art of siRNA administration.

A linker of the invention is a linkage between two atoms that links one chemical group of interest (such as an siRNA) or fragment to another chemical group of interest (such as a targeting group) or fragment via one or more covalent bonds. The labile linkage contains a labile bond. The attachment may optionally include a spacer that increases the distance between the two attached atoms. The spacer may further increase the flexibility and/or length of the connection. Spacers include, but are not limited to, alkyl (e.g., C1-12 straight or linear alkyl), alkenyl (e.g., C2-C8 straight or branched alkenyl), alkynyl (e.g., C2-C8 straight or branched alkynyl), aryl (e.g., C6-14 aryl), aralkyl (e.g., C6-14 arylC 1-12 alkyl), aralkenyl (e.g., C6-14 arylC 2-C8 alkenyl), and aralkynyl (e.g., C6-14 arylC 2-C8 alkynyl); each of which can contain one or more heteroatoms (e.g., N, O and S), heterocycles (e.g., heterocyclyl or heteroaryl containing one or more heteroatoms selected from O, S and N), amino acids, nucleotides, and carbohydrates. The linker contains groups at both ends suitable for attachment (e.g., covalent attachment) to a targeting group as well as to the siRNA. Thus, in some embodiments, the linker of the invention has the following structure: linker 1-spacer-linker 2, which linker 1 may be a group for attachment to a targeting group, such as a carbonyl group for forming an amide group with a targeting group; the linking group 2 can be a group for forming a covalent linkage with the siRNA, such as-O-, thereby forming a linkage with, for example, the phosphate group at the 3' end of the sense strand of the siRNA, and the spacer serves to increase the flexibility and/or length of the linkage.

In some embodiments, the targeting moieties of the present invention bind to a cell surface receptor. For this purpose, any cell surface receptor or biomarker or part thereof is considered suitable. In some embodiments, the bioligand group of the invention specifically binds to a specific receptor of a particular tissue, thereby achieving tissue-specific targeting. In some embodiments, the targeting group of the present invention specifically targets hepatocyte surface receptors and thus specifically targets liver tissue. In some embodiments, the targeting groups of the present invention specifically target cell surface receptors specific for hepatocytes. In some embodiments, the targeting moiety of the present invention comprises (GalNAc), specifically targeting asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes.

In some embodiments, the siRNA conjugates of the present invention have excellent liver targeting specificity, and thus can efficiently deliver the conjugated siRNA to the liver, thereby effectively regulating the expression of a specific gene in the liver cell. Therefore, the siRNA conjugate has wide application prospect.

Pharmaceutical compositions and formulations

The invention also provides a pharmaceutical composition comprising an siRNA or siRNA conjugate described herein, and a pharmaceutically acceptable carrier thereof. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents and/or delivery polymers) are added to a pharmaceutical composition comprising siRNA, thereby forming a pharmaceutical formulation or pharmaceutical composition suitable for in vivo delivery to a subject (including a human).

The pharmaceutical compositions used herein comprise a pharmacologically effective amount of at least one of the therapeutic compounds and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, e.g. PCSK9 siRNA). Excipients are not, or are not intended to, exert a therapeutic effect at the intended dosage. Excipients may serve the following functions: a) Facilitating handling of the drug delivery system during manufacture, b) protecting, supporting or enhancing stability, bioavailability or patient acceptability of the API; c) The product identification is facilitated; and/or d) any other attribute that enhances the overall security, effectiveness, or delivery of the API during storage or use.

Excipients include (but are not limited to): absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextrans, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

In yet another aspect, the present invention provides a pharmaceutical combination for inhibiting PCSK9 gene expression, comprising an siRNA, an siRNA conjugate or a pharmaceutical composition thereof described herein, and one or more additional therapeutic agents for inhibiting PCSK9 gene expression. In some embodiments, the additional therapeutic agent can be a small molecule drug, an antibody fragment, or an siRNA.

Method and use

The invention provides use of an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein in the manufacture of a medicament for the treatment and/or prevention of a PCSK 9-mediated disease or condition.

The present invention provides a method of treating and/or preventing a PCSK 9-mediated disease or disorder, wherein the method comprises administering to a subject in need thereof an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein.

The present invention provides an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein for use in the treatment and/or prevention of a PCSK 9-mediated disease or disorder.

The present invention provides a method of inhibiting PCSK9 gene expression in a subject, the method comprising administering to a subject in need thereof an siRNA, an siRNA conjugate, a pharmaceutical composition or a pharmaceutical combination as described herein.

The invention provides a method of inhibiting expression of a target gene in a cell, the method comprising contacting the cell with a conjugate disclosed herein, or a pharmaceutical composition thereof, for a time sufficient to achieve degradation of an mRNA transcript of the target gene, thereby inhibiting expression of the target gene in the cell. In some embodiments, expression of the target gene is inhibited by at least 30%, at least about 40%, at least about 50%, at least about 60%, or at least 70%.

In some embodiments, the diseases or conditions of the present invention include atherosclerosis, hypercholesterolemia, hypertriglyceridemia, acute coronary syndrome, dyslipidemia, myocardial infarction, coronary artery disease, stroke, coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type ii diabetes, and renal disease.

In some embodiments, the subject has hypercholesterolemia, dyslipidemia, or hyperlipidemia. In some embodiments, the serum cholesterol level of the subject is reduced following administration of a compound or composition described herein.

The invention also provides a method for treating and preventing high cholesterol. In some embodiments, the methods further comprise the step of determining the serum cholesterol level of the subject. Serum cholesterol levels may be determined before, during and/or after said administration.

In some embodiments of the methods of treatment and prevention provided herein, the subject is a mammal, e.g., a primate, rodent, or human.

In some embodiments of the methods of treatment and prevention provided herein, administration of an siRNA conjugate or pharmaceutical composition of the invention results in a reduction in serum cholesterol in the subject.

In some embodiments of the therapeutic and prophylactic methods provided herein, the siRNA, siRNA conjugate, pharmaceutical composition or pharmaceutical combination of the invention is administered parenterally (e.g., subcutaneously, intramuscularly or intravenously) in the form of an injection or infusion solution. In one embodiment, the siRNA, siRNA conjugate, pharmaceutical composition or pharmaceutical combination of the invention is administered subcutaneously.

It is clear to the skilled person that modified nucleotide groups can be introduced into the sirnas according to the invention by using nucleoside monomers with corresponding modifications: methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared by known methods.

The invention in some embodiments utilizes phosphono groups to link the siRNA to the linker. It will be appreciated that the manner of attachment or coupling of such linkers to the siRNA is varied and includes, but is not limited to, direct and indirect coupling, and that indirect coupling may be via a variety of carrier groups and attachment means suitable for coupling.

The invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of applicability will become apparent from the detailed description provided hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are hereby incorporated by reference in their entirety for all purposes.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.

Examples

The invention is illustrated by the following examples, but is not intended to be limiting in any way. Having described the invention in detail, specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the invention.

Example 1: siRNA synthesis

1.1 target sequence screening

siRNA was designed based on PCSK9 whole mRNA sequence, all of which were derived from the NCBI GenBank (https:// www.ncbi.nlm.nih.gov/gene /). All siRNAs were designed to ensure complete identity to human (Gene ID: 255738) and cynomolgus monkey (Gene ID: 102142788) sequences.

The whole sequence is scanned to obtain all potential siRNA sequences with 19-21 nucleotide length, and the sequences of the cynomolgus monkey are simultaneously compared to ensure the matching of the sequences of the cynomolgus monkey. All human/cynomolgus binding sequences were compared to the human full transcriptome mRNA sequence by BLAST and all sirnas that could have off-target effects were removed. Finally, the activity of all siRNA is evaluated by the principle of rational siRNA design, and the molecules with lower theoretical activity are removed.

1.2siRNA Synthesis

After the siRNA design is finished, the siRNA is synthesized by the Kinsley Biotechnology Ltd and annealed to obtain the required siRNA, and the siRNA sequence is shown in Table 1.

Example 2: in vitro Activity assay

2.1 fluorescent quantitative PCR

2.1.1 cell culture and transfection

HepG2 cells or Huh-7 cells (ATCC) were treated with 5% CO using DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, gibco) and double antibody (Gibco) ₂ The cells were cultured at 37 ℃ and resuspended after trypsinization after the growth of the cells was nearly complete coverage of the flask. Resuspended cells were density adjusted and tested at 2X10 ⁴ Per well was inoculated into 96-well plates and siRNA transfection complexes were added in suspension. The siRNA transfection complex was obtained by mixing 0.3 ul/well Liposomal RNAi Max (Thermo) MaxOpti-MEM (Gibco) with siRNA 1:1. After culturing the cells for a certain period of time, dynabeads were used according to the instructions ^TM Extracting with mRNA separation kit (Thermo) to obtain whole mRNA. In the final step of mRNA elution, the TIANGEN first strand synthesis kit was used, which contained 5.2ul 5 Xbuffer, 1.3ul dNTPs (deoxyribonucleic acid triphosphate) and ddH ₂ O make up volume to 19.5ul, 5min at 80 ℃ and quickly transfer 15ul of supernatant on a magnetic stand. The heating process was performed by a Thermo Veriti 96well Thermal Cycler PCR instrument.

Primary cynomolgus monkey hepatocytes were obtained from Miaotong (Shanghai) Biotech Co., ltd and used in combination at 4 ℃Resuscitating in a resuscitating medium, washing, resuspending in a maintenance medium and adjusting to a density of 3X10 ⁴ The wells were inoculated into 96-well plates coated with mating coating media. And adding the siRNA transfection compound after the cells are completely attached to the wall. After a certain period of cell culture Dynabeads were used according to the instructions ^TM mRNA isolation kit (Thermo) extracts whole mRNA. In the final step of mRNA elution, the TIANGEN first strand synthesis kit was used, containing 5.2ul 5 Xbuffer, 1.3ul dNTPs and ddH ₂ O make up volume to 19.5ul, 5min at 80 ℃ and quickly transfer 15ul of supernatant on a magnetic stand. The heating process was performed by a Thermo Veriti 96well Thermal Cycler PCR instrument.

2.1.2 Reverse transcription of cDNA

cDNA Synthesis Using the TIANGEN first Strand Synthesis kit, to 15ul of the supernatant removed in the above example 2.1.1, 2ul of oligo dT (poly-deoxythymidine), 1ul of reverse transcriptase, 0.5ul of RNase inhibitor and H ₂ The O supplement volume is 20ul. cDNA synthesis was carried out using a Thermo Veriti 96well Thermal Cycler PCR instrument at 42 ℃ for 60 minutes, 85 ℃ for 5 minutes, and then stored at 4 ℃ for a period of time.

2.1.3 fluorescent quantitative PCR

Relative mRNA levels of PCSK9 were detected using the Δ Δ Ct method for PCSK9 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) using SYBR green method (TIANGEN) or TaqMan probe method (Thermo). The instrument used was either Applied Biosystem Stepone Plus or QuantStaudio 5 (Applied Biosystem). The reaction conditions are as follows: (1) pre-denaturing at 95 ℃ for 10 minutes; (2) denaturation at 95 ℃ for 15 seconds, annealing extension at 60 ℃ for 30 seconds. Step (2) continued for 40 cycles. The results obtained were normalized using a negative control to obtain relative mRNA levels and knockdown efficiencies. On-demand computing IC ₅₀ Then obtained after four parameter fitting using Graphpad Prism.

2.2 reporter Gene method

The HEK293-psiCheck-PCSK9 cell contains a PCSK9 full-length sequence and a luciferase gene tandem expression system. Using DMEM medium (Gibco) supplemented with 10% FBS (Gibco), 2ug/ml puromycin and diabody (Gibco) in 5% CO ₂ Culturing at 37 deg.C, inoculatingAfter the flask was almost completely covered, the flask was digested with trypsin and resuspended. The resuspended cells were density adjusted and treated at 2X10 ⁴ And/well inoculating to a 96-well plate, and adding the siRNA transfection complex after the cells are completely attached. The siRNA transfection complex was obtained by mixing MaxOpti-MEM (Gibco) containing 0.3 ul/well liposome RNAi Max (Thermo) with siRNA 1:1. After a certain period of cell culture, the cells were lysed and a Renilla substrate (Promega) was added for fluorescence activity detection.

2.3、Stem-loop PCR

The Stem-loop method has been widely used to detect the absolute concentrations of siRNA and miRNA (Curr Protoc Mol biol.2011Jul; chapter 15. Designing corresponding Stem-loop primers aiming at different siRNAs, and carrying out reverse transcription by using the primers to replace oligo dT, and then carrying out siRNA concentration conversion by using a fluorescent quantitative PCR mode and applying a standard curve. The reverse transcription conditions were: (1) 30 seconds at 16 ℃; (2) 30 seconds at 30 ℃,30 seconds at 42 ℃,1 second at 50 ℃, and step (2) lasts for 60 cycles; (3) 5 minutes at 95 ℃. The fluorescent quantitative PCR reaction conditions are as follows: (1) 5 minutes at 95 ℃; (2) 95 ℃ for 5 seconds, 60 ℃ for 10 seconds, and 72 ℃ for 1 second; step (2) continued for 40 cycles.

Example 3: siRNA sequence screening and function identification

3.1 screening of unmodified siRNA sequences

To evaluate the in vitro activity of siRNA on PCSK9, designed siRNA was functionally evaluated in vitro using fluorescent quantitative PCR and reporter gene.

siRNA activity was measured using fluorescent quantitative PCR in HepG2 and Huh-7 cell lines and a reporter gene in HEK293-psiCheck-PCSK9 (RGA) cell line, respectively, and all results are shown in Table 3. Table 3 shows the expression levels of PCSK9mRNA of siRNA versus NC (negative control group).

Table 3: in vitro Activity assay of siRNA

By integrating the in vitro activity, the siRNA can stably and effectively reduce the level of PCSK9mRNA in HepG2 cells, huh-7 cells or RGA cells, and is further optimized on the basis of the molecules.

3.2 screening of modified siRNA sequences

After combining in vitro activity and siRNA modification (synthesized by kasri biotechnology limited), huh7 and HepG2 cells were tested using the fluorescent quantitative PCR method of example 2, HEK293-psiCheck-PCSK9 was tested functionally in vitro using the reporter gene method of example 2, and all results were normalized by negative controls (NC in table 1). The siRNA sequences are shown in Table 2.

The result shows that the siRNA can effectively reduce the level of PCSK9mRNA in HepG2, huh-7 cells or HEK293-psiCheck-PCSK9 cells.

Example 4: synthesis of siRNA conjugates

synthesis of siRNA conjugate 1:

siRNA conjugate 1 was prepared according to the following reaction scheme.

Step 1: preparation of intermediate M1

5-Azidopentanoic acid (263.32mg, 1.84mmol, 1.5eq.) was dissolved in 20mL of dichloromethane, and EDCI (352.64mg, 1.84mmol, 1.5eq.) and HOBT (248.56mg, 1.84mmol, 1.5eq.) were added. And stirred at 25 ℃ for 15min under nitrogen atmosphere. Compound M13 (2.2g, 1.23mmol, 1eq.) was then added to the reaction. The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. TLC (dichloromethane: methanol = 8:1) monitored the completion of consumption of the starting material M13 (see t.p.prakash, m.j.graham, p.p.seth, etc. nucleic Acids Research,2014,42,8796-8807, the same below). 10mL of water was added to the reaction mixture to quench the reaction. The reaction solution was extracted with dichloromethane (10mL. Times.3). The organic phases were combined and washed with aqueous NaCl (15 mL). The organic phase is dried by anhydrous sodium sulfate, and the solvent is dried by spinning to obtain a crude product. After the crude product was subjected to silica gel column chromatography (methanol/dichloromethane =0:100 to 15), white solid M1 was obtained (1.1 g yield 46.7%).

Step 2: preparation of intermediate M2

M1 (800mg, 416.88 μmol,1 eq.), 5-acetylic acid (46.74mg, 416.88 μmol,1 eq.), copper sulfate (79.84mg, 500.25 μmol,1.2 eq.) were added to a suspension of vic sodium (206.47mg, 1.04mmol,2.5 eq.) at 25 ℃ (methanol: water =1, 2ml. After the resulting mixture was warmed to 60 ℃, it was stirred at that temperature for 1h. LC-MS monitors that the consumption of raw material is finished and new product is generated. The solvent in the reaction mixture was dried by spinning, and the obtained residue was subjected to silica gel column chromatography (methanol/dichloromethane =0 = 100 to 15) to obtain M2 as a yellow solid (550 mg, yield 64.9%).

And step 3: preparation of intermediate M3

M2 (350mg, 172.32. Mu. Mol,1 eq.) was dissolved in dichloromethane (10 mL) to give a clear solution. EDCI (49.55mg, 258.47. Mu. Mol,1.5 eq.) and HOBt (34.92mg, 258.47. Mu. Mol,1.5 eq.) were then added to the reaction system. The resulting mixture was stirred at 25 ℃ for 15min under nitrogen atmosphere. Intermediate S2A (86.75mg, 206.78. Mu. Mol,1.2 eq.) was then added (cf. N.H ebert, P.W.Davis, E.L.D.Baets, O.L.Acevedo, tetrahedron Letter,1994,35,9509-9512, the same applies hereinafter). The mixture was stirred at 25 ℃ for 16h. TLC (dichloromethane: methanol = 8:1) monitored complete consumption of starting material M2. The reaction was quenched by the addition of water (24 mL). The reaction was extracted with dichloromethane (24mL. Times.3). The organic phases were combined and washed with aqueous NaCl (30 mL). The organic phase is dried by anhydrous sodium sulfate, and the solvent is dried by spinning to obtain a crude product. After the crude product was subjected to silica gel column chromatography (methanol/dichloromethane (0.1% triethylamine) = 0.

And 4, step 4: preparation of intermediate M4

M3 (350mg, 143.88mmol, 1eq.), succinic anhydride (115.18mg, 1.15mmol, 8eq.), DMAP (35.15mg, 287.75. Mu. Mol,2 eq.) and TEA (349.41mg, 3.45mmol, 479.96. Mu.L, 24 eq.) were dissolved in DCM (10 mL). The resulting mixture was stirred at 20 ℃ for 16h under nitrogen. LC-MS monitors that the consumption of raw material is finished and new product is generated. And (4) spin-drying the solvent in the reaction mixed solution to obtain a crude product. Preparative HPLC separation of the above crude product (carbon-18 column, acetonitrile/0.01% ammonia) gave M4 as a white solid (155 mg, yield 44.1%). HPLC purity: 99.48%, LCMS (ESI): cal.for C ₁₂₀ H ₁₇₄ N ₁₄ O ₄₅ :2531.2,Found[M+H] ⁺ :2532.6。

And 5: preparation of intermediate M5

(I) M4 (92mg, 36.32. Mu. Mol,1 eq.) and HBTU (27.55mg, 72.65. Mu. Mol,2 eq.) were dissolved in 8mL acetonitrile, and N, N-diisopropylethylamine (18.78mg, 145.30. Mu. Mol, 25.31. Mu.L, 4 eq.) was added. After shaking for 3-4min, CPG-amino resin (1.09g, 50. Mu. Mol/g) was added. The resulting mixture was shaken on a shaker at room temperature for 48h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (25 mL). The resulting solid was dried at 45 ℃ for two hours to give a white solid (1.08 g). (II) acetic anhydride (3.64mg, 35.64. Mu. Mol,0.0931 eq.) and pyridine (7.77mg, 98.18. Mu. Mol, 7.90. Mu.L, 0.256 eq.) were dissolved in 10mL acetonitrile, the mixture was shaken well and the above white solid (1.08g, 382.95. Mu. Mol,1 eq.) was added. The resulting mixture was shaken on a shaker at room temperature for 0.5h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The solid obtained is dried at 45 ℃ for two hours, giving 1.08g of solid phase supported product M5 in the form of a white powder. The loading amount was 23.3. Mu. Mol/g.

And 6: preparation of siRNA conjugate 1

R ² siRNA S1 (SEQ ID NO: 59/60) is selected, and siRNA conjugate 1 is obtained after solid phase synthesis and deprotection of a solid phase load product M5 (refer to M.J.Damha, K.K.Ogilvie, methods mol.biol.1993,20,81-114. The same below).

Synthesis of siRNA conjugate 2

siRNA conjugate 2 was prepared according to the following reaction scheme.

Step 1: preparation of intermediate M1

HOBT (120.51mg, 891.91. Mu. Mol,2 eq.), EDCI (170.98mg, 891.91. Mu. Mol,2 eq.) were dissolved in dichloromethane DCM (10 mL), followed by N, N-diisopropylethylamine (288.18mg, 2.23mmol, 388.38. Mu.L, 5 eq.) and then SM1 (145mg, 686.51. Mu. Mol,1.54 eq.). After stirring for 0.5h, M13 (800mg, 445.96. Mu. Mol,1 eq.) was added to the mixture. The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. LC-MS monitors the production of new products. 15mL of water was added to the reaction mixture to quench the reaction. The reaction was extracted with dichloromethane (30mL. Times.2). The organic phases were combined and washed with aqueous NaCl (30 mL). The organic phase is dried by anhydrous sodium sulfate, and the solvent is dried by spinning to obtain a crude product. After the crude product was subjected to silica gel column chromatography (dichloromethane/methanol =99, 1, 95, 90, 10, 80).

And 2, step: preparation of intermediate M2

M1 (400mg, 161.04. Mu. Mol,1 eq.) and 3-mercaptopropionic acid (17.09mg, 161.04. Mu. Mol, 14.03. Mu.L, 1 eq.) were dissolved in dichloromethane (10 mL), and triethylamine (48.89mg, 483.12. Mu. Mol, 67.15. Mu.L, 3 eq.) was added, and the resulting mixture was stirred at 25 ℃ for 0.5h. LC-MS monitors the generation of the target product. The reaction mixture was used directly in the next reaction.

And step 3: preparation of intermediate M3

To a solution of M2 (337.09mg, 161.04. Mu. Mol,1 eq.) in dichloromethane (5 mL) were added EDCI (92.61mg, 483.11. Mu. Mol,3 eq.), HOBT (65.28mg, 483.11. Mu. Mol,3 eq.) and N, N-diisopropylethylamine (124.88mg, 966.22. Mu. Mol, 168.30. Mu.L, 6 eq.), and the mixture was stirred at 30 ℃ for 0.5h under nitrogen. Subsequently, a solution of S2A (135.11mg, 322.07. Mu. Mol,2 eq.) in methylene chloride (3 mL) was added to the above mixture, and stirred at 30 ℃ for 16h. HPLC-MS monitors the formation of the new product. And (4) spin-drying the solvent in the reaction solution to obtain a crude product. After column chromatography of the crude product on silica gel (dichloromethane/methanol =99, 95, 93.

And 4, step 4: preparation of intermediate M4

DMAP (28.79mg, 235.70. Mu. Mol,2 eq.), triethylamine (536.62mg, 5.30mmol, 737.12. Mu.L, 45 eq.) and succinic anhydride (176.90mg, 1.77mmol, 15eq.) were added to a solution of M3 (420.00mg, 117.85. Mu. Mol,1 eq.) in dichloromethane (20 mL) under ice-bath conditions. The obtained mixed solution is stirred for 16 hours at 25-30 ℃ under the nitrogen atmosphere. HPLC-MS monitors the formation of the new product. And (4) spin-drying the solvent in the reaction solution to obtain a crude product. The crude product was separated by preparative HPLC twice (carbon-18 column, acetonitrile/0.01% -ammonia) to give M4 as a white solid (40 mg, yield 44.1%). HPLC purity: 98.83%, LCMS (ESI): cal.for C ₁₂₂ H ₁₇₆ N ₁₂ O ₄₇ S:2594.85,Found[M+H] ⁺ :2595.7。

And 5: preparation of intermediate M5

(I) M4 (80mg, 30.83. Mu. Mol,1 eq.) and HBTU (23.38mg, 61.66. Mu. Mol,2 eq.) were dissolved in 10mL acetonitrile, and N, N-diisopropylethylamine (15.94mg, 123.32. Mu. Mol, 21.48. Mu.L, 4 eq.) was added. After shaking for 3-4min, CPG-amino resin (925.00mg, 50umol/g) was added. The resulting mixture was shaken on a shaker at room temperature for 48h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (25 mL). The resulting solid was dried at 35 ℃ for two hours to give 950mg of a white solid. (II) acetic anhydride (4.85mg, 47.5. Mu. Mol, 4.49. Mu.L, 1.24e-1 eq) and pyridine (11.27mg, 142.5. Mu. Mol, 11.47. Mu.L, 3.72e-1 eq.) were dissolved in 10mL acetonitrile, and the resulting mixture was shaken well and the above white solid was added. The resulting mixture was shaken on a shaker at room temperature for 0.5h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The resulting solid was dried at 40 ℃ for two hours to give a white powdery solid-phase supported product M5 (925 mg). The loading amount was 18.9. Mu. Mol/g.

And 6: preparation of siRNA conjugate 2

R ² siRNA S1 (SEQ ID NO: 59/60) is selected, and a solid phase load product M5 is subjected to solid phase synthesis and deprotection to obtain the siRNA conjugate 2.

Synthesis of siRNA conjugate 3

siRNA conjugate 3 was prepared according to the following reaction scheme.

Step 1: preparation of intermediate M1

After succinic acid Shan Bianzhi (1.44g, 6.93mmol, 1.1eq.), EDCI (1.81g, 9.45mmol, 1.5eq.) and HOBT (1.28g, 9.45mmol, 1.5eq.) were dissolved in 30mL of dichloromethane, 4- (N-Boc-aminomethyl) aniline (1.4g, 6.30mmol, 1eq.) was added. The resulting mixture was stirred at 25 ℃ for 2h. LC-MS monitors that the consumption of raw material is finished and new product is generated. 15mL of water was added to the reaction mixture to quench the reaction. The reaction solution was extracted with dichloromethane (20mL. Times.2). The organic phases were combined and washed with aqueous NaCl (15 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to give a residue. After the residue was subjected to silica gel column chromatography (ethyl acetate/petroleum ether =0 = 100 to 20).

And 2, step: preparation of intermediate M2

M1 was dissolved (1.0 g,2.42mmol, 1eq.) in 10mL of 1, 4-dioxane. A4N HCl dioxane solution (40.00mmol, 10mL, 16.5eq.) was added. The mixture was stirred at 25 ℃ for 1h. LC-MS monitors that the consumption of raw material is finished and new product is generated. The solvent in the reaction mixture was spin dried to obtain 840mg of yellow solid, M2, as the hydrochloride salt. The yield is more than 95 percent.

And 3, step 3: preparation of intermediate M3

M2 hydrochloride (840mg, 2.41mmol, 1eq.), triethylamine (731.03mg, 7.22mmol,1.00mL, 3eq.) were dissolved in 40mL of dichloromethane, and p-nitrophenyl chloroformate (970.77mg, 4.82mmol, 2eq.) was added. The resulting mixture was stirred at 25 ℃ for 1h. LC-MS monitors that the consumption of raw material is finished and new product is generated. 30mL of water was added to the reaction mixture to quench the reaction. The reaction was extracted with dichloromethane (30mL. Times.2). The organic phases were combined and washed with aqueous sodium chloride (30 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to give a residue. The residue was recrystallized from dichloromethane (20 mL) to give M3 as a white solid (800 mg, yield 69.6%).

And 4, step 4: preparation of intermediate M4

M3 (600mg, 1.26mmol, 1eq.) and S2A (527.17mg, 1.26mmol, 1eq.) were dissolved in 25mL of a mixed solution of dichloromethane and tetrahydrofuran (dichloromethane/tetrahydrofuran =4, 1, v/v), and triethylamine (508.64mg, 5.03mmol,698.68 μ L,4 eq.) was added. The mixture was stirred at 25 ℃ for 2h. LC-MS monitors that the consumption of raw material is finished and new product is generated. To the reaction mixture was added 20mL of water to quench the reaction. The reaction solution was extracted with dichloromethane (20mL. Times.2). The organic phases were combined and washed with aqueous NaCl (30 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to give a residue. After the residue was subjected to silica gel column chromatography (methanol/dichloromethane =0 = 100 to 4) to obtain M4 (800 mg, yield 84.0%) as a yellow solid.

And 5: preparation of intermediate M5

M4 (600.00mg, 791.69. Mu. Mol,1 eq.) was dissolved in 20mL of methanol, followed by addition of PtO ₂ (257.14mg, 1.13mmol, 1.43eq.), and the resulting mixture is stirred at 25-30 ℃ for 3h under a hydrogen atmosphere. LC-MS monitors that the consumption of raw material is finished and new product is generated. After the reaction mixture was filtered, 15mL of water was added. The reaction solution was extracted with dichloromethane (20mL. Times.2). The organic phases were combined and washed with aqueous NaCl (30 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to give a residue. After separation of the residue by silica gel column chromatography (methanol: dichloromethane =0:100 to 20, containing 0.1% triethylamine), M5 (500 mg, yield 94.6%) was obtained as a yellow solid.

Step 6: preparation of intermediate M6

M13 (268.65mg, 149.76. Mu. Mol,1 eq.) was dissolved in 10mL of dichloromethane, EDCI (57.42mg, 299.52. Mu. Mol,2.0 eq.) and HOBT (40.47mg, 299.52. Mu. Mol,2.0 eq.) were added, and the mixture was stirred at 25 ℃ for 15min under a nitrogen atmosphere. M5 (100mg, 149.76. Mu. Mol,1 eq.) was then added to the reaction. The resulting mixture was stirred at 25 ℃ for 16h. LC-MS monitors the completion of the consumption of the raw material and the production of a new product. To the reaction solution was added 15mL of water. The reaction solution was extracted with dichloromethane (10mL. Times.2). The organic phases were combined and washed with aqueous NaCl (10 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to give a residue. After separation of the residue by silica gel column chromatography (methanol: dichloromethane =0 = 100 to 100, containing 0.1% triethylamine), white solid M6 was obtained (300 mg, yield 68.0%).

And 7: preparation of intermediate M7

M6 (300mg, 122.77. Mu. Mol,1 eq.), succinic anhydride (122.86mg, 1.23mmol, 10eq.), DMAP (30.00mg, 245.54. Mu. Mol,2 eq.) and TEA (372.69mg, 3.68mmol, 511.93. Mu.L, 30 eq.) were dissolved in 20mL of dichloromethane. The resulting mixture was stirred at 20 ℃ for 16h under nitrogen. LC-MS monitors that the consumption of raw material is finished and new product is generated. And (3) spin-drying the solvent in the reaction mixed solution to obtain an oily substance. Preparative HPLC separation of the above oil (carbon-18 column, acetonitrile/0.01% ammonia) gave M7 as a white solid (53 mg, 37.9% yield). LCMS (ESI): cal.for C ₁₂₁ H ₁₇₁ N ₁₃ O ₄₆ :2542.2,Found[M+H] ⁺ ：2543.2。

And step 8: preparation of intermediate M8

(I) M7 (53mg, 20.84. Mu. Mol,1 eq.) and HBTU (15.80mg, 41.67. Mu. Mol,2 eq.) were dissolved in 10mL acetonitrile, and N, N-diisopropylethylamine (10.77mg, 83.34. Mu. Mol, 14.52. Mu.L, 4 eq.) was added. After shaking for 3-4min, CPG-amino resin (625mg, 50. Mu. Mol/g) was added. The resulting mixture was shaken on a shaker at room temperature for 48h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 ml). The resulting solid was dried at 45 ℃ for two hours to give 620mg of a white solid. (II) acetic anhydride (3.06mg, 30. Mu. Mol,1.37e-1 eq.) and pyridine (4.44mg, 56.15. Mu. Mol, 4.52. Mu.L, 2.56e-1 eq.) were dissolved in 10mL acetonitrile, the mixture was shaken well and the above white solid was added. The resulting mixture was shaken on a shaker at room temperature for 0.5h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The resulting solid was dried at 45 ℃ for 2h to give the product M8 (480 mg) as a white powder as a solid phase. The supported amount was 13.3. Mu. Mol/g.

And step 9: preparation of siRNA conjugate 3

R ² siRNA S1 (SEQ ID NO: 59/60) is selected, and a solid phase load product M8 is subjected to solid phase synthesis and deprotection to obtain the siRNA conjugate 3.

Synthesis of siRNA conjugate 4

siRNA conjugate 4 was prepared according to the following reaction scheme.

Step 1: preparation of intermediate M1

Maleic anhydride (1g, 10.20mmol, 1eq.) was dissolved in 20mL of acetic acid, and tranexamic acid (1.60g, 10.20mmol, 1eq.) was added. The resulting mixture was stirred at 160 ℃ for 6h under nitrogen. TLC monitored the maleic anhydride was almost complete. The reaction solution was spin dried at 60 ℃ and the crude product was chromatographed on silica gel (methanol/dichloromethane, 0-4%) to give M1 as a white solid (1.1 g, 45.5% yield). ¹ H NMR(500MHz,DMSO-d ₆ )δ7.03(s,2H),3.27(d,J＝7.0Hz,2H),2.13(d,J＝11.0Hz,1H),1.90(d,J＝13.0Hz,2H),1.65(d,J＝12.5Hz,2H),1.56(s,1H),1.25(q,J＝12.5Hz,2H),0.97(q,J＝12.5Hz,2H)。

And 2, step: preparation of intermediate M2

After dispersing M1 (79.35mg, 334.47. Mu. Mol,1 eq.) in dichloromethane (10 mL), HBTU (253.69mg, 668.93. Mu. Mol,2 eq.) N, N-diisopropylethylamine (86.45mg, 668.93. Mu. Mol, 116.51. Mu.L, 2 eq.) HOBT (225.97mg, 1.67mmol,5 eq.) and M13 (600mg, 334.47. Mu. Mol,1 eq.) were added. The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. LCMS monitors new product formation. And (4) spin-drying the solvent in the reaction solution to obtain a residue. The residue was subjected to silica gel column chromatography (methanol/dichloromethane, 0 to 20%) to give M2 (300 mg, yield 50.0%) as a pale yellow solid.

And step 3: preparation of intermediate M3

M2 (300mg, 149.02. Mu. Mol,1 eq.) and 3-mercaptopropionic acid (15.82mg, 149.02. Mu. Mol,1 eq.) were dissolved in chloroform (10 mL), and triethylamine (15.08mg, 149.02. Mu. Mol,1 eq.) was added to stir the mixture at 25 ℃ for 2 hours. LC-MS monitors the generation of the target product, and the reaction mixed liquid is directly used for the next reaction.

And 4, step 4: preparation of intermediate M4

EDCI (54.27mg, 283.11. Mu. Mol,2 eq.), HOBT (38.25mg, 283.11. Mu. Mol,2 eq.), N, N-diisopropylethylamine (73.18mg, 566.23. Mu. Mol, 98.62. Mu.L, 4 eq.) and S2A (59.38mg, 141.56. Mu. Mol,1 eq.) were added to a dichloromethane solution (5 mL) of M3 (300mg, 141.56. Mu. Mol,1 eq.) and the resulting mixture was stirred at 25 ℃ for 16h. HPLC-MS monitors the formation of the new product. And (4) spin-drying the solvent in the reaction solution to obtain a crude product. After separation of the crude product by silica gel column chromatography (methanol/dichloromethane, 0-20%), M4 (140 mg, yield 39.2%) was obtained.

And 5: preparation of intermediate M5

M4 (140mg, 55.54. Mu. Mol,1 eq.), triethylamine (252.90mg, 2.50mmol, 45eq.), DMAP (13.57mg, 111.08. Mu. Mol,2 eq.) were mixed with 5mL of dichloromethane, and succinic anhydride (83.37mg, 833.08. Mu. Mol,15 eq.) was added after the mixture was cooled in an ice bath. The resulting mixture was gradually warmed to 25 ℃ and stirred under this nitrogen atmosphere for 16h. LC-MS monitors the production of new products. And (4) spin-drying the solvent in the mixed solution to obtain a crude product. The crude product was isolated by HPLC preparative (carbon)-18 column, acetonitrile/0.01% ammonia) to give M5 (32 mg, yield 22.0%) as a white solid. HPLC purity:>99.9％，LCMS(ESI):Cal.for C ₁₂₄ H ₁₇₈ N ₁₂ O ₄₇ S:2620.89,Found[M-H] ^- :2619.5。

and 6: preparation of intermediate M6

(I) M5 (32mg, 12.21. Mu. Mol,1 eq.) and HBTU (9.26mg, 24.42. Mu. Mol,2 eq.) were dissolved in 5mL acetonitrile and N, N-diisopropylethylamine (6.31mg, 48.84. Mu. Mol, 8.51. Mu.L, 4 eq.) was added. After shaking for 3-4min, CPG-amino resin (366.3mg, 50umol/g) was added. The resulting mixture was shaken on a shaker at room temperature for 48h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The resulting solid was dried at 45 ℃ for two hours to give 360mg of a white solid. (II) the above white solid was mixed with pyridine (391.64mg, 4.95mmol, 398.42. Mu.L, 40 eq.) in 5mL of acetonitrile, and acetic anhydride (252.73mg, 2.48mmol, 20eq.) was added under ice-bath conditions. The resulting mixture was shaken on a shaker at room temperature for 0.5h. Subsequently, the mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The solid obtained is dried at 45 ℃ for 2h, giving 345mg of a white powdery solid-phase supported product M6. The supported amount was 27.0. Mu. Mol/g.

And 7: preparation of siRNA conjugate 4

R ² siRNA S1 (SEQ ID NO: 59/60) is selected, and a solid phase load product M6 is subjected to solid phase synthesis and deprotection to obtain the siRNA conjugate 4.

Synthesis of siRNA conjugate 5

siRNA conjugate 5 was prepared according to the following reaction scheme.

Step 1: preparation of intermediate M1

(S) -2- (((benzyloxy) carbonyl) amino) -5-ureidopentanoic acid (0.8g, 2.59mmol, 1eq.), EDCI (991.60mg, 5.17mmol, 2eq.), N, N-diisopropylethylamine (1.34g, 10.35mmol,1.80mL, 4eq.), HOBt (349.47mg, 2.59mmol, 1eq.) were mixed in 20mL of dichloromethane, and S2A (1.09g, 2.60mmol, 1eq.) was added. The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. LC-MS monitors the production of new products. The reaction mixture was diluted with 20mL of methylene chloride, and 20mL of water was added thereto. Standing and layering to obtain an organic phase. The organic phase is dried by anhydrous sodium sulfate, and the solvent is dried by spinning to obtain a crude product. The crude product was subjected to silica gel column chromatography to give M1 (1.2 g, yield 65.0%).

Step 2: preparation of intermediate M2

M1 (1.2g, 1.69mmol, 1eq.) was dissolved in 10mL of methanol and Pd/C (102.52 mg) was added ₂ Stirred at 25 ℃ for 16h under an atmosphere. LC-MS monitors the generation of the target product. The reaction mixture was filtered to obtain a solution. The solvent was spun dry to give M2 (860 mg) which was used directly in the next reaction.

And step 3: preparation of intermediate M3

Monomethyl azelate (301.61mg, 1.49mmol, 1eq.), HBTU (1.13g, 2.98mmol, 2eq.), N, N-diisopropylethylamine (770.94mg, 5.97mmol,1.04mL, 4eq.) were mixed in 10mL of dichloromethane, followed by addition of M2 (860mg, 1.49mmol, 1eq.). The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. LC-MS monitors the generation of the target product. The reaction mixture was diluted with 20mL of methylene chloride, and 20mL of water was added thereto. Standing and layering to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate and the solvent was spin dried to obtain the crude product. The crude product was chromatographed on a silica gel column (methanol/dichloromethane = 0-6%) to give M3 (680 mg, 59.9% yield).

And 4, step 4: preparation of intermediate M4

M3 (600mg, 788.53. Mu. Mol,1 eq.) was dissolved in 10mL of methanol and 10mL of water, and lithium hydroxide (188.85mg, 7.89mmol, 10eq.) was added. LC-MS monitors the generation of the target product. The solvent was spun off to give M4 (590 mg) which was used directly in the next reaction.

And 5: preparation of intermediate M5

M4 (201.43mg, 267.57. Mu. Mol,1.5 eq.) was dissolved in 5mL of DMF and HOBT (1 eq.), EDCI (2 eq.), DIPEA (2 eq.) were added. After stirring for 0.5h, M13 (320mg, 178.38. Mu. Mol,1 eq.) was added. The resulting mixture was stirred at 25 ℃ for 16h under nitrogen. LC-MS monitors the generation of the target product. After 15mL of water was added to the reaction system, the mixture was extracted with dichloromethane (15mL. Times.3)). After the organic phases are combined, the solvent is dried by spinning to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane (containing 0.1% triethylamine)/methanol = 90.

Step 6: preparation of intermediate M6

M5 (230mg, 91.17. Mu. Mol,1 eq.) was dissolved in 10mL of dichloromethane, and DMAP (22.28mg, 182.34. Mu. Mol,2 eq.) and triethylamine (415.14mg, 4.10mmol, 570.25. Mu.L, 45 eq.) were added. After cooling in an ice bath, succinic anhydride (136.85mg, 1.37mmol, 15eq.) was added to the reaction system. The resulting mixture was gradually warmed to 25 ℃ and stirred under these conditions for 16h. LC-MS monitors the generation of the target product. After concentration of the reaction mixture, the crude product was subjected to preparative HPLC separation (carbon-18 column, acetonitrile/0.01% ammonia) to give M6 (33.8 mg). HPLC purity: 99.83 percent. LCMS (ESI) Cal.for C124H184N14O47:2622.89, found [ 2 ] M-H] ^- :2621.7

And 7: preparation of intermediate M7

(I) M6 (33.8mg, 12.89. Mu. Mol,1 eq.) and HBTU (9.77mg, 25.77. Mu. Mol,2 eq.) were dissolved in 5mL acetonitrile, and N, N-diisopropylethylamine (6.66mg, 51.55. Mu. Mol, 8.98. Mu.L, 4 eq.) was added. Shaking for 3-4min, and adding CPG-amino resin (386.7 mg, 50umol/g). The resulting mixture was shaken on a shaker at room temperature for 48h. The reaction mixture was filtered and the filter cake was washed twice with acetonitrile (25 mL). The resulting solid was dried at 35 ℃ for 2h to give a white solid (382.6 mg). (II) the white solid obtained above was mixed with pyridine (4.54mg, 57.39. Mu. Mol, 4.62. Mu.L, 4.37e-1 eq.) in 10mL of acetonitrile, and acetic anhydride (1.95mg, 19.13. Mu. Mol, 1.81. Mu.L, 1.46e-1 eq.) was added under ice-bath conditions. The resulting mixture was shaken on a shaker at room temperature for 0.5h. The mixture was filtered and the filter cake was washed twice with acetonitrile (10 mL). The resulting solid was dried at 40 ℃ for 2h to give M7 (261.6 mg) as a white solid. The loading amount is 20.5 mu mol/g.

And 8: preparation of siRNA conjugate 5

R ² siRNA S1 (SEQ ID NO: 59/60) or other siRNAs shown in Table 2 is selected, and a solid phase load product M7 is subjected to solid phase synthesis and deprotection to obtain an siRNA conjugate 5 and other siRNA conjugates.

Positive control

The positive control was Inclisiran, reference in the synthesis procedure (CN 104854242B, alnylam), and the specific structure is shown in FIG. 2, wherein the structural formula of L96 is shown as follows:

example 5: in vivo efficacy of PCSK9 siRNA in mice

To evaluate the effect of siRNA on PCSK9 in vivo activity, 42 male PCSK9 humanized mice (southern model) of 6-8 weeks were taken for siRNA conjugate in vivo activity assay, and the mice were randomized into 7 groups of 6 mice each:

g1 normal saline control group (3 mg/kg);

g2 Inclisiran (3 mg/kg) positive control group;

g3 Inclisiran (9 mg/kg) positive control group;

g4 siRNA conjugate 1 (3 mg/kg) treatment group;

g5 siRNA conjugate 1 (9 mg/kg) treatment group;

g6 siRNA conjugate 5 (3 mg/kg) treatment group;

g7 siRNA conjugate 5 (9 mg/kg) treatment group.

Pre-dose serum samples were obtained on day-1 after a 4 hour fast. The siRNA conjugate was diluted with physiological saline and then injected subcutaneously on day 1 according to the experimental design, at doses of 3mg/kg and 9mg/kg, respectively, at a concentration of 1mg/ml, for 1 administration. Plasma was collected after 4 hours of fasting on

days

1,3, 7, 14, 21, 28, 35, 42, 49, respectively.

PCSK9 protein levels in plasma were detected by ELISA methods (R & DSystems) according to the experimental protocols provided by the supplier. Triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c) in serum were detected by a full-automatic biochemical analyzer, which was handed over to Shanghai Bin Yuntian bioscience Co., ltd for detection, and the results are shown in FIG. 1.

PCSK9 protein levels, triglyceride levels, total cholesterol levels, HDL-c levels and LDL-c levels were normalized for each animal. The normalization method is to divide the levels of PCSK9 protein, triglycerides, HDL and total cholesterol in each animal separately at one time point by the animal's pre-treatment expression levels (in this case on day-1) to determine the "normalized to pre-treatment" ratio. Expression at specific time points was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for individual animals by the average "normalized to pre-treatment" ratio of all mice in the saline group.

The results show that the siRNA conjugate can reduce the PCSK9 protein level, TG level and LDL-c level for a long time, and the results are superior to those of the control Inclisiran.

Example 6: in vivo efficacy of PCSK9 siRNA in familial hyperlipidemic rhesus monkeys

To assess the in vivo activity of sirnas on PCSK9, in vivo activity assays were performed using male familial hyperlipidemic PCSK9 rhesus monkeys. Pre-dose serum samples were obtained after a 4 hour fast on days-7 and-14. The above siRNA conjugate was diluted with physiological saline and then injected subcutaneously on day 1 according to the experimental design. Blank saline was used as a negative control. Plasma was collected after 4 hours fasting on days 3, 7, 14, 28, 42, 56, 70, 84, respectively. PCSK9 protein levels in plasma were detected by ELISA following the experimental procedures provided by the supplier (R & D Systems). Serum triglycerides, total cholesterol, high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), apo-A1 and Apo-B were detected using a fully automated biochemical analyzer.

PCSK9 protein levels, triglyceride levels, total cholesterol levels, HDL-c levels and LDL-c levels were normalized for each animal. For normalization, the levels of PCSK9 protein, triglycerides, HDL-c, LDL-c, total cholesterol, apo-A1 and Apo-B for each animal, respectively, at one time point were divided by the animal's pre-treatment expression levels (average of the set of pre-experimental samples) to determine the expression ratio "normalized to pre-treatment". Expression at specific time points was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for individual animals by the average "normalized to pre-treatment" ratio for all animals of the saline group.

The results show that the siRNA conjugate can reduce PCSK9 protein level, TG level and LDL-c level for a long time.

Sequence listing

<110> Shanghai Junshi biomedical science and technology Co., ltd

SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

<120> siRNA for inhibiting PCSK9 gene expression and application thereof

<130> 214741Z11CNCN

<141> 2022-06-21

<150> CN 202110686493.9

<151> 2022-06-21

<160> 58

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 1

ggucaccgac uucgagaaug u 21

<210> 2

<211> 22

<212> RNA

<213> Artificial Sequence

<400> 2

ggcauuucac cauucaaaca gg 22

<210> 3

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 3

aaguuggcug uaaaaaggc 19

<210> 4

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 4

uuuggguguu gcuagcaca 19

<210> 5

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 5

aauucuccga acgugucacg u 21

<210> 6

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

acauucucga agucggugac ctt 23

<210> 7

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

ccuguuugaa uggugaaaug cctt 24

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

gccuuuuuac agccaacuut t 21

<210> 9

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

ugugcuagca acacccaaat t 21

<210> 10

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 10

acgugacacg uucggagaau u 21

<210> 11

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 11

cugugcuagc aacacccaa 19

<210> 12

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 12

cugugcuagc aacacccaa 19

<210> 13

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 13

cugugcuagc aacacccaa 19

<210> 14

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 14

cugugcuagc aacacccaa 19

<210> 15

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 15

cugugcuagc aacacccaa 19

<210> 16

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 16

cugugcuagc aacacccaa 19

<210> 17

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 17

cugugcuagc aacacccaa 19

<210> 18

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 18

cugugcuagc aacacccaa 19

<210> 19

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 19

cugugcuagc aacacccaa 19

<210> 20

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 20

cugugcuagc aacacccaa 19

<210> 21

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 21

cugugcuagc aacacccaa 19

<210> 22

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 22

cugugcuagc aacacccaa 19

<210> 23

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 23

cuagaccugu tuugcuuuug u 21

<210> 24

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 24

cuagaccugu tuugcuuuug u 21

<210> 25

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 25

cuagaccugu tuugcuuuug u 21

<210> 26

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 26

cuagaccugu tuugcuuuug u 21

<210> 27

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 27

cuagaccugu tuugcuuuug u 21

<210> 28

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 28

cuagaccugu tuugcuuuug u 21

<210> 29

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 29

uuggguguug cuagcacagc c 21

<210> 30

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 30

uuggguguug cuagcacagc c 21

<210> 31

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 31

uuggguguug cuagcacagc c 21

<210> 32

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 32

uuggguguug cuagcacagc c 21

<210> 33

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 33

uuggguguug cuagcacagc c 21

<210> 34

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 34

uuggguguug cuagcacagc c 21

<210> 35

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 35

utggguguug cuagcacagc c 21

<210> 36

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 36

uuggguguug cuagcacagc c 21

<210> 37

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 37

uuggguguug cuagcacagc c 21

<210> 38

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 38

uuggguguug cuagcacagc c 21

<210> 39

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 39

uuggguguug cuagcacagc c 21

<210> 40

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 40

uuggguguug cuagcacagc c 21

<210> 41

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 41

uuggguguug cuagcacagc c 21

<210> 42

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 42

uuggguguug cuagcacagc c 21

<210> 43

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 43

uuggguguug cuagcacagc c 21

<210> 44

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 44

uuggguguug cuagcacagc c 21

<210> 45

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 45

uuggguguug cuagcacagc c 21

<210> 46

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 46

uuggguguug cuagcacagc c 21

<210> 47

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 47

uuggguguug cuagcacagc c 21

<210> 48

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 48

uuggguguug cuagcacagc c 21

<210> 49

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 49

uuggguguug cuagcacagc c 21

<210> 50

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 50

uuggguguug cuagcacagc c 21

<210> 51

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 51

uuggguguug cuagcacagc c 21

<210> 52

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 52

uuggguguug cuagcacagc c 21

<210> 53

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 53

acaaaagcaa aacaggucua gaa 23

<210> 54

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 54

acaaaagcaa aacaggucua gaa 23

<210> 55

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 55

acaaaagcaa aacaggucua gaa 23

<210> 56

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 56

acaaaagcaa aacaggucua gaa 23

<210> 57

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 57

acaaaagcaa aacaggucua gaa 23

<210> 58

<211> 23

<212> RNA

<213> Artificial Sequence

<400> 58

acaaaagcaa aacaggucua gaa 23

Claims

1. An siRNA that inhibits expression of a PCSK9 gene comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 contiguous nucleotides differing by 0, 1,2 or 3 nucleotides from any one of the sequences set forth in table 1 or table 2, the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotide complementarity to the antisense strand, and at least one nucleotide in the sense strand and/or the antisense strand is a modified nucleotide.

2. The siRNA of claim 1, wherein the sense strand comprises at least 17 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the sequence of any of the sense strands set forth in table 1 or table 2; preferably, the sense strand of the siRNA comprises the sequence of any one of the sense strands in table 1 and the antisense strand comprises the sequence of any one of the antisense strands in table 1; more preferably, the siRNA is selected from the group consisting of the siRNAs as shown in any of the siRNA numbers in Table 1.

3. The siRNA of claim 1 or 2, wherein the modified nucleotide is selected from: 2 '-O-methyl modified nucleotide, 2' -fluoro modified nucleotide, 2 '-deoxynucleotide, 2' -methoxyethyl modified nucleotide, 2 '-amino modified nucleotide, 2' -alkyl modified nucleotide, 2 '-alkoxy modified nucleotide, 2' -F-arabinose nucleotide, phosphorothioate modified nucleotide, abasic nucleotide, morpholino nucleotide and locked nucleotide.

4. The siRNA of claim 3, wherein said modified nucleotide is selected from the group consisting of: 2' -O-methyl modified nucleotide, 2' -fluoro modified nucleotide, 2' -deoxynucleotide and phosphorothioate modified nucleotide.

5. The siRNA of claim 4, wherein said modified nucleotide comprises:

(4) The 5' end of the sense strand comprises 1 or 2 phosphorothioate modified nucleotides in the 5' to 3' direction; and/or the 5 'end and the 3' end of the antisense strand each independently comprise 1 or 2 phosphorothioate modified nucleotides.

6. The siRNA of any one of claims 1-5, wherein said antisense strand comprises any one of the antisense strand nucleotide sequences set forth in Table 2 and said sense strand comprises any one of the sense strand nucleotide sequences set forth in Table 2.

7. The siRNA of claim 6, wherein said sense and antisense strands comprise or consist of a nucleotide sequence (5 '→ 3') selected from:

XR-1

sense strand mCMUGfUGfmCMUAfGfmCAfmCAfmCAmCMAMmA (SEQ ID NO: 11)

XR-2

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 12)

Antisense chain mU Uf mGGfmGUmGmUmUmGCfUfmAGfmCAfmCAfmG Cf mC (SEQ ID NO: 30)

XR-3

Sense strand mC mU GfmUGfmCUfGfmmCAfmCMAMmA (SEQ ID NO: 13)

XR-4

Sense strand mC mUGfUGfmCUFAfGfmCDAAfmCMMCmCMmCMmmA (SEQ ID NO: 14)

XR-5

Sense strand mCMU GfmUGfmCUAFGfmCGaAfmCMmCMmMamA (SEQ ID NO: 15)

XR-6

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 16)

Antisense chain mUufmGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmGCfmC (SEQ ID NO: 34)

XR-7

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 17)

Antisense strand mUdTMGGfmGUfmGmUmGCfUfmAGfmCAfmCAfmGCfmC (SEQ ID NO: 35)

XR-8

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 18)

Antisense strand mUufmGGfmGUfmGmUmGCfUfmAoGmCAfmGCfmC (SEQ ID NO: 36)

XR-9

Sense chain mCGUGfUGfmCMACfGfmCAfmCMmmmCMAMmA (SEQ ID NO: 19)

Antisense chain mU Uf mGGfmGUmGmUmUmGCfUfmAGfmCAfmCAfmG Cf mC (SEQ ID NO: 37)

XR-10

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmAMmA (SEQ ID NO: 20)

Antisense strand mU UfmGGfmGUfmGmUmUmGCfUfmaGfmCAfmCAfmG Cf mC (SEQ ID NO: 38) XR-11

Sense strand mCMUGfUGfmCMUAfGfmCAfmCMmCMmmCMmMAmA (SEQ ID NO: 21)

Antisense strand mU UfmGGfmGUfmGmUmGCfUfmaGfmmCAfmCGfmCAfmCGfmCAfmGCf mC (SEQ ID NO: 39) XR-12

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 40)

XR-13

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain mUUUUufGfmGGfmUGfUmUmGCfUAfGfCfmACmAGfmCCf (SEQ ID NO: 41)

XR-14

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfmUGfmGGfmUGfmUmUmGCfmUAfGfCfmACfmAGfmCCf (SEQ ID NO: 42)

XR-15

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfmGmGGfmUGfUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 43)

XR-16

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfGfmGfmUmUmGCfUAfGfCfmACfmAGfmcCf (SEQ ID NO: 44)

XR-17

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUmGmUmUmUmGCfUAfGfCfmACmAGfmcCf (SEQ ID NO: 45)

XR-18

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmUmGmCmUAfGfCfmACfmAGfmcCf (SEQ ID NO: 46)

XR-19

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmGCfmUmAGfCfmACfmAGfmcMCf (SEQ ID NO: 47)

XR-20

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmUmGCfUAfGCfmACfmAGfmCCf (SEQ ID NO: 48)

XR-21

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfUAfGfmCMACmAGfmCCf (SEQ ID NO: 49)

XR-22

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfmUAfGfCfmmAGfmcCf (SEQ ID NO: 50)

XR-23

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense strand UfUfGfmGGfmUGfUmUmGCfmUAfGfCfmmmmmmmGmCCf (SEQ ID NO: 51)

XR-24

Sense strand CUGUGCUAGCdAACACCCAA (SEQ ID NO: 22)

Antisense chain UfUfGfmGGfmUGfUmUmUmGCfmUAfGfCfmACfmAGfmCMC (SEQ ID NO: 52)

SR-1

Sense strand mC mUmmGmCCfUGfUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 23)

Antisense strand mA cF mAAfAfAfmGCfmAAfmCAfmGGfUCfUmAMAMmG mA (SEQ ID NO: 53)

SR-2

Sense strand mC mU mAMmCCfUGfmUdTmUmUmGmGmUmUmGmU (SEQ ID NO: 24)

Antisense strand mA mcmAAfmAAfmGCfmAAfmAAfmCMmmGMUmAMOMA mA (SEQ ID NO: 54)

SR-3

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 25)

SR-4

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 26)

Antisense strand mA mmCAfmAAfmGCfmAAfmAAfmCAfmGmUmAMOMG mA (SEQ ID NO: 56)

SR-5

Sense strand mC mU mAMmCCfUGfmUdTmUmUmGmGmUmUmGmU (SEQ ID NO: 27)

Antisense strand mA mmC mAAfmAAfmGCfmAAfmAAfmCMmMAGmGGfUmCUmmA (SEQ ID NO: 57)

SR-6

Sense strand mC mU mAm GmCCfUGfmUdTmUmUmGmCmUmUmGmU (SEQ ID NO: 28)

8. The siRNA of any of claims 1-7, wherein said sense and antisense strands are each independently 17-25 nucleotides in length; preferably, the sense and antisense strands are each independently 19-23 nucleotides in length.

9. An siRNA conjugate comprising the siRNA of any one of claims 1-8 and a targeting group.

10. The siRNA conjugate of claim 9, wherein said targeting group is a ligand that has affinity for an asialoglycoprotein receptor.

11. The siRNA conjugate of claim 9 or 10, wherein the targeting group contains a group from a lipophile selected from cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3- (oleoyl) lithocholic acid, O-3- (oleoyl) cholic acid, dimethoxytribenzyl, and phenoxazine.

12. The siRNA conjugate of claim 9 or 10, wherein said targeting group comprises a group from a carbohydrate selected from the group consisting of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucose, L-fucose, fucosamine, fucose, fucoidan, galactosamine, D-galactosamine, N-acetyl-galactosamine (GalNAc), galactose, glucosamine, N-acetyl-glucosamine, glucitol, glucose-6-phosphate, guloglycoraldehyde, L-glycerol-D-mannose-heptose, glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose-6-phosphate, psicose, quinovose, quinovofuroamine, rhamnose, ribose, ribulose, heptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose.

13. The siRNA conjugate of any of claims 9-12, wherein said targeting group comprises a group from N-acetyl-galactosamine (GalNAc).

14. The siRNA conjugate of any of claims 9 to 13, wherein said targeting group is:

wherein the wavy line indicates where the targeting group is attached to the remainder of the siRNA conjugate.

15. The siRNA conjugate of any of claims 9 to 13, further comprising a linker, said siRNA, said linker and said targeting group being sequentially covalently or non-covalently linked.

16. The siRNA conjugate of claim 15, wherein said linker comprises: reactive groups (such as primary amines and alkynes), alkyl groups, abasic nucleotides, ribitol (abasic ribose) and/or PEG groups; preferably, the linker is a carbonyl group at one end through which the linker is covalently linked to the targeting group and an-O-group at the other end for covalent linkage to the siRNA through a phosphoester linkage (-O-P (O) OH-).

17. The siRNA conjugate of claim 15 or 16, wherein said linker is selected from the group consisting of:

wherein the wavy line indicates the attachment position of the linker to the rest of the siRNA conjugate, wherein the targeting group is attached to the carbonyl group of the linker; the siRNA is connected with the other end of the linker through a phosphate ester bond.

18. The siRNA conjugate of any one of claims 9 to 17, having the structure of formula:

an isomer thereof or a pharmaceutically acceptable salt thereof,

wherein R is ² Is an siRNA as defined in any one of claims 1 to 8;

or R ² Is S1:

19. The siRNA conjugate of any of claims 9-18, wherein said R ² Covalently linked through the 3' end of its sense strand to a targeting group or linker via a phosphono group.

20. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 8 or the siRNA conjugate of any one of claims 9 to 19, an isomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

21. Use of an siRNA according to any of claims 1 to 8, an siRNA conjugate according to any of claims 9 to 20, an isomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 20, for the manufacture of a medicament for the treatment and/or prevention of a PCSK 9-mediated disease or condition.

22. The use of claim 21, wherein the disease or condition comprises atherosclerosis, hypercholesterolemia, hypertriglyceridemia, acute coronary syndrome, dyslipidemia, myocardial infarction, coronary artery disease, stroke, coronary artery disease, cardiovascular disease, diabetes, hyperlipidemia, type ii diabetes, and renal disease.