WO2023134609A1 - 一种抑制PCSK9基因表达的RNAi剂及其应用 - Google Patents

一种抑制PCSK9基因表达的RNAi剂及其应用 Download PDF

Info

Publication number
WO2023134609A1
WO2023134609A1 PCT/CN2023/071189 CN2023071189W WO2023134609A1 WO 2023134609 A1 WO2023134609 A1 WO 2023134609A1 CN 2023071189 W CN2023071189 W CN 2023071189W WO 2023134609 A1 WO2023134609 A1 WO 2023134609A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide sequence
strand consisting
identity
sense strand
antisense strand
Prior art date
Application number
PCT/CN2023/071189
Other languages
English (en)
French (fr)
Inventor
董云霞
邵昱
苏晓晔
王凌宇
Original Assignee
石药集团中奇制药技术（石家庄）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 石药集团中奇制药技术（石家庄）有限公司 filed Critical 石药集团中奇制药技术（石家庄）有限公司
Publication of WO2023134609A1 publication Critical patent/WO2023134609A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the field of molecular biology, relates to a modified double-stranded RNAi agent and its application, in particular to a double-stranded RNAi agent for inhibiting PCSK9 gene expression and its pharmaceutical composition, and the use of the double-stranded RNAi agent or its Use of the pharmaceutical composition for treating diseases mediated by PCSK9 expression.
  • RNA interference widely exists in species in nature. Since Andrew Fire and Craig Mello et al. first discovered RNAi phenomenon in nematodes (Caenorhabditiselegans, C.elegans) in 1998, Tuschl and Phil Sharp et al. confirmed in mammals in 2001 After the existence of RNAi in the human body, a series of progress has been made in the research on the mechanism principle, gene function and clinical application of RNAi. RNAi plays a key role in various body protection mechanisms such as defense against viral infection and transposon jumping (Hutvágner et al., 2001; Elbashire et al., 2001; Zamore 2001). Products developed based on RNAi mechanisms are very promising drug candidates. Small interfering RNA (small interfering RNA, siRNA) can play the role of RNA interference and is the main tool for realizing RNAi.
  • siRNA small interfering RNA
  • Proprotein convertase subtilisin/kexin-9 is a serine protease that indirectly regulates plasma LDL cholesterol levels by controlling the expression of hepatic and extrahepatic LDL receptors (LDLRs) on the plasma membrane.
  • LDLRs extrahepatic LDL receptors
  • PCSK9 inhibitors may be beneficial in reducing the concentration of LDL-C in the blood and in the treatment of PCSK9-mediated diseases, so they are expected to become potential therapeutic targets for controlling hypercholesterolemia and its complications.
  • statins small molecule drugs of statins.
  • studies have shown that patients who are intolerant to statins can cause adverse reactions such as myopathy, such as myalgia and rhabdomyolysis.
  • myopathy such as myalgia and rhabdomyolysis.
  • evolocumab (trade name: Rebaiaan) is already on the market in China, it is expensive to manufacture, and PCSK9 monoclonal antibody is metabolized by the reticuloendothelial system and needs to be injected every 2-4 weeks.
  • small interfering RNA siRNA
  • siRNA small interfering RNA
  • the object of the present invention is to provide a double-stranded RNAi agent that inhibits PCSK9 gene expression, and its pharmaceutical composition, and the above-mentioned double-stranded RNAi agent, and its pharmaceutical composition are effective in inhibiting or reducing PCSK9 gene expression or treating PCSK9 expression-mediated Methods and uses for diseases or conditions.
  • One embodiment of the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of PCSK9 in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary, and the antisense strand is complementary to the antisense strand.
  • the sense strand comprises a sequence complementary to a part of the sequence of the mRNA encoding PCSK9, wherein each strand is 14 to 30 nucleotides in length, and the sense strand nucleotide sequence in the double-stranded RNAi agent is selected from SEQ ID 14 to 30 nucleotide sequences in NO:1 or SEQ ID NO:2.
  • each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
  • the duplex region of the double-stranded RNAi agent can, for example, be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nuclei Nucleotide pair length, 17-21 nucleotide pair length, 17-19 nucleotide pair length, 19-25 nucleotide pair length, 19-23 nucleotide pair length, 19-21 nuclei nucleotide pair length, 21-25 nucleotide pair length, or 21-23 nucleotide pair length.
  • the duplex region is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.
  • the double-stranded RNAi agent can inhibit the expression of PCSK9 gene in human, monkey, rat or mouse.
  • RNAi agents of the present invention include RNAi agents that have a nucleotide overhang at one end (ie, an agent with one overhang and one blunt end) or have nucleotide overhangs at both ends.
  • the double-stranded RNAi agent may contain one or more overhang regions and/or capping groups at the 3' end, 5' end, or both ends of one or both strands.
  • the overhang may be 1-6 nucleotides in length, such as 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length, the overhangs are arbitrarily selected from U, A, G, C, T.
  • the sense strand of the double stranded RNAi agent has 21 nucleotides and the antisense strand has 23 nucleotides.
  • one or more nucleotides of the sense strand and the antisense strand of the double-stranded RNAi agent have one or more modifications selected from the group consisting of: 2'-methoxyethyl , 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, locked nucleic acid modification, ring-opening or non- Locked nucleic acid modification, DNA modification, fluorescent probe modification.
  • both the sense strand and the antisense strand of the double-stranded RNAi agent contain 2'-O-methyl and/or 2'-fluoro modification.
  • the double-stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage, preferably at least one phosphorothioate linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkages are at the 5' and 3' ends of one strand, preferably Preferably, the internucleotide linkages are at the 5' and 3' ends of the sense and antisense strands; more preferably, the internucleotide linkages are at the sense and antisense strands Between the 3 nucleotides of the 5' and 3' ends.
  • the double-stranded RNAi agent comprises: (1) the antisense strand has an overhang of 5'(s)mN(s)mN3' structure at the 3' end; (2) the antisense At least 2, 6, 14, and 16 positions from the 5' end of the sense strand are modified with fluorine, and other positions are modified with methoxy groups as much as possible; (3) Antisense strands are at least two from the 3' end and 5' end Thio modification; (4) The sense strand starts at the 7th position from the 5' end, and the 9-11 position adopts continuous fluorine modification, and the other positions are used for methoxy modification as much as possible; (5) The sense strand starts from the 5' end The end has at least two thio modifications, and the 3' end is covalently coupled with GalNAc.
  • the double-stranded RNAi agent comprises: (1) a sense strand containing 21 nucleotides, consisting of a 2'-fluoro modified region and a 2'-O-methyl modified region Alternate composition, each modified region is 1 to 3 nucleotides in length; the 5' end and the first modified region from the 3' end are modified in the same way; (2) Antisense strand of 23 nucleotides , consisting of 2'-O-methyl modification regions and 2'-fluoro modification regions alternately, each modification region is 1 to 3 nucleotides in length, and the 1st from the 5' end and 3' end
  • the contiguous nucleotide regions to position 3 are all linked by phosphorothioate backbones.
  • the double-stranded RNAi agent is conjugated to at least one ligand selected from the group consisting of cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs , N-acetylgalactosamine derivatives or analogues, N-acetylglucosamine (GalNAc) derivatives or analogues.
  • ligand selected from the group consisting of cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs , N-acetylgalactosamine derivatives or analogues, N-acetylglucosamine (GalNAc) derivatives or analogues.
  • the ligand is attached to the 3' end, the 5' end and/or the middle of the sequence of the double stranded RNAi agent.
  • N-acetylgalactosamine derivatives or analogs are carried out on the 3' end, 5' end and/or the middle of the sequence of the above-mentioned double-stranded RNAi agent ( X) Modification.
  • X double-stranded RNAi agent
  • n is an integer of 1-15.
  • XX is that two adjacent Xs are connected by a phosphodiester bond or a phosphorothioate bond
  • XXX is that two adjacent Xs are connected by a phosphodiester bond or a phosphorothioate bond
  • XXXX is that two adjacent Xs are connected by a phosphodiester bond or a phosphorothioate bond.
  • XXXX is that two adjacent Xs are connected by a phosphodiester bond or a phosphorothioate bond.
  • the values of n in the two X structures are equal; in the XXX structure, the values of n in the three X structures are equal; in the XXXX structure, the values of n in the four X structures are The values are equal; specifically, n is 3 or 1.
  • the ligand is attached to the 3' end of the sense strand.
  • the ligand in the double-stranded RNAi agent, is one or more GalNAc derivatives linked to a monovalent or trivalent branched linker.
  • the double-stranded RNAi agent comprises:
  • the sense strand composed of AmsAfsAmAfAmGfGmCfAmAfCmAmGmAfGmAfGmGfAmCfAmsGmsAm;
  • antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence GfsAmsCfCmUfGmUfUmUfUfGfCmUfUmUfUmGfUmAfAmCf and the nucleotide sequence
  • the sense strand composed of GmsUfsUmAfCmAfAmAfAmGfCmAmAfAmCfAmGfGmUfCmsUmsAm;
  • the sense strand composed of AmsGfsUmUfAmCfAmAfAmAfGmCmAmAfAmAfCmAfGmGfUmsCmsUm;
  • the antisense strand consisting of the nucleotide sequence CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf and the nucleotide sequence
  • the sense strand composed of CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGmsUm;
  • the antisense strand consisting of the nucleotide sequence UfsUmsUfGmUfAmAfCmUfUfGfAmAfGmAfUmAfUmUfUmAf and the nucleotide sequence
  • the sense strand consisting of UmsAfsAmAfUmAfUmCfUmUfCmAmAmGfUmUfAmCfAmAfAmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence UfsUmsGfUmAfAmCfUmUfGfAfAmGfAmUfAmUfUmUfAmUf and the nucleotide sequence
  • the sense strand composed of AmsUfsAmAfAmUfAmUfCmUfUmCmAmAfGmUfUmAfCmAfAmsAmsAm;
  • the antisense strand consisting of the nucleotide sequence UfsGmsUfAmAfCmUfUmGfAfAfGmAfUmAfUmUfUmAfUmUfUmAfUmUfUmUfUf and the nucleotide sequence
  • the sense strand composed of AmsAfsUmAfAmAfUmAfUmAfUmCfUmUmCmAfAmGfUmUfAmCfAmsAmsAm;
  • the antisense strand consisting of the nucleotide sequence GfsUmsAfAmCfUmUfGmAfAfGfAmUfAmUfUmUfAmUfUmCf and the nucleotide sequence
  • the sense strand composed of GmsAfsAmUfAmAfAmUfAmUfCmUmUmCfAmAfGmUfUmAfCmsAmsAm;
  • the antisense strand consisting of the nucleotide sequence AfsUmsAfUmUfUmAfUmUfCfUfGmGfGmUfUmUfUmGfUmAf and the nucleotide sequence
  • the sense strand consisting of UmsAfsCmAfAmAfAmCfCmCfAmGmAmAfUmAfAmAfUmAfUmsCmsUm;
  • the antisense strand consisting of the nucleotide sequence UfsUmsUfAmUfUmCfUmGfGfGfUmUfUmUfGmUfAmGfCmAf and the nucleotide sequence
  • the sense strand composed of UmsGfsCmUfAmCfAmAfAmAfCmCmCmAfGmAfAmUfAmAfAmsUmsAm;
  • the sense strand composed of AmsAfsAmUfGmCfUmAfCmAfAmAmAmCfCmCfAmGfAmAfUmsAmsAm;
  • the antisense strand consisting of the nucleotide sequence CfsUmsGfGmGfUmUfUmUfGfUfAmGfCmAfUmUfUmUfUmAf and the antisense strand consisting of the nucleotide sequence
  • the sense strand composed of UmsAfsAmAfAmAfUmGfCmUfAmCmAmAfAmAfCmCfCmAfGmsAmsAm;
  • the antisense strand consisting of the nucleotide sequence UfsGmsGfGmUfUmUfUmGfUfAfGmCfAmUfUmUfUmUfAmUf and the nucleotide sequence
  • the sense strand composed of AmsUfsAmAfAmAfAmUfGmCfUmAmCmAfAmAfAmCfCmCfAmsGmsAm;
  • the antisense strand consisting of the nucleotide sequence GfsGmsGfCmUfGmAfGmCfUfUfUmAfAmAfAmUfGmGfUmUf and the nucleotide sequence
  • the sense strand composed of AmsAfsCmCfAmUfUmUfUmAfAmAmGmCfUmCfAmGfCmCfCmsCmsAm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of AfsGmsAfCmCfUmGfUmUfUfUfUfGmCfUmUfUmUfGmUfAmAfGmCmAmGmCmCmdGdAGmGmCmUmsGmsCm consists of the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAfsGm;
  • the antisense strand consisting of AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUmUmUmGmUmAmAm and the sense strand consisting of the nucleotide sequence UmsUfsAmCfAmAmAfGmCfAmAmAfCmAfGmGmUmCfUmsAmsGm;
  • the antisense strand consisting of AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmUmGmUmAmAm and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAmAmAfGmCfAmAmAfCmAfGmGmUmCfUmsAmsGm;
  • the antisense strand consisting of AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmUmGmUmAmAm and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAfAfAfAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm;
  • the antisense strand consisting of AmsGmsAmCmCmUmGfUmUfUm(dT)GmCmUmUmUmUmGmUmAmAm and the nucleotide sequence
  • the sense strand consisting of UmsUfsAmCfAfAfAfAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm;
  • the antisense strand consisting of the nucleotide sequence CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf and the nucleotide sequence
  • the sense strand composed of CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGfsUm;
  • the antisense strand consisting of CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUmAmAmCmUmUmGm and the sense strand consisting of the nucleotide sequence CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm;
  • the antisense strand consisting of CmsUmsGmUmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm and the nucleotide sequence
  • the antisense strand consisting of CmsUmsGmUmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm and the nucleotide sequence
  • the sense strand composed of CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm;
  • the antisense strand consisting of CmsUmsGmUmUmUmUmUfGmCfUm(dT)UmUmGmUmAmAmCmUmUmGm and the nucleotide sequence
  • the sense strand composed of CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm;
  • the antisense strand consisting of CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm consists of the nucleotide sequence
  • the sense strand composed of CmsAfsAmGmUmUfAmCfAfAmAmAmAmGmCfAmAfAmAmAmCmAmGmsGmsUm;
  • the antisense strand consisting of CmsUmsGmUmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm and the nucleotide sequence
  • the sense strand consisting of CmsAfsAmGmUmUfAmCmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm; or
  • the antisense strand consisting of CmsUmsGmUmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm and the nucleotide sequence
  • Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2'-O-methyl respectively;
  • Af, Uf, Cf and Gf represent ribonucleotides modified by 2'-fluoro Ribonucleotides A, U, C and G;
  • s means that the two nucleotides before and after are linked by a phosphorothioate backbone.
  • the double-stranded RNAi agent comprises:
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence UfsGmsUfCmCfUmCfUfGfUmUfGmCfCmUfUmUfUmUfUmUf and an antisense strand consisting of a nucleotide sequence identical to the nucleotide sequence
  • AmsAfsAmAfAmGfGmCfAmAfCmAmGmAfGmAfGmGfAmCfAmsGmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence GfsUmsCfCmUfCmUfCmUfGfUfUmGfCmCfUmUfUmUfUmAf and an antisense strand consisting of a nucleotide sequence identical to the nucleotide sequence
  • UmsAfsAmAfAmAfGmGfCmAfAmCmAmGfAmGfAmGfGmAfCmsAmsGm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf and an antisense strand consisting of a nucleotide sequence identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence GfsAmsCfCmUfGmUfUmUfUfGfCmUfUmUfUmGfUmAfAmCf and an antisense strand consisting of a nucleotide sequence identical to the nucleotide sequence
  • GmsUfsUmAfCmAfAmAfAmGfCmAmAfAmCfAmGfGmUfCmsUmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence AfsCmsCfUmGfUmUfUmUfGfCfUmUfUmUfGmUfAmAfCmUf
  • AmsGfsUmUfAmCfAmAfAmAfGmCmAmAfAmAfCmAfGmGfUmsCmsUm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf and an antisense strand having an identity with the nucleotide sequence
  • CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGmsUm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence UfsUmsUfGmUfAmAfCmUfUfGfAmAfGmAfUmAfUmUfUmAf and an antisense strand composed of a nucleotide sequence identical to the nucleotide sequence
  • UmsAfsAmAfUmAfUmCfUmUfCmAmAmGfUmUfAmCfAmAfAmsAmsGm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence UfsUmsGfUmAfAmCfUmUfGfAfAmGfAmUfAmUfUmUfAmUf and an antisense strand composed of a nucleotide sequence identical to the nucleotide sequence
  • AmsUfsAmAfAmUfAmUfCmUfUmCmAmAfGmUfUmAfCmAfAmsAmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • An antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence UfsGmsUfAmAfCmUfUmGfAfAfGmAfUmAfUmUfUmAfUmUfUmAfUmUfUmUfUf
  • AmsAfsUmAfAmAfUmAfUmCfUmUmCmAfAfAmGfUmUfAmCfAmsAmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • GfsUmsAfAmCfUmUfGmAfAfGfAmUfAmUfUfUmUfAmUfUmCf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of a nucleotide sequence
  • GmsAfsAmUfAmAfAmUfAmUfCmUmUmCfAmAfGmUfUmAfCmsAmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • AfsUmsAfUmUfUmAfUmUfCfUfGmGfGmUfUmUfUmGfUmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of the nucleotide sequence
  • UmsAfsCmAfAmAfAmCfCmCfAmGmAmAfUmAfAmAfUmAfUmsCmsUm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • UfsUmsUfAmUfUmCfUmGfGfGfUmUfUmUfGmUfAmGfCmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of the nucleotide sequence
  • UmsGfsCmUfAmCfAmAfAmAfCmCmCmAfGmAfAmUfAmAfAmsUmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • AfsUmsUfCmUfGmGfGmUfUfUfUmGfUmAfGmCfAmUfUmUf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of the nucleotide sequence
  • AmsAfsAmUfGmCfUmAfCmAfAmAmAmCfCmCfAmGfAmAfUmsAmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • CfsUmsGfGmGfUmUfUmUfGfUfAmGfCmAfUmUfUmUfUmAf has at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence consisting of the antisense strand and the nucleotide sequence
  • UmsAfsAmAfAmAfUmGfCmUfAmCmAmAfAmAfCmCfCmAfGmsAmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • UfsGmsGfGmUfUmUfUmGfUfAfGmCfAmUfUmUfUmUfAmUf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of the nucleotide sequence
  • AmsUfsAmAfAmAfAmUfGmCfUmAmCmAfAmAfAmCfCmCfAmsGmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • GfsGmsGfCmUfGmAfGmCfUfUfUmAfAmAfAmUfGmGfUmUf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity and consisting of the nucleotide sequence
  • AmsAfsCmCfAmUfUmUfUmAfAmAmGmCfUmCfAmGfCmCfCmsCmsAm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAmsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAmsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAmsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAmsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAmsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfUfGmCfUmUfUmUfGmUfAmAfGmCmAmGmCmCmdGdAGmGmCmUmsGmsCm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity and consisting of the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmCfUmsAmsGm has a sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • UmsUfsAmCfAmAfAmAfGmCfAmAmAfCmAfGmGfUmC fUmsAfsGm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUmUmGmUmAmAm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence UmsUfsAmCfAmAmAfGmCfAmAmAfCmAfGmGmUmCfU msAmsGm with at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmGmUmUmGmUmAmAmAm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence UmsUfsAmCfAmAmAfGmCfAmAmAfCmAfGmGmUmCfUmsA msGm with at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmGmUmUmGmUmAmAm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence UmsUfsAmCfAfAfAmAfGmCfAmAfAfCmAfGmGfUmCm UmsAmsGm has at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • AmsGmsAmCmCmUmGfUmUfUm(dT)GmCmUmUmUmUmGmUmAmAm has an antisense strand consisting of a nucleotide sequence that is at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identical to the nucleotide sequence
  • UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUm CmUmsAmsGm has Sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmAfCmAfGmAfGmAfGmAfGfsUm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity and composed of a nucleotide sequence with the nucleotide sequence CmsAfsAmGfUmUmAmCfAmAmAmGmCfAmAfAmAmCmAfGms GmsUm has at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfAmAmCmAfGmsG msUm with at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • CmsAfsAmGfUfUfUfAmCfAmAfAfGmCfAmAfAmAfCmAmGmsGmsUm has at least 90%
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUm(dT)UmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • CmsAfsAmGfUfUfUfUfAmCfAmAfAfGmCfAmAfAmAfCmAmA mGmsGmsUm has Sense strand consisting of nucleotide sequences with at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence CmsAfsAmGmUmUfAmCfAfAmAmAmGmCfAmAfAmAmAmCmAmGmsG msUm with at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100%, identical to the nucleotide sequence
  • CmsAfsAmGmUmUfAmAmAmAmGmCfAmAfAmAmCmAmGmsGms Um have at least 90%,
  • the sense strand preferably consists of nucleotide sequences with 95%, 96%, 97%, 98%, 99%, 100% identity;
  • CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm has an antisense strand consisting of a nucleotide sequence of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity and composed of a nucleotide sequence identical to the nucleotide sequence
  • CmsAfsAmGmUmUmAmCmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm has a sense strand consisting of nucleotide sequences of at least 90%, preferably 95%, 96%, 97%, 98%, 99%, 100% identity;
  • Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2'-O-methyl respectively;
  • Af, Uf, Cf and Gf represent ribonucleotides modified by 2'-fluoro Ribonucleotides A, U, C and G;
  • s means that the two nucleotides before and after are linked by a phosphorothioate backbone.
  • RNAi agent in the double-stranded RNAi agent, the structure of the ligand is shown in formula II:
  • the double-stranded RNAi agent has a structure of formula III, wherein X is O or S:
  • the present invention provides the following examples of double-stranded RNAi agents: E1128, E1129, E1164, E1164-GN1, E1164-GN2, E1164-GN3, E1164-GN4, E1164-GN5, E1164-GN6, E1165, E1166, E1168, E1178, E1179, E1180, E1181, E1192, E1195, E1198, E1201, E1202, E0044, 1164E01, 1164E02, 1164E03, 1164E04, 1164E05, 1164E06, 1168E01, 1168E02, 1168E03, 11 68E04, 1168E05, 1168E06, 1168E07, 1168E08, 1168E09.
  • the present invention also includes DNA molecules capable of producing the above double-stranded RNAi agents, vectors capable of expressing the double-stranded RNAi agents, and reagents or kits containing the double-stranded RNAi agents or the DNA molecules or the vectors.
  • the present invention also provides cells comprising the above double-stranded RNAi agent.
  • the present invention additionally provides a pharmaceutical composition comprising the above-mentioned double-stranded RNAi agent.
  • the pharmaceutical composition includes a pharmacologically effective amount of the double-stranded RNAi agent of the present invention and other pharmaceutically acceptable components.
  • Effective amount refers to the dose of double-stranded RNAi that can effectively produce the expected pharmacological therapeutic effect.
  • “Other components” include water, saline, glucose, buffer (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, preservatives or combinations thereof.
  • the present invention also provides a method for inhibiting the expression of PCSK9 in a cell, the method comprising: (a) contacting the cell with the above-mentioned double-stranded RNAi agent or its pharmaceutical composition; The cells are maintained for a period of time sufficient to achieve degradation of the mRNA transcript of the PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cells.
  • the present invention provides the application of the above-mentioned double-stranded RNAi agent or its pharmaceutical composition in inhibiting PCSK9 gene expression or preparing products for inhibiting PCSK9 gene expression, wherein the inhibition of PCSK9 gene expression is to inhibit or reduce the expression of cells in vivo or in vitro.
  • the cells are mammalian cells expressing PCSK9, such as primate cells and human cells.
  • the PCSK9 gene is expressed at a high level in the target cells.
  • the cells are derived from brain, salivary gland, heart, spleen, lung, liver, kidney, intestine, tumor. Further more preferably, the cells are liver cancer cells and cervical cancer cells.
  • the cells are selected from HepG2, HEP3B, Huh7, COS7, 293T, MHCC97H, Hela, primary mouse hepatocytes, and primary human hepatocytes.
  • the final cellular concentration of the double-stranded RNAi agent is 0.1-1000 nM, such as 10-500 nM, 25-300 nM or 50-100 nM.
  • the double-stranded RNAi agent and pharmaceutical composition thereof can be administered by any suitable means, such as parenteral administration, gastrointestinal administration including intramuscular, intravenous, arterial, peritoneal, or subcutaneous administration injection. Modes of administration include, but are not limited to, single administration or multiple administrations.
  • the dosage range is 0.1 mg/kg to 100 mg/kg, 0.5 mg/kg to 50 mg/kg, 2.5 mg/kg to 20 mg/kg, 5 mg/kg to 15 mg/kg, specifically 3 mg/kg, 5 mg/kg, 10mg/kg, 33mg/kg.
  • the present invention also provides that the double-stranded RNAi agent or its pharmaceutical composition can reduce the concentration of low-density lipoprotein (LDL) and/or low-density lipoprotein cholesterol (LDL-C) in serum or prepare for reducing low-density lipoprotein (LDL) in serum.
  • LDL low-density lipoprotein
  • LDL-C low-density lipoprotein cholesterol
  • said reducing serum low-density lipoprotein (LDL) and/or low-density lipoprotein cholesterol (LDL-C) concentration is to reduce low-density lipoprotein (LDL) and/or low-density lipoprotein cholesterol (LDL-C) in human, monkey, rat or mouse serum Or low-density lipoprotein cholesterol (LDL-C) concentration.
  • the present invention also provides the use of the above-mentioned double-stranded RNAi agent or its pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases mediated by PCSK9 expression or alleviating symptoms of diseases mediated by PCSK9 expression .
  • the present invention also provides the above-mentioned double-stranded RNAi agent or its pharmaceutical composition, which is used for preventing and/or treating a disease mediated by PCSK9 expression or for relieving the symptoms of a disease mediated by PCSK9 expression.
  • the present invention also provides a method for preventing and/or treating a disease mediated by PCSK9 expression or a method for alleviating the symptoms of a disease mediated by PCSK9 expression, which comprises administering effective Amount of the double-stranded RNAi agent of the present invention or its pharmaceutical composition.
  • the diseases mediated by PCSK9 expression include cardiovascular diseases, dyslipidemia or tumor diseases.
  • the cardiovascular diseases include atherosclerotic cardiovascular diseases, and abnormal blood lipids include elevated levels of cholesterol and/or triglycerides in serum, elevated low-density lipoprotein cholesterol, or elevated apolipoprotein B (ApoB), specifically Such as hyperlipidemia in mammals, hypercholesterolemia, nonfamilial hypercholesterolemia, polygenic hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia or heterozygous familial Sexual hypercholesterolemia.
  • Said neoplastic diseases such as PCSK9-related melanoma and metastatic liver cancer.
  • a single dose of the pharmaceutical composition can be sustained for at least 3, 5, 7, 10, 14 days or longer with decreased expression of PCSK9.
  • the innovation of the present invention is reflected in: 1.
  • the RNAi molecule screened by high throughput has a comparable or even higher inhibitory activity than AD-60212 (positive control compound); 2.
  • the modified RNAi molecule has high stability and high inhibition active. 3. While maintaining high inhibitory activity and stability, the ligand-modified RNAi molecule also has better liver targeting and the ability to promote cell endocytosis, which can reduce the impact on other tissues or organs and Reducing the amount of RNAi molecules used can achieve the purpose of reducing toxicity and reducing costs; 4.
  • Ligand-modified RNAi molecules can enter target cells and target tissues without transfection reagents, reducing the negative impact of transfection reagents, such as cells or Tissue toxicity, thus providing the possibility for targeted therapy; 5.
  • modified double-stranded RNAi of the present invention has high stability while maintaining high inhibitory activity.
  • PCSK9 refers to the proprotein convertase subtilisin Kexin9 gene or protein. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARCl. Examples of PCSK9 mRNA sequences are readily available using eg GenBank.
  • G", “C”, “A” and “U” generally represent nucleotides containing guanine, cytosine, adenine and uracil as bases, respectively.
  • ribonucleotide or “nucleotide” or “deoxyribonucleotide” may also refer to a modified nucleotide or an alternative replacement moiety.
  • guanine, cytosine, adenine, and uracil can be substituted by other moieties without substantially changing an oligonucleotide (including a nucleotide having such a substituted moiety) base pairing properties. Sequences comprising such replacement moieties are embodiments of the invention.
  • RNAi agent refers to an agent that mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNAi agents direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The RNAi agent modulates, eg, inhibits, expression of PCSK9 in a cell, such as a cell in a subject (eg, a mammalian subject).
  • RISC RNA-induced silencing complex
  • an RNAi agent of the invention comprises a single-stranded RNA that interacts with a target RNA sequence, eg, a PCSK9 target mRNA sequence, to direct cleavage of the target RNA.
  • a target RNA sequence eg, a PCSK9 target mRNA sequence
  • the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA.
  • RNAi agent used in the compositions, uses, and methods of the invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent".
  • double-stranded RNAi agent refers to a complex of ribonucleic acid molecules, which has a double-stranded structure and comprises two antiparallel and substantially complementary nucleic acid strands, which are referred to as relative to the target RNA, i.e. the PCSK9 gene, Has “sense” and “antisense” orientations.
  • double stranded RNA triggers the degradation of target RNA, eg, mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).
  • Antisense strand refers to the strand of a double-stranded RNAi agent that includes a region that is substantially complementary to a target sequence (eg, a human PCSK9 mRNA).
  • Sense strand refers to the RNA strand that contains a region that is substantially complementary to a region of the antisense strand.
  • “Complementary region” refers to the region on the antisense strand that is completely or substantially complementary to a target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be located in an internal or terminal region of the molecule.
  • the term “complementary” refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.
  • nucleotide overhang refers to one or more unpaired portions protruding from the duplex structure of an RNAi agent when a 3' end of one strand of the RNAi agent extends beyond the 5' end of the other strand. nucleotides, or vice versa.
  • Bount end or “blunt end” means that there are no unpaired nucleotides at that end of the double-stranded RNAi agent, i.e., no nucleotide overhangs.
  • a “blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its length, ie, has no nucleotide overhangs at either end of the molecule.
  • PCSK9 in a cell includes inhibiting any PCSK9 gene (e.g., mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, or human PCSK9 gene) and variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene expression.
  • the PCSK9 gene may be a wild-type PCSK9 gene, a mutant PCSK9 gene, or in the case of a genetically manipulated cell, group of cells or organism, a transgenic PCSK9 gene.
  • the cells are mammalian cells expressing PCSK9, such as primate cells and human cells.
  • the PCSK9 gene is expressed at a high level in the target cells.
  • the cells are derived from brain, salivary gland, heart, spleen, lung, liver, kidney, intestine, tumor. Further more preferably, the cells are liver cancer cells and cervical cancer cells. Even more preferably, the cells are selected from HepG2, HEP3B, Huh7, COS7, 293T, MHCC97H, Hela, primary mouse hepatocytes, and primary human hepatocytes.
  • “Inhibition of PCSK9 gene expression” includes any level of inhibition of the PCSK9 gene, such as at least partial inhibition of expression of the PCSK9 gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25% , at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% , at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, or at least about 99%.
  • Contacting a cell with a double-stranded RNAi agent includes contacting a cell by any means possible. Contacting the cell with the double-stranded RNAi agent includes contacting the cell with the RNAi agent in vitro or contacting the cell with the RNAi agent in vivo. This contacting can be done directly or indirectly. Thus, for example, the RNAi agent may come into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may enter into a condition that permits or causes it to subsequently contact the cell.
  • Disease mediated by PCSK9 expression is intended to include any disease associated with the PCSK9 gene or protein. Such diseases may eg be caused by overproduction of PCSK9 protein, by mutation of the PCSK9 gene, by abnormal cleavage of PCSK9 protein, by abnormal interaction between PCSK9 and other proteins or other endogenous or exogenous substances.
  • Hypercholesterolemia refers to a condition characterized by elevated serum cholesterol.
  • Hyperlipidemia refers to a condition characterized by elevated serum lipids.
  • Non-familial hypercholesterolemia refers to a condition characterized by elevated cholesterol that is not caused by a single inherited gene mutation.
  • Polygenic hypercholesterolemia refers to a condition characterized by elevated cholesterol resulting from the influence of multiple genetic factors.
  • FH familial hypercholesterolemia
  • HoFH Homozygous familial hypercholesterolemia
  • HoFH refers to a condition characterized by mutations in the maternal and paternal LDL-R genes.
  • Heterozygous familial hypercholesterolemia (HoFH) refers to a condition characterized by a mutation in the maternal or paternal LDL-R gene.
  • RNAi agents of the invention A wide variety of entities can be conjugated to the RNAi agents of the invention.
  • Preferred moieties are ligands that are covalently coupled, preferably directly or indirectly via an intervening tether.
  • Ligands can typically include therapeutic modifiers, eg, to enhance uptake; diagnostic compounds or reporter groups, eg, to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance.
  • therapeutic modifiers eg, to enhance uptake
  • diagnostic compounds or reporter groups eg, to monitor distribution
  • cross-linking agents eg., to monitor distribution
  • moieties that confer nuclease resistance include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.
  • the ligand can include a naturally occurring substance such as a protein (e.g. human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL) or globulin); a carbohydrate (e.g. a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • HSA human serum albumin
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • globulin e.g. a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, eg a synthetic polyamino acid, an oligonucleotide (eg an aptamer).
  • polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co- -glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol ( PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphospharazine.
  • PLL polylysine
  • poly-L-aspartic acid poly-L-glutamic acid
  • styrene-maleic anhydride copolymer poly(L-lactide-co- -glycolide) copolymer
  • divinyl ether-maleic anhydride copolymer diviny
  • polyamines examples include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine , amidines, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.
  • Ligands may also include targeting moieties, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies, that bind to a given cell type, such as kidney cells.
  • Targeting groups can be thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N- Acetyl-glucosamine polyvalent mannose, polyvalent trehalose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, Lipids, cholesterol, steroids, cholic acid, folate, vitamin B12, biotin, RGD peptides, RGD peptide mimetics or aptamers.
  • ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, thiophyrin (Sapphyrin)), PAHs (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantaneacetic acid, 1- Pyrene butyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, cetyl glycerin, borneol, menthol, 1,3-propanediol, heptadecyl, Palmitic acid, myristic acid, O3-(oleoyl)
  • the ligand can be a protein, such as a glycoprotein, or a peptide, such as a molecule with a specific affinity for a co-ligand, or an antibody, such as binds to a specified cell type (such as a cancer cell, endothelial cell, or bone cells) an antibody.
  • Ligands can also include hormones and hormone receptors. They can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine Multivalent mannose, multivalent fucose or aptamer.
  • the ligand may be, for example, lipopolysaccharide, an activator of p38MAP kinase or an activator of NF- ⁇ B.
  • a ligand can be a substance, eg, a drug, that can increase uptake of an iRNA agent into a cell, eg, by disrupting the cytoskeleton of the cell (eg, by disrupting cellular microtubules, microfilaments, and/or intermediate filaments).
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, red sea spongin A, phalloidin, marine bryosin ( swinholide) A, indanocine or myoservin.
  • the ligand can be any ligand capable of targeting a particular receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters (eg GalNAc clusters, mannose clusters, galactose clusters) or an aptamer. A cluster is a combination of two or more sugar units.
  • These targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL Ligand.
  • the ligands can also be based on nucleic acids, such as an aptamer.
  • the aptamer can be unmodified or have any combination of modifications disclosed herein.
  • FIG. 1 Gene expression of DLR high-throughput screening of PCSK9 candidate sequences.
  • FIG. 1 Gene expression of Top16 candidate modified sequences in Hep3B cells.
  • Figure 3 Free uptake experiment of GalNAc-siRNA in primary human hepatocytes.
  • Figure 4 EC 50 values of GalNAc-siRNA candidate sequences in primary human hepatocytes.
  • Figure 5 IC 50 values of GalNAc-siRNA candidate sequences in HEP3B cells.
  • Figure 6 The binding ability of different GalNAc-modified siRNAs to primary mouse hepatocytes.
  • Figure 7 Liver targeting test of GalNAc-siRNA in vivo.
  • Figure 8 In vivo efficacy test of GalNAc-siRNA mice.
  • FIG. 9 Gene expression of Top2 candidate sequence modification strategies in Hep3B cells.
  • Figure 11 Screening experiments for candidate sequences to reduce mouse PCSK9 protein levels.
  • Figure 12A Changes in LDLc levels caused by candidate sequences in cynomolgus monkeys.
  • Figure 12B Changes in PCSK9 protein levels caused by candidate sequences in cynomolgus monkeys.
  • Figure 12C PK distribution of candidate sequences in cynomolgus monkeys.
  • Figure 13 Toxicology studies of candidate sequences in mice.
  • sequence design method refers to the methods of Elbashir et al.2002; Paddison et al.2002; Reynolds et al.2004; Ui-Tei et al.2004 et al.
  • siRNA synthesis (natural RNA/2'-methoxy or 2'-fluoro modified RNA/GalNAc-RNA)
  • Deprotection was performed using 3% TCA, activation was performed with 0.3 M benzylthiotetrazole in acetonitrile, and capping and oxidation were performed by CAPA/CAPB and 50 mM I2 solutions, respectively.
  • Trityl-off synthesis the solid-phase support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia water was added and heated in an oven at 65° C. for 3 h to remove the protecting group.
  • microplate reader uses a microplate reader to determine the concentration by ultraviolet light, mix equimolar amounts of sense strand and antisense strand into a new centrifuge tube, heat at 95°C for 5min, and slowly anneal to room temperature, and finally use a vacuum concentrator to spin dry at room temperature to obtain the final product.
  • the psicheck-2 plasmid was used to construct a recombinant plasmid (Shanghai Jierui Bioengineering Co., Ltd.), which contained all the target sequences of PCSK9 siRNA to be tested, and the cloning site was the 5'XhoI and 3'NotI sites of the psicheck-2 plasmid.
  • the cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO 2 at 37°C, and plated and transfected when the cells were in the logarithmic growth phase and in good condition (70% confluence). Adjust the cell density, and seed 1.5 ⁇ 10 5 cells per well into a 24-well plate.
  • Prepare transfection complex Mix 250 ⁇ L Opti-MEM, 40ng recombinant plasmid and 5 ⁇ L 10nM siRNA, mix 250 ⁇ L Opti-MEM and 2.5 ⁇ L Lipofectamine 2000 transfection reagent, let stand for 5 minutes, then mix the above two mixtures, let stand for 20 minutes .
  • the above-mentioned transfection complex was added into a 24-well plate, and incubated in a constant temperature incubator at 37° C. for 6 hours in 5% CO 2 . Aspirate the supernatant, add 1 mL of complete medium to each well, and continue culturing for 24 h.
  • control groups were also set up: NC as a negative control (irrelevant siRNA), Lipo group as a transfection reagent control group, and Blank group as an untreated control group (without siRNA). Both the test group and the control group had 3 repetitions.
  • the Dual-Luciferase Reporter Assay System kit (Promega) was used for detection, and the processed cells were lysed and collected according to the kit instructions, and the firefly (Photinus pyralis) luciferase and Renilla reniformis (Renilla reniformis) were sequentially detected with an Infinite Eplex microplate reader (TECAN) ) luciferase fluorescence intensity, calculate the ratio of the fluorescence intensity of Renilla reniformis luciferase to firefly (Photinus pyralis) luciferase, and normalize with the NC group as the control.
  • Table 4 is the result of DLR detection, the average value of the expression level of the dual luciferase reporter gene of the PCSK9 siRNA test group relative to the NC group.
  • serial number 293T-01 293T-02 COS.7-01 COS.7-02 average value U1128 0.2741 0.1768 0.4016 0.4992 0.3379 U1129 0.2375 0.2266 0.5082 0.5239 0.3741 U1164 0.1032 0.1090 0.1519 0.2200 0.1460 U1165 0.1339 0.1347 0.2311 0.3754 0.2188 U1166 0.1121 0.0831 0.1529 0.3702 0.1796 U1168 0.2384 0.0948 0.2239 0.1471 0.1761 U1178 0.2235 0.1579 0.3495 0.3638 0.2737 U1179 0.1220 0.0787 0.1554 0.2810 0.1593 U1180 0.1729 0.1370 0.1666 0.3562 0.2082 U1181 0.3573 0.2243 0.3162 0.4211 0.3297 U1192 0.4567 0.3359 0.2006 0.2764 0.3174 U1195 0.3495 0.1332 0.1928 0.2581 0.2334 U1198 0.2596 0.1193 0.1407 0.1836
  • PCSK9 siRNA DLR screening in COS7 and 293T cells respectively found the top 16 siRNA molecules with high cell activity, which were homologous to human, cynomolgus monkey and rhesus monkey.
  • the sense strand in the first 16 siRNA molecules with higher cell activity corresponds to the position between 3567-3663 or 2483-2505 of PCSK9, and its nucleotide sequences are respectively as SEQ ID NO: 1 and SEQ ID NO: Shown in 2: TCTGTCCTCTCTGTTGCCTTTTTTACAGCCAACTTTTCTAGACCTGTTTTTTGCTTTTGTAACTTGAAGATATTTATTCTGGGTTTTGTAGCATTTTTAT (SEQ ID NO: 1)
  • Hep3B cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 5% CO 2 at 37°C, and plated and transfected when the cells were in the logarithmic growth phase and in good condition (70% confluence) . Adjust the cell density, and seed 1.5 ⁇ 10 5 cells per well into a 24-well plate.
  • Prepare the transfection complex mix 250 ⁇ L Opti-MEM and 5 ⁇ L 10 nM siRNA, mix 250 ⁇ L Opti-MEM and 2.5 ⁇ L Lipofectamine 2000 transfection reagent, let stand for 5 minutes, then mix the above two mixtures and let stand for 20 minutes.
  • the above-mentioned transfection complex was added into a 24-well plate, and incubated in a constant temperature incubator at 37° C. for 6 hours in 5% CO 2 . Aspirate the supernatant, add 1 mL of complete medium to each well, and continue culturing for 24 h.
  • the following control groups were also set up: the NC group was a negative control (irrelevant siRNA), the Lipo group was a transfection reagent control, and the Blank group was an untreated control (without siRNA).
  • Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2'-O-methyl, respectively;
  • Af, Uf, Cf and Gf represent ribonucleotides modified by 2'-fluoro Substituting modified ribonucleotides A, U, C and G;
  • s means that the two nucleotides before and after are connected by a phosphorothioate backbone, and the structure of L96 is as shown in formula IV:
  • the cryopreserved primary human hepatocytes (purchased from the Chinese Academy of Sciences Collection Cell Bank) were recovered, the primary human hepatocytes were adjusted to an appropriate density, and then the primary human hepatocytes were seeded into 96-well plates.
  • GalNAc-siRNA was diluted with PBS to a concentration of 100nM and 1nM, and then a certain volume of diluted GalNAc-siRNA was added to a 96-well plate and incubated with primary human hepatocytes for 48 hours. The supernatant was aspirated, the cells were collected, the total cellular RNA was extracted using a column extraction kit, and the relative expression of PCSK9 mRNA was detected by qPCR. It can be seen from the results ( FIG. 3 ), that GalNAc-siRNA can enter the liver cells through endocytosis and silence the expression of PCSK9 gene, and the inhibitory activity of E1164 and E1168 is relatively high.
  • the primary human hepatocytes were adjusted to a suitable density and seeded in 96-well plates. Dilute E1164 and E1168 with PBS to different concentrations, set the highest final concentration to 100nM, make a 10-fold gradient dilution to a total of 8 concentrations, then take a certain volume of E1164 and E1168 with different concentrations and add them to a 96-well plate to mix with human primary liver Cells were co-incubated for 48 hours. The supernatant was aspirated, the cells were collected, the total cellular RNA was extracted using a column extraction kit, the relative expression of PCSK9 mRNA was detected by qPCR, and the EC 50 value was analyzed and calculated by Graphpad Prism software. From Figure 4, the EC 50 values of E1164 and E1168 in primary human hepatocytes are 5.66nM and 3.45nM, respectively.
  • E1164, E1168 and the positive control AD60212 were diluted to different concentrations with Nuclease-Free Water (Invitrogen), the highest final concentration was set to 100nM, and a total of 8 concentrations were made for 10-fold serial dilution, and Hep3B cells (purchased from the Collection Cell Bank of the Chinese Academy of Sciences) were transfected. ).
  • Nuclease-Free Water Invitrogen
  • Hep3B cells purchased from the Collection Cell Bank of the Chinese Academy of Sciences
  • the IC50 values of E1164, E1168 and positive control AD60212 in Hep3B cells are 0.031nM, 0.005nM and 0.224nM respectively, and the IC50 values of E1164 and E1168 in Hep3B cells are 1/10 of the positive control AD60212. 1/100, significantly better than the positive control AD60212.
  • the liver was removed and placed in Solution III (10% FBS low-glucose DMEM, 4°C) to terminate digestion, the liver capsule was scratched with tweezers, and the liver cells were released by shaking gently. Filter the hepatocytes with a 70 ⁇ m cell strainer, centrifuge at 50 g for 2 min, and discard the supernatant. Resuspend the cells with Solution IV (40% percoll low-glucose DMEM, 4°C), centrifuge at 100g for 2min and discard the supernatant. Add 2% FBS low-glucose DMEM to resuspend the cells, set aside. Cell viability was identified by trypan blue staining.
  • Solution III 50% FBS low-glucose DMEM, 4°C
  • the mean fluorescence intensity MFI of living cells was measured by flow cytometry, and the GraphPad Prism5 software was used for nonlinear fitting and Kd value calculation.
  • the data show that the ligand-modified GalNAc-siRNA promotes endocytosis/uptake by hepatocytes in vitro without adding transfection reagents, and realizes delivery to hepatocytes.
  • GalNAc-siRNAs with different GalNAc structures have different endocytosis and receptor binding abilities (Figure 6), where A1-A6 correspond to E1164-GN1-E1164-GN6 respectively.
  • Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2'-O-methyl, respectively;
  • Af, Uf, Cf and Gf represent ribonucleotides modified by 2'-fluoro Substituting modified ribonucleotides A, U, C and G;
  • s means that the two nucleotides before and after are connected by a phosphorothioate backbone, Cy5 means Cyanine 5 fluorescent dye, and the structure of L96 is shown in formula IV:
  • mice Thirty-six male, 6- to 7-week-old SPF-grade C57 mice (Nanjing Model Organisms Experimental Animal Co., Ltd.) were used in the experiment. They were randomly divided into 6 groups, and the number of animals in each group was 6. 5mg/kg (experimental design see Table 9). Live imaging was performed on all animals 2h, 4h, 24h, and 48h after administration. After euthanasia 48 hours after drug administration, the heart, spleen, lung, liver, and kidney were removed for ex vivo organ imaging (Fig. 7).
  • E1164-GN1 C57 male, six weeks old 1#, 2#, 3#, 4#, 5#, 6# E1164-GN2 C57, male, six weeks old 7#, 8#, 9#, 10#, 11#, 12# E1164-GN3 C57, male, six weeks old 13#, 14#, 15#, 16#, 17#, 18# E1164-GN4 C57, male, six weeks old 19#, 20#, 21#, 22#, 23#, 24# E1164-GN5 C57, male, six weeks old 25#, 26#, 27#, 28#, 29#, 30# E1164-GN6 C57, male, six weeks old 31#, 32#, 33#, 34#, 35#, 36#
  • a total of 50 humanized PCSK9 male and female mice aged 6-8 weeks were used in the experiment. They were randomly divided into groups according to body weight, with 10 mice in each group, 5 male and 5 mice, and a single dose of 3 mg/kg subcutaneously Administration by injection (see Table 10 for administration groups).
  • Table 11 is from the average PCSK9 protein before the normalization to treatment of embodiment 3-three
  • Embodiment 4 optimization of chemical modification platform
  • Am, Um, Cm and Gm represent ribonucleotides A, U, C and G modified by 2'-O-methyl, respectively;
  • Af, Uf, Cf and Gf represent ribonucleotides modified by 2'-fluoro Substituting modified ribonucleotides A, U, C and G;
  • s means that the two nucleotides before and after are connected by a phosphorothioate backbone, and the structure of L96 is shown in formula IV:
  • the protocol of modification is important and some modifications will reduce the inhibitory activity.
  • the preferred modification scheme has high inhibitory activity: (1) the antisense strand has an overhang of 5'(s)mN(s)mN3' structure at the 3' end, which is conducive to loading the antisense strand into RISC and strengthening RNAi interfere with activity without affecting stability.
  • the antisense strand is modified with fluorine at least at positions 2, 6, 14, and 16 from the 5' end, and the other positions are modified with methoxy as much as possible, which is conducive to RISC binding; (3) The antisense strand is modified from the 3' At least two thio modifications from the 5' end and the 5' end are conducive to maintaining the stability of the nucleic acid; (4) The sense strand is the 7th position from the 5' end, and the 9-11 positions are continuously modified with fluorine, and the other positions are as far as possible The use of methoxy modification is beneficial to RISC binding; (5) At least two sulfur modifications from the 5' end of the sense strand, and the 3' end is covalently coupled with GalNAc. Next, the U1168 sequence was selected for screening of different chemical modifications.
  • mice A total of 50 humanized PCSK9 male and female mice aged 6-8 weeks (Nanjing Model Organisms Experimental Animal Co., Ltd.) were used in the experiment. They were randomly divided into groups according to body weight, with 10 mice in each group, 5 male and 5 mice, and a single dose of 3 mg/kg subcutaneously Administration by injection (see Table 13 for administration groups).
  • mice showed ( Figure 11, Table 15), the positive control AD60212 reduced the PCSK9 protein level by 80% on the 10th day, continued to rebound after the 31st day, and returned to the pre-drug level on the 73rd day; while the candidate In the molecule, 1168E06 and 1168E07 were less effective than the positive control, and returned to the pre-drug level on the 59th day; the 1168E08 molecule showed the lowest point on the 13th day, which decreased by 80%, and then continued to rebound on the 38th day, and returned to the level on the 80th day
  • the pre-drug level is about a week behind the positive control rebound cycle.
  • Embodiment 6 detection of effectiveness in vivo in cynomolgus monkeys
  • the concentration of LDL-C in serum was detected by Toshiba TBA120 blood biochemical analyzer, and compared between groups.
  • PCSK9 protein in serum was detected by Monkey Proprotein convertase subtilisin/kexin type 9 (PCSK9) ELISA kit (CUSABIO), and compared between groups.
  • the serum collected from cynomolgus monkeys at different times was quantitatively analyzed by mass spectrometry, and the drug concentration levels at different times were obtained by calibration of the standard curve of the standard.
  • Embodiment 7 mouse toxicology study

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种抑制PCSK9基因表达的RNAi剂及其应用，本发明涉及一种修饰的双链RNAi剂及其应用，具体涉及一种抑制PCSK9基因表达的双链RNAi剂及其药物组合物，以及使用所述双链RNAi剂或其药物组合物用于治疗由PCSK9表达介导的疾病的应用。

Description

一种抑制PCSK9基因表达的RNAi剂及其应用 技术领域
本发明属于分子生物学领域，涉及一种修饰的双链RNAi剂及其应用，具体涉及一种抑制PCSK9基因表达的双链RNAi剂及其药物组合物，以及使用所述双链RNAi剂或其药物组合物用于治疗患有由PCSK9表达介导的疾病的应用。
背景技术
RNA干扰(RNAi)广泛存在于自然界的物种中，自Andrew Fire和Craig Mello等人1998年在线虫(Caenorhabditiselegans，C.elegans)中首次发现RNAi现象，Tuschl和Phil Sharp等人2001年证实在哺乳动物中也存在RNAi之后，关于RNAi的机制原理、基因功能和临床应用等研究取得了一系列的进展。RNAi在防御病毒感染，防转座子跳跃等多种机体保护机制中发挥关键作用(Hutvágneretal.,2001；Elbashiretal.,2001；Zamore 2001)。基于RNAi机制开发的产品是十分有前景的候选药物。小干扰RNA(small interferingRNA，siRNA)能够发挥RNA干扰作用，是实现RNAi的主要工具。
前蛋白转化酶枯草杆菌蛋白酶/kexin-9(也称为PCSK9)是一种丝氨酸蛋白酶，其通过控制质膜上肝脏和肝外LDL受体(LDLR)的表达而间接调节血浆LDL胆固醇水平。PCSK9蛋白表达降低会增加LDLR受体的表达，从而降低血浆LDL胆固醇和由此导致的高胆固醇血症和/或动脉粥样硬化以及由此引起的并发症。同时，研究发现小鼠PCSK9敲除后，血中胆固醇水平降低，以及表现出对他汀类药物在降低血液胆固醇中增强的敏感性。以上研究显示，PCSK9的抑制剂对于血液中LDL-C浓度的降低，以及对治疗PCSK9介导的疾病可能是有益的，因此有望成为控制高胆固醇血症及其并发症潜在的治疗靶点。
目前胆固醇血症及其并发症的临床治疗手段主要还是以他汀类小分子药物为主，研究表明对他汀类药物不耐受患者，可引起肌肉病等不良反应，如肌肉痛、横纹肌溶解。虽然目前中国已有依洛尤单抗(evolocumab，商品名瑞百安)上市，但是其造价昂贵，且PCSK9单抗经网状内皮***代谢每隔2-4周就需要注射。研究表明，小干扰RNA(siRNA)可特异性沉默PCSK9基因，从而抑制其蛋白表达，降低低密度脂蛋白(LDL-c)，同时在欧盟和美国获批的Inclisiran药物由于其持久的疗效(每半年皮下注射一针)正在为高胆固 醇血症患者带来希望。因此，开发一种高效的沉默PCSK9的抑制剂，将为长效治疗高胆固醇血症提供有效的手段，使其具有更好的疗效、特异性、稳定性、靶向性或耐受性等。
发明内容
本发明的目的是提供一种抑制PCSK9基因表达的双链RNAi剂、及其药物组合物，以及上述双链RNAi剂、及其药物组合物在抑制或降低PCSK9基因表达或治疗PCSK9表达介导的疾病或症状的方法和用途。
本发明一个实施例中提供一种双链RNAi剂，其能够抑制细胞中PCSK9的表达，其中所述双链RNAi剂包含有义链和反义链，其中有义链与反义链互补，反义链包含与编码PCSK9的mRNA的一部分序列互补的序列，其中每个链在长度上为14至30个核苷酸，并且所述双链RNAi剂中有义链核苷酸序列选自SEQ ID NO:1或SEQ ID NO:2中的14至30个核苷酸序列。
例如，每条链(有义链或反义链)可以在14-30个核苷酸长度、17-30个核苷酸长度、25-30个核苷酸长度、27-30个核苷酸长度、17-23个核苷酸长度、17-21个核苷酸长度、17-19个核苷酸长度、19-25个核苷酸长度、19-23个核苷酸长度、19-21个核苷酸长度、21-25个核苷酸长度或21-23个核苷酸长度之间。
该双链RNAi剂的双链体区可以，例如，在14-30个核苷酸对长度、17-30个核苷酸对长度、27-30个核苷酸对长度、17-23个核苷酸对长度、17-21个核苷酸对长度、17-19个核苷酸对长度、19-25个核苷酸对长度、19-23个核苷酸对长度、19-21个核苷酸对长度、21-25个核苷酸对长度或21-23个核苷酸对长度之间。
在另一个实例中，该双链体区具有选自15、16、17、18、19、20、21、22、23、24、25、26以及27个核苷酸对长度。
本发明的一个实施例中，所述双链RNAi剂能够抑制人、猴、大鼠或小鼠的PCSK9基因表达。
本发明的RNAi剂包括在一端处具有核苷酸突出端(即，具有一个突出端和一个平端的试剂)或在两端处都具有核苷酸突出端的RNAi剂。
在一个实施例中，所述双链RNAi剂可以在一条链或两条链的3'端、5'端、或两端处含有一个或多个突出端区和/或封端基团。突出端可以具有1-6个核苷酸长度、例如2-6 个核苷酸长度、1-5个核苷酸长度、2-5个核苷酸长度、1-4个核苷酸长度、2-4个核苷酸长度、1-3个核苷酸长度、2-3个核苷酸长度、或1-2个核苷酸长度，突出端任意选自U、A、G、C、T。
在一个实施例中，所述双链RNAi剂的有义链具有21个核苷酸，所述反义链具有23个核苷酸。
在另一个实例中，所述的双链RNAi剂的有义链和反义链的一个或多个核苷酸上具有选自下组的一个或多个修饰：2'-甲氧基乙基、2'-O-烷基、2'-O-烯丙基、2'-C-烯丙基、2'-氟、2'-脱氧、2'-羟基、锁核酸修饰、开环或非锁核酸修饰、DNA修饰、荧光探针修饰。
在本发明的一个实施例中，所述双链RNAi剂的有义链和反义链均含有2'-O-甲基和/或2'-氟修饰。
在本发明的另一个实例中，所述双链RNAi剂，进一步包含至少一个硫代磷酸酯或甲基膦酸酯核苷酸间键合，优选包含至少一个硫代磷酸酯键合。
在本发明的另一个实例中，所述的双链RNAi剂中，所述硫代磷酸酯或甲基膦酸酯核苷酸间键合是在一条链的5'和3'末端处，优选地，所述核苷酸间键合是在有义链和反义链的5'和3'末端处；更优选地，所述核苷酸间键合是在有义链和反义链的5'和3'末端的3个核苷酸间。
在本发明的另一个实施例中，所述双链RNAi剂包括：(1)反义链在3'端处具有5'(s)mN(s)mN3'结构的突出端；(2)反义链从5'端起至少第2，6，14，16位使用氟代修饰，其他位置尽可能采用甲氧基修饰；(3)反义链从3'端和5'端起至少两个硫代修饰；(4)正义链从5'端起第7位，且9-11位采用连续的氟代修饰，其他位置尽可能用于甲氧基修饰；(5)有义链从5'端起至少两个硫代修饰，3'端采用GalNAc共价偶联。
在本发明的另一个实施例中，所述双链RNAi剂包括：(1)含21个核苷酸的有义链，由2'-氟代修饰区和2'-O-甲基修饰区交替组成，每个修饰区的长度为1至3个核苷酸；5'末端和3'末端起的第一个修饰区的修饰方式相同；(2)含23个核苷酸的反义链，由2'-O-甲基修饰区和2'-氟代修饰区交替组成，每个修饰区的长度为1至3个核苷酸，且从5'末端和3'末端起的第1至3位的连续的核苷酸区，均由硫代磷酸骨架连接。
在本发明的一个实施例中，所述双链RNAi剂与至少一个配体缀合，所述配体选自由胆固醇、生物素、维生素、半乳糖衍生物或类似物、乳糖衍生物或类似物、N-乙酰半乳糖胺衍生物或类似物、N-乙酰葡萄糖胺(GalNAc)衍生物或类似物组成的组。
在一些实施方案中，所述配体连接到所述双链RNAi剂的3’末端、5’末端和/或序列中间。
在一些实施方案中，对上述双链RNAi剂的3’末端、5’末端和/或序列中间进行1-5个、2-4个或3个N-乙酰半乳糖胺衍生物或类似物(X)修饰。具体的，单个所述N-乙酰半乳糖胺衍生物的结构如式I所示：
Figure PCTCN2023071189-appb-000001
其中，n为1-15的整数。
在本发明的一个实施例中，XX是相邻的两个X通过磷酸二酯键或硫代磷酸二酯键相连，XXX是相邻的两个X通过磷酸二酯键或硫代磷酸二酯键相连，XXXX是相邻的两个X通过磷酸二酯键或硫代磷酸二酯键相连。所述XX结构中，两个X结构中n的取值相等；在所述XXX结构中，三个X结构中n的取值相等；在所述XXXX结构中，四个X结构中n的取值相等；具体的，n为3或1。
优选地，所述配体连接到有义链的3’端。
在本发明的一个实例中，所述双链RNAi剂中，所述配体是与一种单价或三价支链连接物连接的一种或多种GalNAc衍生物。
在本发明的一个实例中，所述双链RNAi剂包括：
(1)由核苷酸序列UfsGmsUfCmCfUmCfUmCfUfGfUmUfGmCfCmUfUmUfUmUf组成的反义链和由核苷酸序列
AmsAfsAmAfAmGfGmCfAmAfCmAmGmAfGmAfGmGfAmCfAmsGmsAm组成的有义链；
(2)由核苷酸序列GfsUmsCfCmUfCmUfCmUfGfUfUmGfCmCfUmUfUmUfUmAf组成的反义链和由核苷酸序列
UmsAfsAmAfAmAfGmGfCmAfAmCmAmGfAmGfAmGfGmAfCmsAmsGm组成的有义链；
(3)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(4)由核苷酸序列GfsAmsCfCmUfGmUfUmUfUfGfCmUfUmUfUmGfUmAfAmCf组成的反义链和由核苷酸序列
GmsUfsUmAfCmAfAmAfAmGfCmAmAmAfAmCfAmGfGmUfCmsUmsAm组成的有义链；
(5)由核苷酸序列AfsCmsCfUmGfUmUfUmUfGfCfUmUfUmUfGmUfAmAfCmUf组成的反义链和由核苷酸序列
AmsGfsUmUfAmCfAmAfAmAfGmCmAmAfAmAfCmAfGmGfUmsCmsUm组成的有义链；
(6)由核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf组成的反义链和由核苷酸序列
CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGmsUm组成的有义链；
(7)由核苷酸序列UfsUmsUfGmUfAmAfCmUfUfGfAmAfGmAfUmAfUmUfUmAf组成的反义链和由核苷酸序列
UmsAfsAmAfUmAfUmCfUmUfCmAmAmGfUmUfAmCfAmAfAmsAmsGm组成的有义链；
(8)由核苷酸序列UfsUmsGfUmAfAmCfUmUfGfAfAmGfAmUfAmUfUmUfAmUf组成的反义链和由核苷酸序列
AmsUfsAmAfAmUfAmUfCmUfUmCmAmAfGmUfUmAfCmAfAmsAmsAm组成的有义链；
(9)由核苷酸序列UfsGmsUfAmAfCmUfUmGfAfAfGmAfUmAfUmUfUmAfUmUf组成的反义链和由核苷酸序列
AmsAfsUmAfAmAfUmAfUmCfUmUmCmAfAmGfUmUfAmCfAmsAmsAm组成的有义链；
(10)由核苷酸序列GfsUmsAfAmCfUmUfGmAfAfGfAmUfAmUfUmUfAmUfUmCf组成的反义链和由核苷酸序列
GmsAfsAmUfAmAfAmUfAmUfCmUmUmCfAmAfGmUfUmAfCmsAmsAm组成的有义链；
(11)由核苷酸序列AfsUmsAfUmUfUmAfUmUfCfUfGmGfGmUfUmUfUmGfUmAf组成的反义链和由核苷酸序列
UmsAfsCmAfAmAfAmCfCmCfAmGmAmAfUmAfAmAfUmAfUmsCmsUm组成的有义链；
(12)由核苷酸序列UfsUmsUfAmUfUmCfUmGfGfGfUmUfUmUfGmUfAmGfCmAf组成的反义链和由核苷酸序列
UmsGfsCmUfAmCfAmAfAmAfCmCmCmAfGmAfAmUfAmAfAmsUmsAm组成的有义链；
(13)由核苷酸序列AfsUmsUfCmUfGmGfGmUfUfUfUmGfUmAfGmCfAmUfUmUf组成的反义链和由核苷酸序列
AmsAfsAmUfGmCfUmAfCmAfAmAmAmCfCmCfAmGfAmAfUmsAmsAm组成的有义链；
(14)由核苷酸序列CfsUmsGfGmGfUmUfUmUfGfUfAmGfCmAfUmUfUmUfUmAf组成的反义链和由核苷酸序列
UmsAfsAmAfAmAfUmGfCmUfAmCmAmAfAmAfCmCfCmAfGmsAmsAm组成的有义链；
(15)由核苷酸序列UfsGmsGfGmUfUmUfUmGfUfAfGmCfAmUfUmUfUmUfAmUf组成的反义链和由核苷酸序列
AmsUfsAmAfAmAfAmUfGmCfUmAmCmAfAmAfAmCfCmCfAmsGmsAm组成的有义链；
(16)由核苷酸序列GfsGmsGfCmUfGmAfGmCfUfUfUmAfAmAfAmUfGmGfUmUf组成的反义链和由核苷酸序列
AmsAfsCmCfAmUfUmUfUmAfAmAmGmCfUmCfAmGfCmCfCmsCmsAm组成的有义链；
(17)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(18)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(19)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(20)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(21)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(22)由核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAfGmCmAmGmCmCmdGdAGmGmCmUmsGmsCm组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm组成的有义链；
(23)由核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf组成的反义链和由核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAfsGm组成的有义链；
(24)由核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUmUmUmGmUmAmAm组成的反义链和由核苷酸序列UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm组成的有义链；
(25)由核苷酸序列
AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm组成的反义链和由核苷酸序列
UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm组成的有义链；
(26)由核苷酸序列
AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm组成的反义链 和由核苷酸序列
UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm组成的有义链；
(27)由核苷酸序列
AmsGmsAmCmCmUmGfUmUfUm(dT)GmCmUmUmUmUmGmUmAmAm组成的反义链和由核苷酸序列
UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm组成的有义链；
(28)由核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf组成的反义链和由核苷酸序列
CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGfsUm组成的有义链；
(29)由核苷酸序列
CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm组成的有义链；
(30)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列
CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm组成的有义链；
(31)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列
CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm组成的有义链；
(32)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUm(dT)UmUmGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列
CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm组成的有义链；
(33)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm组成的反义链和 由核苷酸序列
CmsAfsAmGmUmUfAmCfAfAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm组成的有义链；
(34)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列
CmsAfsAmGmUmUfAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm组成的有义链；或
(35)由核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm组成的反义链和由核苷酸序列
CmsAfsAmGmUmUmAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm组成的有义链；
其中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接。
在本发明的一些实例中，所述双链RNAi剂包括：
(1)由与核苷酸序列UfsGmsUfCmCfUmCfUmCfUfGfUmUfGmCfCmUfUmUfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsAfsAmAfAmGfGmCfAmAfCmAmGmAfGmAfGmGfAmCfAmsGmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(2)由与核苷酸序列GfsUmsCfCmUfCmUfCmUfGfUfUmGfCmCfUmUfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsAfsAmAfAmAfGmGfCmAfAmCmAmGfAmGfAmGfGmAfCmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(3)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(4)由与核苷酸序列GfsAmsCfCmUfGmUfUmUfUfGfCmUfUmUfUmGfUmAfAmCf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
GmsUfsUmAfCmAfAmAfAmGfCmAmAmAfAmCfAmGfGmUfCmsUmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(5)由与核苷酸序列AfsCmsCfUmGfUmUfUmUfGfCfUmUfUmUfGmUfAmAfCmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsGfsUmUfAmCfAmAfAmAfGmCmAmAfAmAfCmAfGmGfUmsCmsUm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(6)由与核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(7)由与核苷酸序列UfsUmsUfGmUfAmAfCmUfUfGfAmAfGmAfUmAfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsAfsAmAfUmAfUmCfUmUfCmAmAmGfUmUfAmCfAmAfAmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(8)由与核苷酸序列UfsUmsGfUmAfAmCfUmUfGfAfAmGfAmUfAmUfUmUfAmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsUfsAmAfAmUfAmUfCmUfUmCmAmAfGmUfUmAfCmAfAmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(9)由与核苷酸序列UfsGmsUfAmAfCmUfUmGfAfAfGmAfUmAfUmUfUmAfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsAfsUmAfAmAfUmAfUmCfUmUmCmAfAmGfUmUfAmCfAmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(10)由与核苷酸序列
GfsUmsAfAmCfUmUfGmAfAfGfAmUfAmUfUmUfAmUfUmCf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
GmsAfsAmUfAmAfAmUfAmUfCmUmUmCfAmAfGmUfUmAfCmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(11)由与核苷酸序列
AfsUmsAfUmUfUmAfUmUfCfUfGmGfGmUfUmUfUmGfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsAfsCmAfAmAfAmCfCmCfAmGmAmAfUmAfAmAfUmAfUmsCmsUm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(12)由与核苷酸序列
UfsUmsUfAmUfUmCfUmGfGfGfUmUfUmUfGmUfAmGfCmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsGfsCmUfAmCfAmAfAmAfCmCmCmAfGmAfAmUfAmAfAmsUmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(13)由与核苷酸序列
AfsUmsUfCmUfGmGfGmUfUfUfUmGfUmAfGmCfAmUfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsAfsAmUfGmCfUmAfCmAfAmAmAmCfCmCfAmGfAmAfUmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(14)由与核苷酸序列
CfsUmsGfGmGfUmUfUmUfGfUfAmGfCmAfUmUfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsAfsAmAfAmAfUmGfCmUfAmCmAmAfAmAfCmCfCmAfGmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(15)由与核苷酸序列
UfsGmsGfGmUfUmUfUmGfUfAfGmCfAmUfUmUfUmUfAmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsUfsAmAfAmAfAmUfGmCfUmAmCmAfAmAfAmCfCmCfAmsGmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(16)由与核苷酸序列
GfsGmsGfCmUfGmAfGmCfUfUfUmAfAmAfAmUfGmGfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的反义链和由与核苷酸序列
AmsAfsCmCfAmUfUmUfUmAfAmAmGmCfUmCfAmGfCmCfCmsCmsAm具有至少90％，优选具有95％、96％、97％、98％、99％同一性的核苷酸序列组成的有义链；
(17)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(18)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(19)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序 列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(20)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(21)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(22)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAfGmCmAmGmCmCmdGdAGmGmCmUmsGmsCm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列
UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(23)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAfsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(24)由与核苷酸序列
AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm具有 至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(25)由与核苷酸序列
AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(26)由与核苷酸序列
AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(27)由与核苷酸序列
AmsGmsAmCmCmUmGfUmUfUm(dT)GmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(28)由与核苷酸序列
CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGfsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(29)由与核苷酸序列
CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷 酸序列CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(30)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(31)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(32)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUm(dT)UmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(33)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGmUmUfAmCfAfAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(34)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGmUmUfAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
(35)由与核苷酸序列
CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列
CmsAfsAmGmUmUmAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
其中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接。
在本发明的另一个实例中，所述双链RNAi剂中，所述配体结构如式II所示：
Figure PCTCN2023071189-appb-000002
Figure PCTCN2023071189-appb-000003
本发明的一个实例中，所述双链RNAi剂具有式III结构，其中X是O或S：
Figure PCTCN2023071189-appb-000004
本发明提供以下双链RNAi剂实例：E1128、E1129、E1164、E1164-GN1、E1164-GN2、E1164-GN3、E1164-GN4、E1164-GN5、E1164-GN6、E1165、E1166、E1168、E1178、E1179、E1180、E1181、E1192、E1195、E1198、E1201、E1202、E0044、1164E01、1164E02、1164E03、1164E04、1164E05、1164E06、1168E01、1168E02、1168E03、1168E04、1168E05、1168E06、1168E07、1168E08、1168E09。
本发明还包括能够产生上述双链RNAi剂的DNA分子、能够表达所述双链RNAi剂的载体以及含有所述双链RNAi剂或者所述DNA分子或所述载体的试剂或试剂盒。
本发明还提供了包含上述双链RNAi剂的细胞。
本发明另外提供了包含上述双链RNAi剂的药物组合物。
所述药物组合物包括药理学有效量的本发明的双链RNAi剂和其他药学可接受的组分。“有效量”是指能有效产生预期的药理学治疗效果的双链RNAi剂量。“其他组分”包括水、盐水、葡萄糖、缓冲液(如PBS)，赋形剂、稀释剂、崩解剂、结合剂、润滑剂、甜味剂，调味剂，防腐剂或其组合。
本发明还提供了一种抑制细胞中PCSK9表达的方法，该方法包括：(a)使该细胞与上述的双链RNAi剂或其药物组合物接触；(b)将步骤(a)中产生的细胞维持一段时间，该时间足以获得PCSK9基因的mRNA转录本的降解，由此抑制该细胞中PCSK9基因的表达。
本发明提供了上述的双链RNAi剂或其药物组合物在抑制PCSK9基因表达或者制备用于抑制PCSK9基因表达的产品中的应用，其中，所述抑制PCSK9基因表达为抑制或降低体内或体外细胞中人、猴、大鼠或小鼠的PCSK9基因表达水平。所述细胞为表达PCSK9的哺乳动物细胞，如灵长类动物细胞、人细胞。较好是，靶细胞中PCSK9基因高水平表达。更好的是，细胞来源于脑、唾液腺、心脏、脾脏、肺脏、肝脏、肾脏、肠道、肿瘤。进一步更好的是，细胞为肝癌细胞，***细胞。再进一步更好的是，细胞选自HepG2，HEP3B，Huh7，COS7，293T，MHCC97H，Hela，小鼠原代肝细胞，人原代肝细胞。在本发明的一些实例中，双链RNAi剂的细胞终浓度为0.1-1000nM，如10-500nM，25-300nM或50-100nM。
在本发明的一些实例中，所述双链RNAi剂及其药物组合物可以通过任何合适的手段来施用，如胃肠外给药，胃肠给药包括肌肉、静脉、动脉、腹膜、或皮下注射。给药方式包括但不限于单次施用或多次施用。给药剂量范围为0.1mg/kg至100mg/kg，0.5mg/kg至50mg/kg、2.5mg/kg至20mg/kg、5mg/kg至15mg/kg，具体如3mg/kg、5mg/kg、10mg/kg、33mg/kg。
本发明还提供了所述双链RNAi剂或其药物组合物在降低血清中低密度脂蛋白(LDL)和/或低密度脂蛋白胆固醇(LDL-C)浓度或者制备用于降低血清中低密度脂蛋白(LDL)和/或低密度脂蛋白胆固醇(LDL-C)浓度的产品中的应用。
其中，所述降低血清中低密度脂蛋白(LDL)和/或低密度脂蛋白胆固醇(LDL-C)浓度为降低人、猴、大鼠或小鼠血清中低密度脂蛋白(LDL)和/或低密度脂蛋白胆固醇(LDL-C)浓度。
血清LDL(低密度脂蛋白)或LDL-C(低密度脂蛋白胆固醇)的浓度或含量至少减少了5％、10％、15％、20％、25％、30％、35％、40％、50％。
本发明还提供了上述的双链RNAi剂或其药物组合物在制备用于预防和/或治疗患有由PCSK9表达介导的疾病或缓解由PCSK9表达介导的疾病的症状的药物中的用途。
本发明还提供了上述的双链RNAi剂或其药物组合物，其用于预防和/或治疗患有由PCSK9表达介导的疾病或用于缓解由PCSK9表达介导的疾病的症状。
本发明也提供了一种预防和/或治疗患有由PCSK9表达介导的疾病的方法或用于缓解由PCSK9表达介导的疾病的症状的方法，其包括向有需要的受试者施用有效量的本发明所述的双链RNAi剂或其药物组合物。
本发明中，其中所述由PCSK9表达介导的疾病包括心血管疾病、血脂异常或肿瘤性疾病。所述心血管疾病包括动脉粥样硬化性心血管疾病，血脂异常包括血清中胆固醇和/或甘油三酯水平升高、低密度脂蛋白胆固醇升高或载脂蛋白B(ApoB)升高，具体如哺乳动物的高脂血症，高胆固醇血症、非家族性高胆固醇血症、多基因高胆固醇血症、家族性高胆固醇血症、纯合性家族性高胆固醇血症或杂合性家族性高胆固醇血症。所述肿瘤性疾病如与PCSK9有关的黑色素瘤和转移性肝癌。
在一些实施方案中，单剂量的药物组合物可以长久持续，PCSK9表达下降持续至少3、5、7、10、14天或更长时间。
本发明的创新性体现在：1、经高通量筛选的RNAi分子具有和AD-60212(阳性对照化合物)相当甚至较高的抑制活性；2、经修饰的RNAi分子具有高稳定性和高抑制活性。3、经配体修饰的RNAi分子在保持了较高的抑制活性和稳定性的同时，还具有较好的肝靶向性和促进细胞内吞的能力，可降低对其他组织或器官的影响以及减少RNAi分子使用量，可达到减轻毒性和降低成本的目的；4、配体修饰的RNAi分子无需转染试剂即可进入靶细胞和靶组织，降低了转染试剂的负性影响，如细胞或组织毒性，从而为靶向治疗提供可能；5、恒河猴药效学实验证明，候选序列和阳性对照AD-60212相比，在PCSK9蛋白和LDL-c(低密度脂蛋白)水平最高分别降低90％和65％，LDL-c药效持续性延长一周，效果显著。
需要说明的是，尽管能够尝试进行许多修饰来改善双链RNAi的性能，然而这些尝试通常很难阐释既介导RNA干扰又在血清中具有提高的稳定性(例如，具有对核酸酶的增加的抗性和/或延长的持续时间)。本发明的经修饰的双链RNAi具有高稳定性的同时保持了高的抑制活性。
定义
“PCSK9”是指前蛋白转化酶枯草杆菌蛋白酶Kexin9基因或蛋白。PCSK9也称为FH3、HCHOLA3、NARC-1、或NARCl。PCSK9mRNA序列的实例使用例如GenBank可易于获得。
“G”、“C”、“A”以及“U”通常分别代表包含鸟嘌呤、胞嘧啶、腺嘌呤以及尿嘧啶作为碱基的核苷酸。然而，应理解术语“核糖核苷酸”或“核苷酸”或“脱氧核糖核苷酸”还可以指一种经修饰的核苷酸或一种替代性的置换部分。技术人员应很好地意识到，鸟嘌呤、胞嘧啶、腺嘌呤以及尿嘧啶可以被其他部分置换而基本上不改变一种寡核苷酸(包括一种具有这种置换部分的核苷酸)的碱基配对特性。包含此类置换部分的序列是本发明的实施例。
“RNAi剂”，是指介导通过RNA诱导沉默复合物(RISC)途径的RNA转录本靶向切割剂。RNAi剂通过已知为RNA干扰(RNAi)的过程指导mRNA的序列特异性降解。RNAi剂调节，例如抑制，PCSK9在细胞如受试者(如哺乳动物受试者)体内的细胞中的表达。
在一个实施例中，本发明的RNAi剂包括与靶RNA序列，例如PCSK9靶mRNA序列相互作用以指导靶RNA切割的单链RNA。
在另一个实施例中，RNAi剂可以是引进细胞或生物体内以抑制靶mRNA的单链siRNA。
在另一个实施例中，本发明组合物、用途、和方法中使用的“RNAi剂”是双链RNA，并且在此是指“双链RNAi剂”。术语“双链RNAi剂”，是指核糖核酸分子的复合体，其具有双链结构，包含两条反向平行的和基本上互补的核酸链，被称为相对于靶RNA，即PCSK9基因，具有“有义”和“反义”定向。在本发明的一些实施例中，双链RNA通过转录后基因沉默机制(在此称为RNA干扰或RNAi)触发靶RNA例如mRNA的降解。
“反义链”是指一种双链RNAi剂的、包括与一种标靶序列(例如一种人类PCSK9mRNA)基本上互补的一个区的链。
“有义链”指的是含有与反义链区域基本上互补的区域的RNA链。
“互补区域”是指反义链上与靶mRNA序列完全或基本互补的区域。在互补区域与靶序列不完全互补的情况下，错配可以位于分子的内部或末端区域中。如此处所使用，术语“互补”是指第一多核苷酸在某些条件例如严格条件下与第二多核苷酸杂交的能力。
一个“核苷酸突出端”是指当一种RNAi剂的一条链的一个3'端延伸超出另一条链的5'端时从该RNAi剂的双链体结构突出的一个或多个不成对的核苷酸，或反之亦然。“平 端”或“平末端”意指在该双链RNAi剂的那端处不存在不成对的核苷酸，即无核苷酸突出端。一种“平末端的”RNAi剂是一种在其整个长度上都是双链、即在该分子的任一端处都无核苷酸突出端的dsRNA。
“抑制细胞中PCSK9表达”包括抑制任何PCSK9基因(例如小鼠PCSK9基因、大鼠PCSK9基因、猴PCSK9基因、或人类PCSK9基因)以及PCSK9基因的变体(例如天然存在的变体)或突变体的表达。因此，该PCSK9基因可以是野生型PCSK9基因、突变PCSK9基因、或在遗传操作的细胞、细胞群组或生物体的情形下的转基因PCSK9基因。
所述细胞为表达PCSK9的哺乳动物细胞，如灵长类动物细胞、人细胞。较好是，靶细胞中PCSK9基因高水平表达。更好的是，细胞来源于脑、唾液腺、心脏、脾脏、肺脏、肝脏、肾脏、肠道、肿瘤。进一步更好的是，细胞为肝癌细胞，***细胞。再进一步更好的是，细胞选自HepG2，HEP3B，Huh7，COS7，293T，MHCC97H，Hela，小鼠原代肝细胞，人原代肝细胞。
“抑制PCSK9基因表达”包括任何水平的PCSK9基因的抑制，例如至少部分抑制PCSK9基因的表达，如抑制至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％。
“使细胞与双链RNAi剂接触”包括通过任何可能手段使细胞接触。使细胞与双链RNAi剂接触包括使细胞在体外与该RNAi剂接触或使细胞在体内与该RNAi剂接触。该接触可以直接或间接地进行。因此，例如RNAi剂可通过执行该方法的个体与细胞的物理接触，或者可替代地，RNAi剂可进入许可或导致它后来接触到该细胞的一种情况。
“由PCSK9表达介导的疾病”旨在包括任何与PCSK9基因或蛋白相关的疾病。这种疾病可以例如由PCSK9蛋白的过量产生、由PCSK9基因突变、由PCSK9蛋白的异常裂解、由PCSK9与其他蛋白质或其他内源或外源物质之间的异常相互作用引起。
“高胆固醇血症”是指以增高的血清胆固醇为特征的状况。“高脂血症”是指以增高的血清脂质为特征的状况。“非家族性高胆固醇血症”是指不是由单一遗传性基因突变导致的以增高的胆固醇为特征的状况。“多基因高胆固醇血症”是指由多种遗传因子影响导致 的、以增高的胆固醇为特征的状况。“家族性高胆固醇血症(familial hypercholesterolemia，FH)”是指以LDL-受体(LDL-R)中的突变、显著增高的LDL-C和动脉粥样硬化的过早发作(premature onset)为特征的常染色体显性代谢病症。“纯合性家族性高胆固醇血症(homozygous familial hypercholesterolemia，HoFH)”是指以母体和父体LDL-R基因的突变为特征的状况。“杂合性家族性高胆固醇血症(heterozygous familial hypercholesterolemia，HoFH)”是指以母体或父体LDL-R基因的突变为特征的状况。
多种多样的实体可以偶联到本发明的RNAi剂。优选的部分是优选地直接地或者经由一个介入系栓物间接地共价偶联的配体。
配体通常可以包括治疗改性剂，例如用于增强摄取；诊断化合物或报道基团，例如用于监测分布；交联剂；以及赋予核酸酶抗性的部分。一般实例包括脂质、类固醇、维生素、糖、蛋白质、肽、多胺以及肽模拟物。
配体可以包括一种天然存在的物质，如一种蛋白质(例如人血清白蛋白(HSA)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)或球蛋白)；一种碳水化合物(例如一种右旋糖酐、支链淀粉、甲壳质、壳聚糖、菊糖、环糊精或透明质酸)；或一种脂质。该配体还可以是一种重组或合成分子，如一种合成聚合物，例如一种合成聚氨基酸、一种寡核苷酸(例如一种适体)。聚氨基酸的实例包括以下聚氨基酸：聚赖氨酸(PLL)、聚L-天冬氨酸、聚L-谷氨酸、苯乙烯-马来酸酐共聚物、聚(L-丙交酯-共-乙交酯)共聚物、二乙烯基醚-马来酐共聚物、N-(2-羟丙基)甲基丙烯酰胺共聚物(HMPA)、聚乙二醇(PEG)、聚乙烯醇(PVA)、聚氨酯、聚(2-乙基丙烯酸)、N-异丙基丙烯酰胺聚合物或聚膦嗪。聚胺的实例包括：聚乙烯亚胺、聚赖氨酸(PLL)、精胺、亚精胺、聚胺、假肽-聚胺、肽模拟聚胺、树枝状聚合物聚胺、精氨酸、脒、鱼精蛋白、阳离子脂质、阳离子卟啉、聚胺的季盐、或α螺旋肽。
配体也可以包括靶向基团，例如与指定的细胞类型如肾细胞结合的细胞或组织靶向剂，例如凝集素、糖蛋白、脂质或蛋白质，例如抗体。靶向基团可以是促甲状腺激素、促黑激素、凝集素、糖蛋白、表面活性蛋白A、粘蛋白碳水化合物、多价乳糖、多价半乳糖、N-乙酰基-半乳糖胺、N-乙酰基-葡糖胺多价甘露糖、多价海藻糖、糖基化的聚氨基酸、多价半乳糖、转铁蛋白、双膦酸盐、聚谷氨酸盐、聚天冬氨酸盐、脂质、胆固醇、类固醇、胆酸、叶酸盐、维生素B12、生物素、RGD肽、RGD肽模拟物或适体。
配体的其他实例包括染料、嵌入剂(例如吖啶)、交联剂(例如补骨脂素、丝裂霉素C)、卟啉(TPPC4、德克萨斯卟啉(texaphyrin)、噻啉(Sapphyrin))、多环芳烃(例如，吩嗪、二氢吩嗪)、人工核酸内切酶或螯合剂(例如EDTA)、亲脂性分子，例如，胆固醇、胆酸、金刚烷乙酸、1-芘丁酸、二氢睾酮、1,3-双-O(十六烷基)甘油、香叶基氧己基、鲸蜡基甘油、冰片、薄荷醇、1,3-丙二醇、十七烷基、棕榈酸、肉豆蔻酸、O3-(油酰)石胆酸、O3-(油酰)胆烯酸、二甲氧基三苯甲基、或吩噁嗪肽缀合物(例如，触角足肽、Tat肽)、烷基化剂、磷酸酯、氨基、巯基、PEG(例如，PEG-40K)、MPEG、[MPEG] 2、聚氨基、烷基、取代的烷基、放射标记的标记物、酶、半抗原(例如生物素)、转运/吸收促进剂(例如，阿司匹林、维生素E、叶酸)、合成性核糖核酸酶(例如，咪唑、双咪唑、组胺、咪唑聚类、吖啶-咪唑缀合物、四氮杂大环类的Eu 3+络合物)、二硝基苯基、HRP或AP。
配体可以是蛋白质，例如糖蛋白，或肽，例如对一种共配体具有一种特异性亲和力的分子，或抗体，例如结合到一种指定细胞类型(如一种癌细胞、内皮细胞或骨细胞)的一种抗体。配体也可以包括激素和激素受体。它们还可以包括非肽类种类，如脂质、凝集素、碳水化合物、维生素、辅因子、多价乳糖、多价半乳糖、N-乙酰基-半乳糖胺、N-乙酰基-葡糖胺多价甘露糖、多价岩藻糖或适体。该配体可以是例如脂多糖，p38MAP激酶的活化剂或NF-κB的活化剂。
配体可以是可以例如通过破坏细胞的细胞骨架(例如，通过破坏细胞微管、微丝和/或中间丝)增加iRNA剂摄入细胞中的物质，例如，药物。药物可以例如是泰素(taxon)、长春新碱、长春碱、松胞菌素、诺考达唑、促微丝聚合剂(japlakinolide)、红海海绵素A、鬼笔环肽、海洋苔藓素(swinholide)A、茚满诺星(indanocine)或myoservin。
配体可以是能够靶向一种特定受体的任何配体。实例是：叶酸盐、GalNAc、半乳糖、甘露糖、甘露糖-6P、糖簇(如GalNAc簇、甘露糖簇、半乳糖簇)或一种适体。一个簇是两个或更多个糖单元的组合。这些靶向配体还包括整合素受体配体、趋化因子受体配体、转铁蛋白、生物素、血清素受体配体、PSMA、内皮素、GCPII、生长抑素、LDL以及HDL配体。这些配体还可以基于核酸，例如一种适体。该适体可以是未被修饰的或具有在此披露的修饰的任何组合。
附图说明
图1：DLR高通量筛选PCSK9候选序列的基因表达。
图2：Top16候选修饰序列在Hep3B细胞中的基因表达。
图3：GalNAc-siRNA在人原代肝细胞中的自由摄取实验。
图4：GalNAc-siRNA候选序列在人原代肝细胞中的EC 50值。
图5：GalNAc-siRNA候选序列在HEP3B细胞中的IC 50值。
图6：不同GalNAc修饰的siRNA与小鼠原代肝细胞的结合能力。
图7：GalNAc-siRNA体内肝脏靶向性试验。
图8：GalNAc-siRNA小鼠体内药效试验。
图9：Top2候选序列修饰策略在Hep3B细胞中的基因表达。
图10：1168序列不同化学修饰的小鼠体内药效实验。
图11：候选序列降低小鼠PCSK9蛋白水平的筛选实验。
图12A：候选序列在食蟹猴体内导致的LDLc水平的变化。
图12B：候选序列在食蟹猴体内导致的PCSK9蛋白水平变化。
图12C：候选序列在食蟹猴体内的PK分布。
图13：候选序列在小鼠体内的毒理研究。
具体实施方式
实施例1、PCSK9-siRNA活性筛选
一、siRNA设计
根据人PCSK9mRNA序列，选择不同位点设计多种PCSK9 siRNAs，设计的所有单个siRNA均能靶向靶基因的所有转录本(如表1)，以上序列(如表2)经序列相似性软件比对与其他所有非靶标基因序列有最低同源性。序列设计方法参考Elbashir et al.2002；Paddison et al.2002；Reynoldset al.2004；Ui-Tei et al.2004等人的方法。
表1靶基因
靶基因 物种 GeneID NM_ID
PCSK9 Homosapiens(人) 255738 NM_174936.3
表2高通量筛选序列
Figure PCTCN2023071189-appb-000005
Figure PCTCN2023071189-appb-000006
Figure PCTCN2023071189-appb-000007
Figure PCTCN2023071189-appb-000008
Figure PCTCN2023071189-appb-000009
二、siRNA合成(天然RNA/2’-甲氧基或2’-氟修饰RNA/GalNAc-RNA)
本发明中仅含核糖核苷酸或2’-甲氧基或2’-氟修饰寡核苷酸按照理论产量1μmol合成规格完成，选用1μmol通用的Frit载体(
Figure PCTCN2023071189-appb-000010
逗点生物)或者GalNAc CPG(药明康德/Glen research，载量30umol/g)在LK-192X合成仪上制备所有的寡核苷酸。所有亚磷酰胺单体(上海兆维)均按照1:20(g/mL)的无水乙腈溶剂稀释，偶联时间为3min，共两次偶联。使用3％TCA进行脱保护，采用0.3M苄硫基四唑乙腈溶液进行活化，并通过CAPA/CAPB和50mM I 2溶液分别进行盖帽和氧化。在去三苯甲基合成(Trityl-off synthesis)后，将固相载体转移至2mL离心管中，加入1.2mL氨水置于65℃烘箱中加热3h，脱去保护基。然后冷却至室温，真空浓缩30min，将溶液通过0.22um滤膜过滤至进样瓶中，采用半制备反相纯化仪进行单链纯化，洗脱梯度为7％～30％(ACN:100mM TEAA)， 时间10min，流速：5mL/min，纯化制备后真空浓缩，室温旋干。最后用水溶解样品，在GE Hi-Trap脱盐柱上将每种溶液脱盐以洗脱最终的寡聚核苷酸产物。分别使用ESI-MS和IEX HPLC确认所有的特性和纯度。采用酶标仪紫外定浓，将等摩尔量的有义链和反义链混合并至新的离心管中，95℃加热5min，并缓慢退火至室温，最后采用真空浓缩仪室温旋干得到最终产品。
三、PCSK9-siRNA在体外psicheck-2***中的抑制活性检测
1、构建检测质粒
采用psicheck-2质粒构建重组质粒(上海捷瑞生物工程有限公司)，含有所有待测PCSK9 siRNA的目标序列，克隆位点为psicheck-2质粒的5’XhoI、3’NotI位点。
2、PCSK9 siRNA和重组质粒共转染不同细胞
所有细胞均购于中科院典藏细胞库；其他试剂均可商购。
表3细胞名称与种类
细胞名称 Cos7 293T Hep3B
细胞种类 肝癌细胞 肝癌细胞 肝癌细胞
细胞在含10％胎牛血清的DMEM培养基中，于5％CO 2、37℃恒温培养箱中培养，待细胞处于对数生长期且状态良好(70％汇合度)时铺板转染。调整细胞密度，每孔1.5×10 5个细胞铺种到24孔板中。配制转染复合物：将250μL Opti-MEM，40ng重组质粒和5μL 10nM siRNA混合，250μL Opti-MEM和2.5μL Lipofectamine 2000转染试剂混合，静置5min，然后将以上两个混合物混合，静置20min。将上述转染复合物加入24孔板中，于5％CO 2、37℃恒温培养箱中孵育6h。吸出上清，每孔加入1mL完全培养基，继续培养24h。
每次细胞转染除了试验组，还设置如下对照组：NC为阴性对照(不相关siRNA)、Lipo组为转染试剂对照组、Blank组未处理对照组(不加siRNA)。试验组和对照组均有3次重复。
3、DLR检测分析
使用Dual-Luciferase Reporter Assay System试剂盒(Promega)进行检测，根据试剂盒说明书裂解收集处理细胞，用Infinite Eplex酶标仪(TECAN)依次检测萤火虫(Photinus pyralis)萤光素酶和海肾(Renilla reniformis)萤光素酶的荧光强度，计算海肾(Renilla reniformis)萤光素酶与萤火虫(Photinus pyralis)萤光素酶荧光强度的比值，并以NC组为对照做归一化处理。表4为DLR检测的结果，PCSK9 siRNA试验组相对NC组的双荧光素酶报告基因表达水平的平均值。
表4 DLR检测结果
编号 293T-01 293T-02 COS.7-01 COS.7-02 平均值
U1128 0.2741 0.1768 0.4016 0.4992 0.3379
U1129 0.2375 0.2266 0.5082 0.5239 0.3741
U1164 0.1032 0.1090 0.1519 0.2200 0.1460
U1165 0.1339 0.1347 0.2311 0.3754 0.2188
U1166 0.1121 0.0831 0.1529 0.3702 0.1796
U1168 0.2384 0.0948 0.2239 0.1471 0.1761
U1178 0.2235 0.1579 0.3495 0.3638 0.2737
U1179 0.1220 0.0787 0.1554 0.2810 0.1593
U1180 0.1729 0.1370 0.1666 0.3562 0.2082
U1181 0.3573 0.2243 0.3162 0.4211 0.3297
U1192 0.4567 0.3359 0.2006 0.2764 0.3174
U1195 0.3495 0.1332 0.1928 0.2581 0.2334
U1198 0.2596 0.1193 0.1407 0.1836 0.1758
U1201 0.0937 0.0534 0.1363 0.2310 0.1286
U1202 0.1146 0.0656 0.1850 0.3287 0.1735
U0044 0.1095 0.0775 0.1868 1.0693 0.3608
如图1所示，分别在COS7、293T细胞中进行的PCSK9 siRNA DLR筛选发现了细胞活性较高的前16条siRNA分子，且为人、食蟹猴、恒河猴同源。细胞活性较高的前16条siRNA分子中有义链对应于PCSK9的介于3567-3663之间或2483-2505之间的位置，其核苷酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示：TCTGTCCTCTCTGTTGCCTTTTTACAGCCAACTTTTCTAGACCTGTTTTGCTTTTGTAACTTGAAGATATTTATTCTGGGTTTTGTAGCATTTTTAT(SEQ ID NO:1)
TGGGGCTGAGCTTTAAAATGGTT(SEQ ID NO:2)。
四、PCSK9 siRNA qPCR筛选
1、PCSK9 siRNA转染Hep3B细胞
Hep3B细胞在含10％胎牛血清的DMEM培养基中，于5％CO 2、37℃恒温培养箱中培养，待细胞处于对数生长期且状态良好时(70％汇合度)时铺板转染。调整细胞密度，每孔1.5×10 5个细胞铺种到24孔板中。配制转染复合物：将250μL Opti-MEM和5μL 10nM siRNA混合，250μL Opti-MEM和2.5μL Lipofectamine 2000转染试剂混合，静置5min，然后将以上两个混合物混合，静置20min。将上述转染复合物加入24孔板中，于5％CO 2、37℃恒温培养箱中孵育6h。吸出上清，每孔加入1mL完全培养基，继续培养24h。每次细胞转染除了试验组，还设置如下对照组：NC组为阴性对照(不相关siRNA)、Lipo组为转染试剂对照、Blank组为未处理对照(不加siRNA)。
2、实时荧光定量PCR分析：
转染24h后裂解细胞，柱提法试剂盒(诺唯赞)提取细胞总RNA。以β-actin基因为内参基因，用Taqman探针法，使用CFX96荧光定量PCR仪(Bio-Rad)进行实时荧光定量PCR反应。使用引物为：
表5引物序列信息
引物名称 序列(5'to3')
H-ACTB-FO-3 TGCCGACAGGATGCAGAAG(SEQ ID NO:239)
H-ACTB-RE-3 GCCGATCCACACGGAGTACT(SEQ ID NO:240)
H-ACTB-PR-3-FAM ATCAAGATCATTGCTCCTCCTGAGCGC(SEQ ID NO:241)
H-PCSK9V1-FO-1 GATCCTGCATGTCTTCCA(SEQ ID NO:242)
H-PCSK9V1-RE-1 GTCCTCCTCGATGTAGTC(SEQ ID NO:243)
H-PCSK9V1-PR1-1-JOE CCTTCTTCCTGGCTTCCTGGT(SEQ ID NO:244)
3、数据分析
PCR反应结束后，用2–ΔΔCt(Livak)方法，以参照基因作为标准进行相对定量分析。表6和图2结果表明，前16对siRNA序列中0.1nM的U1164、U1168在Hep3B细胞中抑制活性较高，比阳性对照AD60212抑制活性高23.2％、17.5％。
表6前16对siRNA序列抑制活性
Figure PCTCN2023071189-appb-000011
实施例2、PCSK9-siRNA的优化
一、人原代肝细胞自由摄取评价GalNAc-siRNA抑制PCSK9的活性
为进一步确认前16对高活性的siRNA分子，我们分别对其进行序列修饰优化(表7)， 其合成步骤同实施例1，并用人原代肝细胞自由摄取评价其抑制活性。
表7前16条高活性的siRNA修饰序列
Figure PCTCN2023071189-appb-000012
Figure PCTCN2023071189-appb-000013
在上表中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接，L96的结构如式IV所示：
Figure PCTCN2023071189-appb-000014
1、16对GalNAc-siRNA修饰序列对人原代肝细胞自由摄取PCSK9的抑制活性
复苏冻存的人原代肝细胞(购于中科院典藏细胞库)，将人原代肝细胞调整到合适的密度，然后将人原代肝细胞铺种到96孔板中。将GalNAc-siRNA用PBS稀释成100nM、1nM浓度，然后取一定体积稀释的GalNAc-siRNA加入到96孔板中与人原代肝细胞共孵育48小时。吸出上清，收集细胞，使用柱提法试剂盒提取细胞总RNA，qPCR检测PCSK9mRNA相对表达量。由结果可知(图3)，GalNAc-siRNA可通过内吞方式进入肝细胞并沉默PCSK9基因的表达，E1164、E1168抑制活性较高。
2、人原代肝细胞自由摄取的EC 50值
将人原代肝细胞调整到合适的密度铺种到96孔板中。将E1164、E1168用PBS梯度稀释成不同浓度，设置最高终浓度为100nM，做10倍梯度稀释共8个浓度，然后取一定体积 不同浓度的E1164、E1168加入到96孔板中与人原代肝细胞共孵育48小时。吸出上清，收集细胞，使用柱提法试剂盒提取细胞总RNA，qPCR检测PCSK9mRNA相对表达量，应用Graphpad Prism软件分析计算EC 50值。由图4所知，E1164，E1168在人原代肝细胞的EC 50值分别为5.66nM和3.45nM。
二、转染Hep3B细胞的IC 50值
将E1164，E1168和阳性对照AD60212用Nuclease-Free Water(Invitrogen)梯度稀释成不同浓度，设置最高终浓度为100nM，做10倍梯度稀释共8个浓度，转染Hep3B细胞(购于中科院典藏细胞库)。转染、定量PCR检测分析步骤参见实施例1，应用GraphpadPrism软件分析计算IC 50值。由图5所知，E1164，E1168和阳性对照AD60212在Hep3B细胞的IC 50值分别为0.031nM，0.005nM和0.224nM，E1164和E1168在Hep3B细胞的IC 50值是阳性对照AD60212的1/10、1/100，显著优于阳性对照AD60212。
实施例3、GalNAc靶向结构的筛选
一、小鼠原代肝细胞分离
麻醉小鼠，剪开皮肤和肌肉层，暴露肝脏，将灌注导管***门静脉，下腔静脉剪开小口，准备肝脏灌注。40℃预热perfusion Solution I(Hank’s，0.5mM EGTA，pH 8)和perfusion Solution II(Low-glucose DMEM，100U/mL Type IV，pH7.4)，37℃perfusion Solution I沿门静脉插管灌注肝脏，流速7mL/min，灌注5min，肝脏变灰白色为止。接着用37℃perfusion Solution II灌注肝脏，流速7mL/min，灌注7min。灌注完成后，取下肝脏置于Solution III(10％FBS low-glucose DMEM，4℃)终止消化，镊子划破肝脏包膜，轻轻抖动释放肝细胞。用70μm细胞滤器过滤肝细胞，50g离心2min后弃上清。用Solution IV(40％percoll low-glucose DMEM，4℃)重悬细胞，100g离心2min后弃上清。加入2％FBS low-glucose DMEM重悬细胞，备用。台盼蓝染色鉴定细胞活力。
二、测定GalNAc结合曲线和K d值
将新鲜分离的小鼠原代肝细胞铺到96孔板中，2×10 4个/孔，100μl/孔。每孔分别加GalNAc-siRNA(见表8)。每条GalNAc-siRNA设置终浓度20nM。4℃孵育2h后50g离心2min，弃上清。10μg/ml PI重悬细胞，染色10min后50g离心2min。用预冷的PBS洗细胞，50g离心2min后弃上清。PBS重悬细胞。流式细胞仪测定活细胞平均荧光强度MFI，GraphPad Prism5软件进行非线性拟合及Kd值计算。数据表明，配体修饰的GalNAc-siRNA在不加转 染试剂的情况下促使体外肝细胞内吞/摄取，实现了对肝细胞的递送。同时不同GalNAc结构的GalNAc-siRNA呈现出的细胞内吞和与受体结合能力有一定的差异(图6)，其中A1-A6分别对应E1164-GN1—E1164-GN6。
表8不同GalNAc修饰的siRNA
Figure PCTCN2023071189-appb-000015
在上表中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接，Cy5表示Cyanine 5荧光染料，L96结构如式IV所示：
Figure PCTCN2023071189-appb-000016
X具有式I结构：
Figure PCTCN2023071189-appb-000017
其中n＝3，XX中，相邻的两个X通过磷酸二酯键或硫代磷酸二酯键相连；X和XXX均通过磷酸二酯键或硫代磷酸二酯键与siRNA有义链核苷酸序列的5'、3'末端或者中间的核苷酸连接。
试验采用雄性、6～7周龄的SPF级C57小鼠36只(南模生物实验动物有限公司)，随机分成6组，各组动物数分别为6只，皮下注射给药，给药剂量为5mg/kg(实验设计见表9)。给药后2h、4h、24h、48h对所有动物进行活体成像。药后48小时安乐死后，取出心脏、脾脏、肺脏、肝脏、肾脏进行离体器官成像(图7)。
表9肝靶向实验设计
组别 动物 小鼠编号
E1164-GN1 C57，雄性，六周龄 1#、2#、3#、4#、5#、6#
E1164-GN2 C57，雄性，六周龄 7#、8#、9#、10#、11#、12#
E1164-GN3 C57，雄性，六周龄 13#、14#、15#、16#、17#、18#
E1164-GN4 C57，雄性，六周龄 19#、20#、21#、22#、23#、24#
E1164-GN5 C57，雄性，六周龄 25#、26#、27#、28#、29#、30#
E1164-GN6 C57，雄性，六周龄 31#、32#、33#、34#、35#、36#
活体成像和离体成像分析(图7)结果显示，顺序自左至右，自上而下，依次对应1#-36#小鼠，如第一行代表1#-12#小鼠。给药后2h，不同GalNAc修饰的E1164组肝脏的荧光强度均集中在肝脏部位。在24h后，大部分GalNAc序列被代谢，荧光强度表明，GalNAc不同结构对肝脏均有一定靶向性。
三、GalNAc结构体内药效实验
实验用6-8周龄的人源化PCSK9雌雄小鼠(南模生物实验动物有限公司)，共50只，根据体重随机分组，每组10只，雌雄各5只，单次3mg/kg皮下注射给药(给药分组见表10)。 第-7、-3、4、7、11、14、18、21、25、32、39天(采血前禁食6小时，第一剂量为第0天)采血，眼球后部放血收集0.1ml血液，收集的血液分离血清使用Human PCSK9ELISA Kit(SinoBiological)检测血清中PCSK9蛋白的表达水平，并作组间比较。
表10皮下注射siRNA药物小鼠实验方案
组别 动物 小鼠编号
NC 人源化PCSK9，雌雄各5只，六周龄 #1-#10
E1164-GN1 人源化PCSK9，雌雄各5只，六周龄 #11-#20
E1164-GN2 人源化PCSK9，雌雄各5只，六周龄 #21-#30
E1164-GN3 人源化PCSK9，雌雄各5只，六周龄 #31-#40
E1164-GN4 人源化PCSK9，雌雄各5只，六周龄 #41-#50
表11来自实施例3-三的标准化至治疗前的平均PCSK9蛋白
Figure PCTCN2023071189-appb-000018
实验结果表明(图8，表11)，E1164-GN1、E1164-GN2、E1164-GN3、E1164-GN分子中，第四天PCSK9蛋白最高降低80％，第39天仍然能降低到60％左右；同时结果表明，连续串联3个GalNAc结构以及偶联三叉戟GalNAc结构均能实现PCSK9蛋白的降低和持久性，3’端偶联GalNAc结构比5’端药效更加持久和滞后反弹。
实施例4、化学修饰平台的优化
一、抑制活性测定
我们分别对U1164和U1168进行序列修饰优化(表12)，对不同位置进行氟代和甲氧基修饰组合，总的修饰策略反义链尽可能采用甲氧基修饰替代，其合成、转染Hep3B细胞、定量PCR检测分析步骤同实施例1，转染终浓度为0.1nM。
表12候选序列的siRNA修饰策略组合
Figure PCTCN2023071189-appb-000019
Figure PCTCN2023071189-appb-000020
Figure PCTCN2023071189-appb-000021
在上表中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接，L96结构如式IV所示：
Figure PCTCN2023071189-appb-000022
如图9所示，修饰的方案是重要的，某些修饰会降低抑制活性。优选的修饰方案具有高的抑制活性：(1)反义链在3'端处具有5'(s)mN(s)mN3'结构的突出端，有利于反义链加载到RISC中，加强RNAi的干扰活性而不影响稳定性。(2)反义链从5'端起至少第2，6，14，16位使用氟代修饰，其他位置尽可能采用甲氧基修饰，有利于RISC结合；(3)反义链从3'端和5'端起至少两个硫代修饰有利于保持核酸的稳定性；(4)正义链从5'端起第7位，且9-11位采用连续的氟代修饰，其他位置尽可能用于甲氧基修饰有利于RISC结合；(5)有义链从5'端起至少两个硫代修饰，3'端采用GalNAc共价偶联。接下来选择U1168序列进行不同化学修饰筛选。
二、1168序列不同化学修饰体内药效实验
实验用6-8周龄的人源化PCSK9雌雄小鼠(南模生物实验动物有限公司)，共25只，根据体重随机分组，每组5只，单次3mg/kg皮下注射给药(给药分组见表12)。第-7、-3、4、7、11、14、18、21、25天(采血前禁食6小时，第一剂量为第0天)采血，眼球后部放血收集0.1ml血液，收集的血液分离血清使用Human PCSK9 ELISA Kit(SinoBiological)检测血清中PCSK9蛋白的表达水平，并作组间比较。
表13皮下注射siRNA药物小鼠实验方案
组别 动物 小鼠编号
NC 人源化PCSK9，雌雄共5只，六周龄 #1-#5
E1168 人源化PCSK9，雌雄共5只，六周龄 #6-#10
1168E07 人源化PCSK9，雌雄共5只，六周龄 #11-#15
1168E08 人源化PCSK9，雌雄共5只，六周龄 #16-#20
1168E09 人源化PCSK9，雌雄共5只，六周龄 #21-#25
实验结果表明(图10)，不同化学修饰的1168序列在第11天达到最低点，PCSK9蛋白下降了接近90％左右，而随着氟代修饰碱基数目的减少，PCSK9蛋白反弹减缓，持续性增加，1168E08及E09序列药效持久。
实施例5、小鼠体内有效性检测
实验用6-8周龄的人源化PCSK9雌雄小鼠(南模生物实验动物有限公司)，共50只，根据体重随机分组，每组10只，雌雄各5只，单次3mg/kg皮下注射给药(给药分组见表13)。第-6、-3、3、6、10、13、17、20、24、31、38、45、52、59、66、73、80天(采血前禁食6小时，第一剂量为第0天)采血，眼球后部放血收集0.1ml血液，收集的血液分离血清使用Human PCSK9 ELISA Kit(SinoBiological)检测血清中PCSK9蛋白的表达水平，并作组间比较。
表14皮下注射siRNA药物小鼠实验方案
组别 动物 小鼠编号
NC 人源化PCSK9，雌雄各5只，六周龄 #1-#10
1168 E06 人源化PCSK9，雌雄各5只，六周龄 #11-#20
1168 E07 人源化PCSK9，雌雄各5只，六周龄 #21-#30
1168 E08 人源化PCSK9，雌雄各5只，六周龄 #31-#40
AD60212 人源化PCSK9，雌雄各5只，六周龄 #41-#50
表15来自实施例5的标准化至治疗前的平均PCSK9蛋白
Figure PCTCN2023071189-appb-000023
小鼠药效结果表明(图11，表15)，阳性对照AD60212在第10天PCSK9蛋白水平降 低了80％水平，持续到第31天后开始出现反弹，第73天恢复到药前水平；而候选分子中1168E06，1168E07比阳性对照药效较差，第59天恢复到药前水平；1168E08分子第13天出现最低点，降低了80％，随后持续到第38天出现反弹，第80天恢复到药前水平，比阳性对照反弹周期滞后一周左右。
实施例6、食蟹猴体内有效性检测
实验用8只食蟹猴(北京昭衍生物技术有限公司)，按照体重随机分配到阳性对照组和实验测试组(AD60212，1168E06，1168E07，1168E08，四个测试化合物)共4组，每组2只动物，雌雄各一只。分别给与一次5mg/kg药物皮下注射，在第-1，2，5，8，11，15，18，22，25，29，32，36，43，50，57，64，71，78，85，92，99，106，113，120天(采血前禁食过夜，第一剂量为第0天)前肢头静脉采血，分离血清用于PCSK9蛋白及LDL-C(低密度脂蛋白胆固醇)检测，在第120天后将继续检测PCSK9蛋白及LDL-C直至恢复到药前水平。
一、LDL-C检测
1、方法
使用Toshiba TBA120血生化仪检测血清中LDL-C的浓度，并作组间比较。
2、结果
表16来自实施例6的标准化至治疗前的平均LDL-c水平
  1168E06 1168E07 1168E08 AD60212
第2天 1.09 1.11 0.80 1.02
第5天 0.75 0.81 0.67 0.71
第8天 0.90 0.76 0.71 0.70
第11天 0.77 0.48 0.35 0.43
第15天 0.74 0.42 0.37 0.51
第18天 0.69 0.39 0.30 0.38
第22天 0.72 0.40 0.36 0.49
第25天 0.65 0.40 0.27 0.33
第29天 1.00 0.34 0.34 0.39
第32天 0.92 0.38 0.33 0.34
第36天 0.77 0.44 0.29 0.42
第43天 1.07 0.40 0.37 0.48
第50天 0.83 0.46 0.34 0.52
第57天 0.83 0.43 0.39 0.50
第64天 0.71 0.37 0.32 0.44
第71天 0.91 0.44 0.36 0.54
第78天 0.82 0.46 0.39 0.53
第85天 0.88 0.58 0.39 0.65
第92天 0.88 0.53 0.38 0.61
第99天 0.79 0.56 0.46 0.64
第106天 0.85 0.64 0.36 0.59
第113天 0.83 0.52 0.43 0.71
第120天 0.92 0.69 0.60 0.80
实验结果(图12A，表16表明，与给药前相比，1168E06，1168E07，1168E08，AD60212组给药后11天血清LDL-C水平分别下降了23％，52％，65％和57％，随后，1168E07和1168E08分子降低LDL-C效果较阳性对照显著，一直持续到100天开始反弹。
二、PCSK9蛋白检测
1、方法
使用Monkey Proprotein convertase subtilisin/kexin type 9(PCSK9)ELISA kit(CUSABIO)检测血清中PCSK9蛋白的表达水平，并作组间比较。
2、结果
表17来自实施例6的标准化至治疗前的平均PCSK9蛋白
  1168E06 1168E07 1168E08 AD60212
第2天 0.83 0.50 0.95 0.56
第5天 0.79 0.37 0.82 0.63
第8天 0.23 0.28 0.26 0.28
第11天 0.50 0.15 0.21 0.31
第15天 0.29 0.08 0.11 0.17
第18天 0.36 0.13 0.24 0.24
第22天 0.31 0.08 0.16 0.17
第25天 0.25 0.09 0.19 0.18
第29天 0.30 0.09 0.12 0.17
第32天 0.28 0.08 0.13 0.16
第36天 0.28 0.09 0.14 0.19
第43天 0.10 0.11 0.07 0.12
第50天 0.50 0.15 0.13 0.24
第57天 0.18 0.06 0.12 0.15
第64天 0.57 0.14 0.25 0.40
结果(图12B，表17)表明，与给药前相比，1168E06，1168E07，1168E08，AD60212组给药后15天血清PCSK9蛋白水平下降到最低点，分别降低了71％，92％，89％，83％，并维持在低位。随后，1168E07以及1168E08分子降低PCSK9蛋白的效果较阳性对照显著，一直持续到57天开始反弹。
三、药代动力学测试
将不同时间收集到食蟹猴的血清通过质谱定量分析，并通过标准品的标准曲线标定得到不同时间的药物浓度水平。
实验结果(图12C)表明，阳性对照AD60212以及1168E08序列的正义链、反义链的药代动力学曲线相似，2h到达峰值，24h检测不到siRNA浓度。
实施例7、小鼠毒理研究
实验采用雄性、6～7周龄的SPF级C57小鼠(南模生物实验动物有限公司)，共21只，按照体重随机分配到空白对照组、阳性对照组和实验测试组(NC、AD60212，1168E08)共3组，每组7只，单次400mg/kg皮下注射给药。第72、96、120h(采血前禁食6小时，第一剂量为第0天)采血，眼球后部放血收集0.1ml血液，收集的血液使用Toshiba TBA120血生化仪检测血清中ALT和AST的浓度变化，并作组间比较。
小鼠实验结果表明(图13)，阳性对照AD60212以及候选分子1168E08在400mpk的剂量下，血清中ALT和AST变化表现良好，无明显毒性。

Claims (19)

  1. 一种双链RNAi剂，其能够抑制细胞中的PCSK9的表达，其中所述双链RNAi剂包含有义链和反义链，其中有义链与反义链互补，反义链包含与编码PCSK9的mRNA一部分序列互补的序列，其中每个链在长度上为14至30个核苷酸，并且所述双链RNAi剂中有义链核苷酸序列选自SEQ ID NO:1或SEQ ID NO:2中的14至30个核苷酸序列。
  2. 权利要求1所述的双链RNAi剂，其特征在于，所述有义链和反义链具有17-30个核苷酸，优选具有17-25个核苷酸，更优选具有19-23个核苷酸。
  3. 权利要求2所述的双链RNAi剂，其特征在于，所述有义链具有21个核苷酸，所述反义链具有23个核苷酸。
  4. 权利要求1-3任一项所述的双链RNAi剂，其特征在于，所述有义链和反义链的一个或多个核苷酸上具有选自下组的一个或多个修饰：2'-甲氧基乙基、2'-O-烷基、2'-O-烯丙基、2'-C-烯丙基、2'-氟、2'-脱氧、2'-羟基、锁核酸修饰、开环或非锁核酸修饰、DNA修饰、荧光探针修饰。
  5. 权利要求4所述的双链RNAi剂，其特征在于，所述修饰为2'-O-甲基和/或2'-氟修饰。
  6. 权利要求1-5任一项所述的双链RNAi剂，其特征在于，所述双链RNAi剂进一步包含至少一个硫代磷酸酯或甲基膦酸酯核苷酸间键合，优选包含至少一个硫代磷酸酯键合。
  7. 权利要求6所述的双链RNAi剂，其特征在于，所述硫代磷酸酯或甲基膦酸酯核苷酸间键合是在一条链的5'和3'末端处，优选地，所述核苷酸间键合是在有义链和反义链的5'和3'末端的3个核苷酸间。
  8. 根据权利要求4-7任一项所述的双链RNAi剂，其特征在于：所述双链RNAi剂包括：(1)反义链在3'端处具有5'(s)mN(s)mN3'结构的突出端；(2)反义链从5'端起至少第2，6，14，16位使用氟代修饰，其他位置采用甲氧基修饰；(3)反义链从3'端和5'端起至少两个硫代修饰；(4)正义链从5'端起第7位，且9-11位采用连续的氟代修饰，其他位置采用甲氧基修饰；(5)有义链从5'端起至少两个硫代修饰。
  9. 权利要求1-8任一项所述的双链RNAi剂，其特征在于，所述有义链与至少一个配体缀合，所述配体选自由胆固醇、生物素、维生素、半乳糖衍生物或类似物、乳糖衍 生物或类似物、N-乙酰半乳糖胺衍生物或类似物、N-乙酰葡萄糖胺衍生物或类似物组成的组；优选地，所述配体连接到该有义链的3’端。
  10. 权利要求9所述的双链RNAi剂，其特征在于，所述配体是与一种单价或三价支链连接物连接的一种或多种GalNAc衍生物。
  11. 权利要求1-10任一项所述的双链RNAi剂，其特征在于，所述双链RNAi剂包括：
    (1)由与核苷酸序列UfsGmsUfCmCfUmCfUmCfUfGfUmUfGmCfCmUfUmUfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列AmsAfsAmAfAmGfGmCfAmAfCmAmGmAfGmAfGmGfAmCfAmsGmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (2)由与核苷酸序列GfsUmsCfCmUfCmUfCmUfGfUfUmGfCmCfUmUfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsAfsAmAfAmAfGmGfCmAfAmCmAmGfAmGfAmGfGmAfCmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (3)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (4)由与核苷酸序列GfsAmsCfCmUfGmUfUmUfUfGfCmUfUmUfUmGfUmAfAmCf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列GmsUfsUmAfCmAfAmAfAmGfCmAmAmAfAmCfAmGfGmUfCmsUmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (5)由与核苷酸序列AfsCmsCfUmGfUmUfUmUfGfCfUmUfUmUfGmUfAmAfCmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组 成的反义链和由与核苷酸序列AmsGfsUmUfAmCfAmAfAmAfGmCmAmAfAmAfCmAfGmGfUmsCmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (6)由与核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (7)由与核苷酸序列UfsUmsUfGmUfAmAfCmUfUfGfAmAfGmAfUmAfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsAfsAmAfUmAfUmCfUmUfCmAmAmGfUmUfAmCfAmAfAmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (8)由与核苷酸序列UfsUmsGfUmAfAmCfUmUfGfAfAmGfAmUfAmUfUmUfAmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列AmsUfsAmAfAmUfAmUfCmUfUmCmAmAfGmUfUmAfCmAfAmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (9)由与核苷酸序列UfsGmsUfAmAfCmUfUmGfAfAfGmAfUmAfUmUfUmAfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列AmsAfsUmAfAmAfUmAfUmCfUmUmCmAfAmGfUmUfAmCfAmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (10)由与核苷酸序列GfsUmsAfAmCfUmUfGmAfAfGfAmUfAmUfUmUfAmUfUmCf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列GmsAfsAmUfAmAfAmUfAmUfCmUmUmCfAmAfGmUfUmAfCmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (11)由与核苷酸序列AfsUmsAfUmUfUmAfUmUfCfUfGmGfGmUfUmUfUmGfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsAfsCmAfAmAfAmCfCmCfAmGmAmAfUmAfAmAfUmAfUmsCmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (12)由与核苷酸序列UfsUmsUfAmUfUmCfUmGfGfGfUmUfUmUfGmUfAmGfCmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsGfsCmUfAmCfAmAfAmAfCmCmCmAfGmAfAmUfAmAfAmsUmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (13)由与核苷酸序列AfsUmsUfCmUfGmGfGmUfUfUfUmGfUmAfGmCfAmUfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列AmsAfsAmUfGmCfUmAfCmAfAmAmAmCfCmCfAmGfAmAfUmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (14)由与核苷酸序列CfsUmsGfGmGfUmUfUmUfGfUfAmGfCmAfUmUfUmUfUmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsAfsAmAfAmAfUmGfCmUfAmCmAmAfAmAfCmCfCmAfGmsAmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (15)由与核苷酸序列UfsGmsGfGmUfUmUfUmGfUfAfGmCfAmUfUmUfUmUfAmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列AmsUfsAmAfAmAfAmUfGmCfUmAmCmAfAmAfAmCfCmCfAmsGmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (16)由与核苷酸序列GfsGmsGfCmUfGmAfGmCfUfUfUmAfAmAfAmUfGmGfUmUf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序 列AmsAfsCmCfAmUfUmUfUmAfAmAmGmCfUmCfAmGfCmCfCmsCmsAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (17)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (18)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (19)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (20)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (21)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (22)由与核苷酸序列 AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAfGmCmAmGmCmCmdGdAGmGmCmUmsGmsCm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (23)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUfUmUfGmUfAmAf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAfAmAfGmCfAmAmAmAfCmAfGmGfUmCfUmsAfsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (24)由与核苷酸序列AfsGmsAfCmCfUmGfUmUfUfUfGmCfUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (25)由与核苷酸序列AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAmAmAmAfGmCfAmAmAmAfCmAfGmGmUmCfUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (26)由与核苷酸序列AmsGmsAmCmCmUmGfUmUfUmUmGmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (27)由与核苷酸序列AmsGmsAmCmCmUmGfUmUfUm(dT)GmCmUmUmUmUmGmUmAmAm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列UmsUfsAmCfAfAfAmAfGmCfAmAfAmAfCmAfGmGfUmCmUmsAmsGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (28)由与核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUfAmAfCmUfUmGf具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUfAmCfAmAfAmAmGmCfAmAfAmAfCmAfGmsGfsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (29)由与核苷酸序列CfsUmsGfUmUfUmUfGmCfUfUfUmUfGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (30)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUmUmAmCfAmAfAmAmGmCfAmAfAmAmCmAfGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (31)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUmUmUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm具 有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (32)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUm(dT)UmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGfUfUfAmCfAmAfAmAfGmCfAmAfAmAfCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (33)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGmUmUfAmCfAfAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    (34)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGmUmUfAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；或
    (35)由与核苷酸序列CmsUmsGmUmUmUmUfGmCfUfUfUmUmGmUmAmAmCmUmUmGm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的反义链和由与核苷酸序列CmsAfsAmGmUmUmAmCmAmAmAmAmGmCfAmAfAmAmCmAmGmsGmsUm具有至少90％，优选具有95％、96％、97％、98％、99％、100％同一性的核苷酸序列组成的有义链；
    其中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接
  12. 权利要求9-10任一项所述的双链RNAi剂，其特征在于，所述配体结构如式II所示：
    Figure PCTCN2023071189-appb-100001
  13. 权利要求9-12任一项所述的双链RNAi剂，其中所述双链RNAi剂具有式III结构，其中X是O或S：
    Figure PCTCN2023071189-appb-100002
  14. 权利要求11所述的双链RNAi剂，其中所述双链RNAi剂具有：
    (1)由核苷酸序列SEQ ID NO:3组成的反义链和由核苷酸序列SEQ ID NO:4组成的有义链；
    (2)由核苷酸序列SEQ ID NO:5组成的反义链和由核苷酸序列SEQ ID NO:6组成的有义链；
    (3)由核苷酸序列SEQ ID NO:7组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (4)由核苷酸序列SEQ ID NO:9组成的反义链和由核苷酸序列SEQ ID NO:10组成的有义链；
    (5)由核苷酸序列SEQ ID NO:11组成的反义链和由核苷酸序列SEQ ID NO:12组成的有义链；
    (6)由核苷酸序列SEQ ID NO:13组成的反义链和由核苷酸序列SEQ ID NO:14组成的有义链；
    (7)由核苷酸序列SEQ ID NO:15组成的反义链和由核苷酸序列SEQ ID NO:16组成的有义链；
    (8)由核苷酸序列SEQ ID NO:17组成的反义链和由核苷酸序列SEQ ID NO:18组成的有义链；
    (9)由核苷酸序列SEQ ID NO:19组成的反义链和由核苷酸序列SEQ ID NO:20组成的有义链；
    (10)由核苷酸序列SEQ ID NO:21组成的反义链和由核苷酸序列SEQ ID NO:22组成的有义链；
    (11)由核苷酸序列SEQ ID NO:23组成的反义链和由核苷酸序列SEQ ID NO:24组成的有义链；
    (12)由核苷酸序列SEQ ID NO:25组成的反义链和由核苷酸序列SEQ ID NO:26组成的有义链；
    (13)由核苷酸序列SEQ ID NO:27组成的反义链和由核苷酸序列SEQ ID NO:28组成的有义链；
    (14)由核苷酸序列SEQ ID NO:29组成的反义链和由核苷酸序列SEQ ID NO:30组成的有义链；
    (15)由核苷酸序列SEQ ID NO:31组成的反义链和由核苷酸序列SEQ ID NO:32组成的有义链；
    (16)由核苷酸序列SEQ ID NO:33组成的反义链和由核苷酸序列SEQ ID NO:34组成的有义链；
    (17)由核苷酸序列SEQ ID NO:35组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (18)由核苷酸序列SEQ ID NO:36组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (19)由核苷酸序列SEQ ID NO:37组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (20)由核苷酸序列SEQ ID NO:38组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (21)由核苷酸序列SEQ ID NO:39组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (22)由核苷酸序列SEQ ID NO:40组成的反义链和由核苷酸序列SEQ ID NO:8组成的有义链；
    (23)由核苷酸序列SEQ ID NO:7组成的反义链和由核苷酸序列SEQ ID NO:41组成的有义链；
    (24)由核苷酸序列SEQ ID NO:42组成的反义链和由核苷酸序列SEQ ID NO:43组成的有义链；
    (25)由核苷酸序列SEQ ID NO:44组成的反义链和由核苷酸序列SEQ ID NO:43组成的有义链；
    (26)由核苷酸序列SEQ ID NO:44组成的反义链和由核苷酸序列SEQ ID NO:45组成的有义链；
    (27)由核苷酸序列SEQ ID NO:46组成的反义链和由核苷酸序列SEQ ID NO:45组成的有义链；
    (28)由核苷酸序列SEQ ID NO:13组成的反义链和由核苷酸序列SEQ ID NO:47组成的有义链；
    (29)由核苷酸序列SEQ ID NO:48组成的反义链和由核苷酸序列SEQ ID NO:49组成的有义链；
    (30)由核苷酸序列SEQ ID NO:50组成的反义链和由核苷酸序列SEQ ID NO:49组成的有义链；
    (31)由核苷酸序列SEQ ID NO:50组成的反义链和由核苷酸序列SEQ ID NO:51组成的有义链；
    (32)由核苷酸序列SEQ ID NO:52组成的反义链和由核苷酸序列SEQ ID NO:51组成的有义链；
    (33)由核苷酸序列SEQ ID NO:53组成的反义链和由核苷酸序列SEQ ID NO:54组成的有义链；
    (34)由核苷酸序列SEQ ID NO:53组成的反义链和由核苷酸序列SEQ ID NO:55组成的有义链；或
    (35)由核苷酸序列SEQ ID NO:53组成的反义链和由核苷酸序列SEQ ID NO:56组成的有义链；
    其中，Am、Um、Cm和Gm分别表示经2'-O-甲基修饰的核糖核苷酸A、U、C和G；Af、Uf、Cf和Gf分别表示经2'-氟代修饰的核糖核苷酸A、U、C和G；s表示前后两个核苷酸由硫代磷酸骨架连接，Cy5表示Cyanine 5荧光染料，L96具有式IV结构：
    Figure PCTCN2023071189-appb-100003
    X具有式I结构：
    Figure PCTCN2023071189-appb-100004
    其中，n为3。。
  15. 包含权利要求1-14中任一项所述的双链RNAi剂的细胞。
  16. 包含权利要求1-15中任一项所述的双链RNAi剂的药物组合物。
  17. 一种抑制细胞中PCSK9表达的方法，该方法包括：(a)使该细胞与权利要求1-16中任一项所述的双链RNAi剂或权利要求16所述的药物组合物接触；(b)将步骤(a)中产生的细胞维持一段时间，该时间足以获得PCSK9基因的mRNA转录本的降解，由此抑制该细胞中PCSK9基因的表达。
  18. 权利要求1-14中任一项所述的双链RNAi剂或权利要求16所述的药物组合物在制备用于治疗患有由PCSK9表达介导的疾病的药物中的用途。
  19. 如权利要求18所述的用途，其中所述疾病包括心血管疾病或肿瘤性疾病，所述心血管疾病选自高脂血症、高胆固醇血症、非家族性高胆固醇血症、多基因高胆固醇血症、家族性高胆固醇血症、纯合性家族性高胆固醇血症或杂合性家族性高胆固醇血症；所述肿瘤性疾病选自与PCSK9有关的黑色素瘤或转移性肝癌。
PCT/CN2023/071189 2022-01-13 2023-01-09 一种抑制PCSK9基因表达的RNAi剂及其应用 WO2023134609A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202210038513 2022-01-13
CN202210038513.6 2022-01-13
CN202211092636 2022-09-08
CN202211092636.4 2022-09-08

Publications (1)

Publication Number Publication Date
WO2023134609A1 true WO2023134609A1 (zh) 2023-07-20

Family

ID=87280151

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/071189 WO2023134609A1 (zh) 2022-01-13 2023-01-09 一种抑制PCSK9基因表达的RNAi剂及其应用

Country Status (1)

Country Link
WO (1) WO2023134609A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101589143A (zh) * 2006-11-27 2009-11-25 Isis药物公司 用于治疗高胆固醇血症的方法
CN104854242A (zh) * 2012-12-05 2015-08-19 阿尔尼拉姆医药品有限公司 PCSK9 iRNA组合物及其使用方法
CN109957565A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种修饰的siRNA分子及其应用
CN109957567A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
CN112368381A (zh) * 2018-04-18 2021-02-12 迪克纳制药公司 用于治疗高胆固醇血症和相关病况的pcsk9靶向寡核苷酸
CN113286888A (zh) * 2019-05-22 2021-08-20 苏州瑞博生物技术股份有限公司 核酸、药物组合物与缀合物及制备方法和用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101589143A (zh) * 2006-11-27 2009-11-25 Isis药物公司 用于治疗高胆固醇血症的方法
CN104854242A (zh) * 2012-12-05 2015-08-19 阿尔尼拉姆医药品有限公司 PCSK9 iRNA组合物及其使用方法
CN109957565A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种修饰的siRNA分子及其应用
CN109957567A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种抑制PCSK9基因表达的siRNA分子及其应用
CN112368381A (zh) * 2018-04-18 2021-02-12 迪克纳制药公司 用于治疗高胆固醇血症和相关病况的pcsk9靶向寡核苷酸
CN113286888A (zh) * 2019-05-22 2021-08-20 苏州瑞博生物技术股份有限公司 核酸、药物组合物与缀合物及制备方法和用途

Similar Documents

Publication Publication Date Title
JP7420391B2 (ja) 十分に安定化された非対称ｓｉｒｎａ
US20200263179A1 (en) Compositions and methods for inhibiting gene expression of lpa
KR101857707B1 (ko) 아포지질단백질 c-iii 발현을 조절하는 조성물 및 방법
CN105018492B (zh) 不对称干扰rna的组合物及其用途
CN111107853A (zh) 用于抑制载脂蛋白C-III (APOC3)的表达的RNAi试剂和组合物
KR20070059187A (ko) 이중 가닥 리보핵산에 의한 염증성 질환의 치료 방법
JP4505749B2 (ja) Ｂｃｌ−２の発現抑制をするオリゴ二本鎖ＲＮＡとそれを含有する医薬組成物
JP2008289488A (ja) 化学的に修飾した短干渉核酸（ｓｉＮＡ）を用いる遺伝子発現のＲＮＡ干渉媒介性阻害
EP1942943A2 (en) Peptide-dicer substrate rna conjugates as delivery vehicles for sirna
JP2007525192A (ja) 化学修飾した低分子干渉核酸（ｓｉＮＡ）を使用する遺伝子発現のＲＮＡ干渉仲介抑制
JP2021525508A (ja) 核酸治療薬のための調節可能な共カップリングポリペプチドナノ粒子送達系の組成物および方法
JP2023550485A (ja) Ｄｇａｔ２調節のためのオリゴヌクレオチド
WO2013056670A1 (zh) 小干扰RNA及其应用和抑制plk1基因表达的方法
US9758782B2 (en) Inhibition of microRNA for treatment of sepsis
WO2023134609A1 (zh) 一种抑制PCSK9基因表达的RNAi剂及其应用
WO2015051135A2 (en) Organic compositions to treat hepcidin-related diseases
US20100280097A1 (en) Compositions comprising hif-1 alpha sirna and methods of use thereof
US20100279919A1 (en) Compositions comprising human integrin-linked kinase-sirna and methods of use thereof
KR102630164B1 (ko) 펩티드 핵산 복합체를 유효성분으로 함유하는 비알콜성 지방간염 예방 또는 치료용 조성물
US20210087221A1 (en) Spherical nucleic acids with tailored and active protein coronae
US20230257753A1 (en) Products and compositions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23739951

Country of ref document: EP

Kind code of ref document: A1