WO2024032608A1 - Molécule d'arnsi pour réguler l'activité du gène angptl3 - Google Patents

Molécule d'arnsi pour réguler l'activité du gène angptl3 Download PDF

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WO2024032608A1
WO2024032608A1 PCT/CN2023/111739 CN2023111739W WO2024032608A1 WO 2024032608 A1 WO2024032608 A1 WO 2024032608A1 CN 2023111739 W CN2023111739 W CN 2023111739W WO 2024032608 A1 WO2024032608 A1 WO 2024032608A1
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seq
sirna
antisense strand
nhc
chain
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PCT/CN2023/111739
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Chinese (zh)
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黄金宇
岳明
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大睿生物医药科技(上海)有限公司
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    • 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
    • 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

  • This disclosure relates to the field of RNA interference.
  • Angiopoietins are a family of secreted growth factors. Together with its corresponding endothelial-specific receptors, angiopoietin plays an important role in angiogenesis.
  • Angiopoietin-like 3 also known as angiopoietin-like 3, ANGPTL3 or angiopoietin 5, ANGPT5
  • ANGPTL3 angiopoietin-like 3
  • ANGPT5 angiopoietin 5
  • Angiopoietin-like 3 or ANGPTL3 is a lipid metabolism regulator that can regulate VLDL triglycerides (TG) by inhibiting the catalytic activity of lipoprotein lipase (LPL).
  • TG VLDL triglycerides
  • LPL lipoprotein lipase
  • the APOEKO mouse model with hypl mutation (apoEKO-hypl) has reduced Angptl3 expression.
  • This mouse model exhibited significant reductions in VLDL TG, VLDL cholesterol, and plasma apoB levels, and post-heparin plasma LPL and hepatic lipase activities were significantly increased in apoEKO-hypl mice, indicating enhanced lipid metabolism (Ando et al., (2003) J. Lipid Res, 44: 1216-1223).
  • Human ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thr
  • siRNA has great potential for development as a new treatment method.
  • siRNA acts on intracellular mRNA. Compared with traditional small molecule drugs, it can directly silence target genes, so it can fundamentally prevent the occurrence and development of diseases more efficiently. develop.
  • due to the poor stability of siRNA it is easily degraded by nucleases in the body, is not easily absorbed by tissues, is difficult to be taken up by cells, and is prone to off-target effects, which limits its clinical application.
  • siRNA that can effectively inhibit the expression of ANGPTL3 gene in cells.
  • the present disclosure provides new small interfering RNA (siRNA), kits and pharmaceutical compositions for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, and the siRNA, kits or pharmaceutical compositions are effective in inhibiting the expression of angiopoietin-like 3 (ANGPTL3). Or methods for reducing ANGPTL3 gene expression or treating diseases or symptoms related to ANGPTL3 expression.
  • the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29 A nucleotide sequence of at least 15 consecutive nucleotides.
  • the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
  • the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
  • the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
  • one or both of the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, e.g., the sense strand and the One or both antisense strands contain a 3' overhang and/or a 5' overhang of at least 1 nucleotide.
  • the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand comprises a 3' overhang and/or a 5' overhang of 2 nucleotides. end.
  • the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides.
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
  • the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, and at least 17 contiguous nucleotides
  • the nucleotide sequence of the acid, the nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-14.
  • the siRNA comprises paired sense and antisense strand sequences as shown in Table 3.
  • substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1.
  • the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides.
  • the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.
  • the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the antisense strand contains
  • the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand of the siRNA is conjugated to the ligand moiety.
  • the 3' end of the sense strand is conjugated to the ligand moiety.
  • the 5' end of the sense strand is conjugated to the ligand moiety.
  • the ligand moiety includes a conjugation group represented by formula (X'):
  • L 1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation ligand targets asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • the conjugation group is selected from Table 1:
  • the conjugation group is selected from Table 2:
  • the ligand comprised in the siRNA has the following structure:
  • the ligand comprised in the siRNA has the following structure:
  • the ligand comprised in the siRNA of the present disclosure has the following structure:
  • the ligand comprised in the siRNA of the present disclosure has the following structure:
  • the present disclosure provides a cell containing the siRNA described in the present disclosure.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the siRNA or cells of the disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a kit comprising the siRNA, cells or pharmaceutical composition of the present disclosure.
  • the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising: The subject is administered a siRNA, cell, or pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
  • the disease associated with ANGPTL3 expression is the metabolic disease or cardiovascular disease.
  • the metabolic disease or the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfattyemia or metabolic syndrome or a combination thereof.
  • NAFLD non-alcoholic fatty liver disease
  • the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia.
  • the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
  • the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
  • the disease associated with ANGPTL3 expression is diabetes, which is type 2 diabetes or type 2 diabetes with dyslipidemia.
  • methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA, cells, or pharmaceutical composition to the subject by subcutaneous administration, topical administration, or intravenous administration.
  • the subject is a human subject.
  • siRNA refers to a class of double-stranded RNA molecules that can mediate silencing of a target RNA that is complementary to it (eg, mRNA, eg, the transcript of a gene encoding a protein).
  • siRNA is usually double-stranded, including an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand.
  • mRNA is also referred to herein as the mRNA to be silenced.
  • Such genes are also called target genes.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNA other than mRNA e.g. tRNA
  • viral RNA can also be targeted.
  • antisense strand refers to a strand of siRNA that contains a region that is completely or substantially complementary to a target sequence.
  • the term "complementary region” refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotide of the 5' and/or 3' end. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region.” For example, in a siRNA containing a 19nt strand, some mismatches can be tolerated at position 19 (from 5' to 3').
  • stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours.
  • complementary sequences may also include or be formed entirely from non-Watson-Crick base pairs and/or from non-natural and processed base pairs.
  • Base pairs formed by modified nucleotides include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.
  • a polynucleotide that is “at least partially complementary” or “substantially complementary” to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding ANGPTL3). glycosides.
  • a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding ANGPTL3.
  • sense strand refers to a strand of siRNA that includes a region that is substantially complementary to a region that is the antisense strand as the term is defined herein.
  • Nucleoside is a compound composed of two substances: purine base or pyrimidine base, and ribose or deoxyribose.
  • Nucleoside is a compound composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphate.
  • Olionucleotide refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides.
  • Base is the basic unit for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, also known as “nitrogen-containing bases”.
  • the capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA (eg, siRNA). Nucleotide overhangs exist, for example when the 3'-end of one strand of siRNA extends beyond the 5'-end of the other strand or vice versa.
  • the siRNA can comprise an overhang having at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. many.
  • Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs (including deoxynucleotides/nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the overhanging nucleotide or nucleotides may be present on the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.
  • siRNAs of the present disclosure include siRNAs with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.
  • nucleotides of the iRNAs of the present disclosure are modified.
  • All nucleotides are modified nucleotides, and/or substantially all nucleotides in the antisense strand are modified nucleotides, and/or substantially all nucleotides in both the sense and antisense strands are Acids are all modified nucleotides.
  • all nucleotides of the iRNA of the disclosure are modified nucleotides.
  • nucleotides of the sense strand are modified nucleotides
  • all nucleotides of the antisense strand are modified nucleotides
  • all nucleotides of both the sense strand and the antisense strand are all modified nucleotides.
  • substantially all nucleotides are modified means that the siRNAs of the present disclosure are mostly, but not entirely, modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.
  • Modified nucleotides herein include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleic acids).
  • nucleotides 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides , nucleotides containing phosphorothioate groups, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleosides Acids, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, linked to cholesteryl derivatives or dodecyl dodecyl amido groups Terminal nucleotides on the group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleo
  • the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.
  • the 2'-deoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • Nucleotide containing a phosphorothioate group refers to a nucleotide in which one or more oxygen atoms on the phosphate are replaced by sulfur atoms.
  • Modification of phosphorothioate inter-nucleotide bonding refers to the modification in which two adjacent nucleotides on the left and right are connected through phosphorothioate.
  • a "ligand moiety” refers to a chemical moiety that conjugates to an siRNA and is capable of altering the distribution, targeting, or lifetime of the siRNA.
  • ligand is a selected target (e.g. molecule, cell or cell type, compartment (e.g. cell or organ compartment, tissue, organ or area of the body) provides enhanced affinity.
  • the ligand moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand moiety to ASGPR mediates internalization via clathrin-coated vesicles. Maturation of endosomes results in a decrease in lysosomal pH, which promotes dissociation of ligand-receptor complexes, thereby releasing siRNA. Conjugation of a ligand moiety targeting the asialoglycoprotein receptor (ASGPR) on hepatocytes results in siRNA efficacy and stability in vivo or intracellularly. This facilitates subcutaneous administration of the siRNA.
  • ASGPR asialoglycoprotein receptor
  • inhibitortion is used interchangeably with “reduction,” “silencing,” “downregulation,” and other similar terms and includes any level of inhibition.
  • the phrase "inhibiting the expression of ANGPTL3" is intended to mean inhibiting the expression of any ANGPTL3 gene as well as variants or mutants of the ANGPTL3 gene.
  • the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or in the case of a genetically manipulated cell, cell population, or organism, a transgenic ANGPTL3 gene.
  • “Inhibition of ANGPTL3 gene expression” includes any level of inhibition of the ANGPTL3 gene, such as at least partial inhibition of ANGPTL3 gene expression.
  • ANGPTL3 gene expression can be assessed based on the level or change in level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels, ANGPTL3 protein levels, or lipid levels. This level can be assessed in individual cells or in a group of cells (including, for example, a sample derived from a subject).
  • Inhibition can be assessed by a reduction in absolute or relative levels of one or more variables associated with ANGPTL3 expression compared to control levels.
  • the control level may be any type of control level utilized in the art, such as a pre-dose baseline level or from a similar untreated or control (e.g., buffer only control or inert control) subject, cell , or the level determined by the sample.
  • “Hydroxy protecting group” refers to a group that can protect the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group.
  • Mainly include silane-type protective groups, acyl-type protective groups or ether-type protective groups, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac ), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc
  • Halo or "halogen” refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • C 1-6 haloalkyl refers to the above-mentioned “C 1-6 alkyl” which is substituted by one or more halogen groups. In some embodiments, C 1-4 haloalkyl is particularly preferred, with C 1-2 haloalkyl being more preferred. Exemplary haloalkyl groups include, but are not limited to: -CF 3 , -CH 2 F, -CHF 2 , -CHFCH 2 F, -CH 2 CHF 2 , -CF 2 CF 3 , -CCl 3 , -CH 2 Cl , -CHCl 2 , 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. Haloalkyl groups may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • C 1-6 alkylene refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred.
  • the unsubstituted alkylene group includes, but is not limited to: methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), ethylene Base (-CH 2 CH 2 CH 2 CH 2 -), pentylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), hexylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -) ,etc.
  • alkylene groups substituted by one or more alkyl (methyl) include, but are not limited to: substituted methylene (-CH(CH 3 )- , -C(CH 3 ) 2 -), substituted ethylene (-CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )-, -C(CH 3 ) 2 CH 2 -, -CH 2 C(CH 3 ) 2- ), substituted propylene (-CH(CH 3 )CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH(CH 3 ) -, -C(CH 3 ) 2 CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 -, -CH 2 CH 2 C(CH 3 ) 2 -), etc.
  • treatment refers to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease.
  • treatment may include treatment of a disease or condition (eg, cancer) in a mammal, particularly a human, and includes: (a) prophylaxis in a subject susceptible to the disease but who has not yet been diagnosed with the disease.
  • the occurrence of the disease or disease symptoms (for example, including diseases that may be related to or caused by the primary disease); (b) inhibit the disease, that is, prevent its progression; (c) alleviate the disease, that is, cause the regression of the disease.
  • Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty.
  • Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination.
  • treating includes administering an siRNA or pharmaceutical composition disclosed in the present disclosure to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (eg, cancer).
  • a disease eg, cancer
  • therapeutic effect refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.
  • terapéuticaally effective amount refers to an amount that, when administered to a subject to treat a disease, is sufficient to effect treatment of a disease.
  • the term "subject” refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired.
  • "Mammal” for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.
  • the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the length of the antisense strand is each independently 15-30 nucleotides, and the antisense strand includes at least 15 consecutive nucleotide sequences of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 The nucleotide sequence of a nucleotide.
  • siRNA small interfering RNA
  • the double-stranded region formed by the sense strand and antisense strand is completely complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain 1, 2, 3, 4, or 5 non-complementary sites.
  • the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
  • the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.
  • the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.
  • the sense strand and the antisense strand comprise a 3' overhang and/or a 5' overhang having at least 1 nucleotide, for example one or both of the sense strand and the antisense strand or Both contain 3' overhangs and/or 5' overhangs with at least 1 nucleotide.
  • the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand includes a 3' overhang of 2 nucleotides. Overhangs and/or 5' overhangs.
  • the sense strand and the antisense strand are the same length.
  • the entire length of the sense strand is complementary to the entire length of the antisense strand to form a double strand, ie, has blunt ends.
  • the sense strand and the antisense strand are the same length, and a part of the sense strand is complementary to a part of the antisense strand, that is, both the sense strand and the antisense strand have a 5' overhang.
  • the sense strand and the antisense strand are different lengths.
  • the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.
  • the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides.
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.
  • the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, at least 17 contiguous nucleotides acid nucleotide sequence, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse
  • the sense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-1093.
  • the siRNA comprises paired sense strand sequences and reverse strand sequences as shown in Table 3 sense strand sequence.
  • substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and/or at least 80% of the nucleotides of the antisense strand are modified nucleotides.
  • all nucleotides of the sense strand and/or all nucleotides of the antisense strand are modified nucleotides.
  • Modifications of nucleotides described in the present disclosure may be modifications on the phosphate group, ribose group and/or base group of the nucleotide.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
  • the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.
  • the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides.
  • the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.
  • the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.
  • the sense strand and/or antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides and/or cytosine deoxyribonucleotides glycosides.
  • the sense strand and/or antisense strand comprise thymidine deoxyribonucleotides.
  • the sense strand comprises thymidine deoxyribonucleotides.
  • the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and/or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification. Modified nucleotide sequence.
  • the siRNA includes the paired modified sense strand sequence and the modified antisense strand sequence shown in any one of Table 6 of the specification.
  • the siRNAs of the present disclosure are further conjugated to a ligand moiety comprising N-acetylgalactosamine.
  • the sense strand of the siRNA is conjugated to the ligand moiety.
  • the 3' end of the sense strand is conjugated to the ligand moiety.
  • the 5' end of the sense strand is conjugated to the ligand moiety.
  • the ligand moiety includes a conjugation group represented by formula (X'):
  • L 1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugating group is represented by Formula (I'):
  • L 1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, - (OCH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond, -C(O)NH- or -NHC(O)-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O-
  • L’ is a chemical bond
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):
  • L 1 is -CH 2 O- or -NHC(O)-;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O-
  • R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond or -C(O)NH-
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the conjugation group is represented by Formula (II'-1) or Formula (II'-2):
  • L 1 is -CH 2 O- or -CH 2 O-CH 2 CH 2 O-;
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -NHC(O)-
  • L’ is a chemical bond or -C(O)NH-
  • R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a - ;
  • L 2 is a chemical bond
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is -CH 2 O- or -NHC(O)-;
  • L’ is a chemical bond or -C(O)NH-
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is -CH 2 - or -C(O)-;
  • L 3 is -(NHCH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c -;
  • c 1, 2, 3, 4 or 5;
  • L is -CH 2 O- or -NHC(O)-;
  • R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • T is -M-, -CH 2 -M- or -C(O)-M-, where M is
  • L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -CH 2 O-;
  • L' is a chemical bond or -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • conjugation group is represented by Formula (III'-1), Formula (III'-2) or Formula (III'-3):
  • L 1 is -CH 2 O- or -NHC(O)-;
  • L 2 is -CH 2 CH 2 C(O)-
  • L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -CH 2 O-;
  • R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
  • L 2 is a chemical bond
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -NHC(O)-
  • L’ is a chemical bond
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • L 1 is -CH 2 -, -CH 2 O- or -C(O)-;
  • L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond or -NHC(O)-
  • L’ is a chemical bond
  • R' is H, a hydroxyl protecting group or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • T is as defined in the embodiment above.
  • L 1 is a chemical bond, -CH 2 -, -CH 2 CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-( CH 2 NHC(O)) a -;
  • L 2 is a chemical bond or -CH 2 CH 2 C(O)-;
  • L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;
  • L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;
  • b 1, 2, 3, 4 or 5;
  • c 1, 2, 3, 4 or 5;
  • d 1, 2, 3, 4, 5, 6, 7 or 8;
  • L is a chemical bond, -CH 2 O- or -NHC(O)-;
  • L' is -O(CH 2 CH 2 O) e -;
  • e 1, 2, 3, 4 or 5;
  • T is a chemical bond, -CH 2 -, -C(O)-, -M-, -CH 2 -M- or -C(O)-M-;
  • R 1 and R 2 together form -CH 2 CH 2 O- or -CH 2 CH(R)-O-, and R 3 is H;
  • R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;
  • R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH 2 CH 2 C(O)OH or 4,4'-dimethoxytrityl;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • conjugating group is selected from the following:
  • conjugating group is selected from the following:
  • the ligand targets asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • said ligand has the following structure:
  • said ligand has the following structure:
  • said ligand has the following structure:
  • said ligand has the following structure:
  • the siRNA of the present disclosure can inhibit ANGPTL3 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, At least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • Inhibition of ANGPTL3 gene expression may be manifested by a decrease in the amount of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from the subject) in which the ANGPTL3 gene is transcribed and the cells or these cells have been treated (e.g., by contacting the cell or cells with an siRNA of the present disclosure, or by administering an siRNA of the present disclosure to a subject in which these cells are present or previously present, such that contact with the first cell or cells ANGPTL3 gene expression is inhibited compared to a second group or group of cells (one or more control cells) that are substantially the same but have not been so treated.
  • the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the following formula.
  • inhibition of ANGPTL3 gene expression may be assessed in terms of reduction in parameters functionally related to ANGPTL3 gene expression, such as lipid levels, cholesterol levels, e.g., LDLc levels.
  • ANGPTL3 gene silencing can be determined in any cell that expresses ANGPTL3 constitutively or by genome engineering and by any assay known in the art.
  • the liver is the main site of ANGPTL3 expression.
  • Other important sites of expression include the pancreas, kidney, and intestine.
  • Inhibition of expression of ANGPTL3 protein may be manifested by a decrease in the level of ANGPTL3 protein expressed by a cell or population of cells (eg, the level of protein expressed in a sample derived from a subject).
  • a cell or population of cells eg, the level of protein expressed in a sample derived from a subject.
  • inhibition of protein expression levels in treated cells or populations of cells can similarly be expressed as a percentage of the levels of the protein in control cells or populations of cells.
  • Control cells or cell populations that may be used to assess inhibition of ANGPTL3 gene expression include cells or cell populations that have not been contacted with the siRNA of the present disclosure.
  • the control cells or population of cells may be derived from an individual subject (eg, a human or animal subject) prior to treatment of the subject with siRNA.
  • the present disclosure provides cells containing the siRNA of the present disclosure, wherein the siRNA of the present disclosure is capable of being transcribed in the cell.
  • compositions comprising the siRNA or cells of the present disclosure, and optionally a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for contact with tissues of human subjects and animal subjects without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates the administration of siRNA or cells containing the siRNA to the human body and/or facilitates its absorption or effect.
  • diluents excipients such as water, fillers such as starch, sucrose, etc.
  • binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone
  • wetting agents such as glycerin
  • disintegrants such as agar, carbonic acid Calcium and sodium bicarbonate
  • absorption accelerators such as quaternary ammonium compounds
  • surfactants such as cetyl alcohol
  • adsorption carriers such as kaolin and bentonite
  • lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc.
  • other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.
  • a pharmaceutical composition containing the siRNA or cells of the present disclosure may include a pharmaceutically acceptable diluent or sustained-release matrix in which the siRNA of the present disclosure is embedded.
  • kits comprising the siRNA or cells described in the present disclosure.
  • kits for using the siRNA described in the present disclosure and/or performing the methods of the present disclosure include one or more siRNAs or cells described in the present disclosure and may further include instructions for use. Instructions for inhibiting the expression of ANGPTL3 in the cell by contacting the cell with the siRNA described in the present disclosure in an amount effective to inhibit the expression of ANGPTL3 may be recorded in the instructions for use.
  • the kit of the present disclosure may also include means (e.g., an injection device) for contacting the cells with the siRNA of the present disclosure or with Tools for measuring the inhibitory effect of ANGPTL3 (e.g., a device for measuring inhibition of ANGPTL3 mRNA or protein).
  • a device for measuring inhibition of ANGPTL3 may comprise a device for obtaining a sample (eg, like a plasma sample) from a subject.
  • the kit of the present disclosure may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject.
  • Device or device for determining a therapeutically effective amount or a prophylactically effective amount may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject.
  • the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, fat pad weight in a subject, the method comprising administering to the subject siRNA, cells, or pharmaceutical compositions of the present disclosure.
  • the present disclosure provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.
  • the disease associated with ANGPTL3 expression is cardiovascular disease.
  • the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or metabolic syndrome or its combination.
  • the disease associated with ANGPTL3 expression is a dyslipidemia, and the dyslipidemia is hyperlipidemia.
  • hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
  • the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.
  • the disease associated with ANGPTL3 expression is diabetes, and the diabetes Is type 2 diabetes or type 2 diabetes with dyslipidemia.
  • methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA or pharmaceutical composition to the subject including subcutaneous administration or intravenous administration.
  • the subject is a human patient.
  • the present disclosure also relates to the siRNA, cells or pharmaceutical compositions of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject.
  • the present disclosure also relates to the use of the siRNA, cells, or pharmaceutical compositions of the present disclosure in the preparation of a medicament for treating a disease or symptom associated with ANGPTL3 expression in a subject.
  • the medicine of the present disclosure can be prepared into emulsions, microemulsions, and microparticles.
  • RNA sequence provided by the present disclosure targets the human ANGPTL3 gene (or target gene, target mRNA sequence, target sequence).
  • Tables 4 to 6 show modified RNA sequences used in the present disclosure.
  • the A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.
  • d indicates that the nucleotide adjacent to the right is a deoxyribonucleotide.
  • dA, dT, dG and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide and cytosine deoxyribonucleotide respectively.
  • i inosine ribonucleotide
  • m indicates that the adjacent nucleotide to its left is a 2'-OCH 3 modified nucleotide.
  • Am, Um, Gm and Cm represent 2'-OCH 3 modified A, U, G and C.
  • f indicates that the adjacent nucleotide to its left is a 2'-F modified nucleotide.
  • Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.
  • s or "s-" means that two adjacent nucleotides and/or delivery vectors are connected through phosphorothioate.
  • VP indicates that the nucleotide adjacent to the right side is a vinylphosphonate-modified nucleotide, which is well known in the art, see, for example, PCT Publication Nos. WO2011139702, WO2013033230 and WO2019105419.
  • IB stands for reverse abasic deoxyribonucleotide, which can include the following three structures depending on its position/connection method in siRNA (respectively for the 5' end, middle and 3' end of the nucleic acid chain):
  • L96 represents a GalNAc delivery vector of the following structure, which is well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or a phosphorothioate group, see for example PCT Publication Nos. WO2009073809 and WO2009082607.
  • NAG37 represents a GalNAc delivery vector of the structure well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or phosphorothioate group, see for example PCT Publication No. WO2018044350
  • GL6 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
  • GL12 represents a GalNAc delivery vector of the following structure, where Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA
  • STM1 represents the nucleotide substitution of the following structure. Depending on the position of STM1 in the nucleic acid chain, Can be linked to adjacent nucleotides, 3' end structures or 5' end structures
  • Huh7 cell line was purchased from Nanjing Kebai, product number CBP60202;
  • Hep3B cell line was purchased from Nanjing Kebai, product number CBP60197;
  • PHH cells were purchased from Shanghai Xuanyi, product number QYLF-HPMC;
  • HEK293A cell line was purchased from Nanjing Kebai, product number CBP60436;
  • Balb/c mice were from Zhejiang Vitong Lever, product number Balb/c.
  • the filter cake was ground with water (1.45 L) at 25°C for 30 minutes. The mixture was filtered and the filter cake was washed with water (175 mL x 3), and the filter cake was collected to obtain compound 2A as a white solid (about 580 g).
  • reaction solution was concentrated under reduced pressure, and the crude product obtained was prepared by preparative high-performance liquid chromatography (preparative-HPLC, column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: water-ACN; B%: 17% -57%, 5min) to obtain white solid compound E7 (53.0mg, yield 17.08%, purity 78.94%).
  • reaction mixture was slowly poured into a stirred cold 0.5 M aqueous HCl solution (230 mL), stirred for 10 minutes, a white solid formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L).
  • the combined organic phases were washed with 5% NaHCO 3 (aq.) (200 mL), dried (Na 2 SO 4 ), and concentrated by evaporation under pressure.
  • compound 1-7 (8.00g, 24.4mmol) was added to HCl (100mL, 12M), and the reaction was carried out at 50°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 1-8 (about 3.60g) as a colorless oil. ,HCl salt),
  • siRNAs of the present disclosure are prepared using the solid-phase phosphoramidite method, which is well known in the art. Specific methods can be found, for example, in PCT publication numbers WO2016081444 and WO2019105419, and are briefly described below.
  • the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. monomer.
  • Each connection of a nucleoside monomer or nucleotide analog monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5 ⁇ mol are as follows:
  • the nucleoside monomer or nucleotide analog monomer is provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions of each step are the same, that is, the temperature is 25°C, and 3% trichloroacetic acid-dichloromethane solution is used for deprotection.
  • the activator used in the coupling reaction was 0.25 mol/L ETT-acetonitrile solution, coupled 2 times; the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10: 14:76, v/v/v), blocked twice; oxidized using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; used sulfide 0.2mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v), sulfide 2 times.
  • the nucleotide monomer of IB was purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP-040.
  • the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. single object.
  • Each linkage of a nucleoside monomer or nucleotide analog monomer involves deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions of 5 ⁇ mol of oligonucleotide for the antisense strand are the same as those for the sense strand.
  • a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, and the products can be collected and tubed.
  • a gel packing purification column can be used for desalting, and the elution system is pure water.
  • Compound E13 was connected to the CPG carrier in a manner similar to the method for connecting compound E7 to the CPG carrier.
  • the GL6 solid-phase carrier prepared in Section 2.1.1 of Example 4 is used as the initial cycle, and the nucleosides are connected one by one from the 3'-5' direction in the sequence of the sense strand nucleotides. monomer.
  • Each connection of a nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions for an oligonucleotide with a synthesis scale of 5 ⁇ mol are as follows:
  • the nucleoside monomer is provided with a 0.05 mol/L acetonitrile solution.
  • the reaction conditions for each step are the same, that is, the temperature is 25°C.
  • the temperature is 25°C.
  • the capping agent used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/ v), cap twice; use 0.05mol/L iodine for oxidation /tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; for sulfide, use 0.2 mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v). Thiogeneration 2 times.
  • a blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction in the order of the antisense strand nucleotide arrangement.
  • Each connection of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfation.
  • the synthesis conditions of 5 ⁇ mol of oligonucleotide of the antisense strand are the same as those of the sense strand.
  • a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, the products can be collected and tubed, and a gel packing purification column can be used for desalting.
  • the elution system is pure water.
  • L96-conjugated siRNA and NAG37-conjugated siRNA were obtained in a similar manner.
  • the Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested, resuspended, counted, and plated in a 96-well plate at 100 ⁇ L/well and 1 ⁇ 10 4 cells/well. Transfection was performed 18 hours later.
  • RNAiMAX (Thermo, 13778150)
  • 14.1 ⁇ L Opti-MEM and dilute 0.9 ⁇ L RNAiMAX (Thermo, 13778150)
  • mix gently by pipetting and let stand at room temperature for 5 minutes.
  • Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
  • Denaturation reaction mixture To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 ⁇ L of Oligo dT Primer, 1 ⁇ L of dNTP Mixture, and 12.5 ⁇ L of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture Cool quickly on ice for 2 minutes.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 ⁇ L of 5 ⁇ Prime Script II Buffer, 0.5 ⁇ L of RNase Inhibitor, and 1 ⁇ L of PrimeScript II RTase.
  • reaction program is: (50°C, 2min) ⁇ 1Cycle; (95°C, 20s) ⁇ 1Cycle; (95°C, 1s; 60°C, 24s) ⁇ 40Cycles.
  • ⁇ Ct [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
  • the final concentration of siRNA was 1 nM for high-throughput screening of cell line activity of siRNA compounds.
  • the experimental screening results are shown in Table 8.
  • the starting concentration of siRNA was 10 nM, and 10-fold gradient dilution was performed to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Hep3B cell line activity of siRNA was screened.
  • the screening results are shown in Table 9, in which columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
  • PHH cells (Shanghai Xuanyi, Cat. No. QYLF-HPMC) were revived at 37°C, added to the recovery medium, centrifuged, resuspended and counted. PHH cells were plated in a 96-well plate at 90 ⁇ L/well, 2 ⁇ 10 4 cells/well; complete medium was replaced after 4 hours, and transfection was performed after 18 hours.
  • siRNA stock solution was diluted with Opti-MEM. Add 198 ⁇ L Opti-MEM to 2 ⁇ L siRNA stock solution, mix by pipetting, and use it as the first concentration point. Perform corresponding gradient dilutions according to actual experimental needs.
  • RNAiMAX (Thermo, 13778150)
  • Opti-MEM dilute 0.9 ⁇ L RNAiMAX
  • 15 ⁇ L of the prepared RNAi-MAX mixture and 15 ⁇ L of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let it stand at room temperature for 10 minutes.
  • Cell RNA was extracted using a nucleic acid extractor (Auto-pure96, Hangzhou Aosheng) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).
  • Denaturation reaction mixture To prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 ⁇ L of Oligo dT Primer, 1 ⁇ L of dNTP Mixture, and 12.5 ⁇ L of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture on ice to cool quickly for 2 minutes.
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 ⁇ L of 5 ⁇ Prime Script II Buffer, 0.5 ⁇ L of RNase Inhibitor, and 1 ⁇ L of PrimeScript II RTase.
  • reaction program is: (50°C, 2min) ⁇ 1Cycle; (95°C, 20s) ⁇ 1Cycle; (95°C, 1s; 60°C, 24s) ⁇ 40Cycles.
  • ⁇ Ct [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].
  • the starting concentration of siRNA was 10 nM, and it was diluted 10 times to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM).
  • the activity of siRNA in human liver primary cells was screened. The screening results are shown in Table 11. , where columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.
  • the corresponding antisense strand off-target plasmid was designed based on the siRNA sequence, and the psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Bioengineering (Shanghai) Co., Ltd., and the recombinant plasmid was diluted to 1000ng/ ⁇ L for later use.
  • HEK293A cell (Nanjing Kebai, Cat. No. CBP60436) resuspension was plated, with 8 ⁇ 10 3 cells/well.
  • siRNA preparation Dilute siRNA 3 times starting from a final concentration of 40nM, with a total of 11 concentration points (10nM, 3.3333nM, 1.1111nM, 0.37037nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM, 0.00051nM, 0.0 0017 nM).
  • the preparation volume for a single well is 0.01 ⁇ L/well for plasmid and 8.99 ⁇ L/well for Opti-MEM.
  • Lipo mixture Preparation of Lipo mixture: Add 0.2 ⁇ L of Lipo 2000 and 9.8 ⁇ L of Opti-MEM to each well to dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM to obtain a Lipo mixture, and let it stand at room temperature for 5 minutes.
  • Lipo 2000 Lipofectamine TM 2000 transfection reagent, Thermo, 11668019
  • the fluorescence activity is measured by a microplate reader, and the collected Renilla signals are normalized by the Firefly signal standard.
  • the inhibitory effect of siRNA is obtained by comparing the unprocessed results (residual inhibitory activity). The calculation process is as follows:
  • Ratio Renilla (Renilla luciferase)/Firefly (firefly luciferase).
  • the remaining inhibition rate (RatiosiRNA/Ratiocontrol) * 100%, take the average of two duplicate wells: Ratiocontrol is the Ratio value of the control well (excluding siRNA) (take the average of two duplicate wells).
  • IC50 Half maximal inhibitory concentration
  • results of the off-target activity screening of siRNA's psiCHECK2 GSSM-5Hits are shown in Table 12, in which columns 2-12 are the remaining inhibition rates and column 13 is the IC50 value.
  • the results show that DR000405, DR000424, DR000430, DR000442, DR000447, DR000619, and DR000621 of the present disclosure have lower off-target activity against HEK293A cells compared to the positive control DR001483.
  • mice Using high-pressure tail vein injection, six- to eight-week-old female Balb/c mice were treated with double-gene stabilized
  • the transfection system performs in vivo transfection modeling. Piggy-Bac transposon plasmids containing target gene cDNA sequences (Genbank registration number NM_014495.2) with different mass ratios are transferred through the tail vein through a 27-gauge needle within 5-7 seconds.
  • the delivery solution (total volume is 10% of the animal body weight, Mirusbio-MIR 5240) was injected into the mice, and after injection, they were returned to the cage for observation for 30 min. Taking the day of modeling as day 0, serum was obtained at various time points after modeling (day 7 to day 35) for detecting SEAP expression levels.
  • Dual-gene stable transfection system including Piggy-Bac auxiliary plasmid and Piggy-Bac transposon plasmid, in which Piggy-Bac auxiliary plasmid provides Piggy-Bac transposase; Piggy-Bac transposon plasmid uses Piggy-Bac transposon Based on it, it contains a dual-gene expression element, which contains the secreted alkaline phosphatase gene (SEAP) and the target gene (ANGPTL3). SEAP and ANGPTL3 are co-expressed.
  • SEAP secreted alkaline phosphatase gene
  • ANGPTL3 target gene
  • CSPD substrate and reaction buffer diluent at a ratio of 1:20 to form a reaction solution.
  • siRNA compounds The effect of siRNA compounds on inhibiting the expression of target genes was evaluated by measuring SEAP expression levels in serum. The lower the SEAP chemiluminescence value, the better the siRNA compound is at inhibiting the expression of the target gene. Select the test sample that can inhibit the expression level of SEAP as a nucleic acid drug.
  • each mouse was given a single subcutaneous administration according to Table 13: 200 ⁇ l of physiological saline containing 3 mg/kg (mpk) RNAi reagent; or 200 ⁇ l of physiological saline without RNAi reagent was used as a control (vehicle) .
  • the HDI model screening results are shown in Table 14.

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Abstract

L'invention concerne un petit ARN interférent (ARNsi) pour inhiber l'expression de la protéine de type angiopoïétine 3 (ANGPTL3) dans une cellule, une cellule comprenant l'ARNsi, et un procédé pour traiter une maladie ou un symptôme associé à l'expression d'ANGPTL3 chez un sujet à l'aide de l'ARNsi ou de la cellule.
PCT/CN2023/111739 2022-08-08 2023-08-08 Molécule d'arnsi pour réguler l'activité du gène angptl3 WO2024032608A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130023579A1 (en) * 2010-01-08 2013-01-24 Isis Pharmaceuticals, Inc. Modulation of angiopoietin-like 3 expression
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
WO2020099482A2 (fr) * 2018-11-13 2020-05-22 Lipigon Pharmaceuticals Ab Oligonucléotides d'angptl3 influençant la régulation du métabolisme des acides gras
CN111343994A (zh) * 2017-09-14 2020-06-26 箭头药业股份有限公司 用于抑制血管生成素-样3 (ANGPTL3)的表达的RNAi剂和组合物以及使用方法
WO2022068923A1 (fr) * 2020-09-30 2022-04-07 纳肽得(青岛)生物医药有限公司 Arnsi de l'angiopoïétine-like 3 (angptl3) et son utilisation
WO2022079222A1 (fr) * 2020-10-16 2022-04-21 Sanofi Nouvelles compositions d'arn et méthodes d'inhibition d'angptl3

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130023579A1 (en) * 2010-01-08 2013-01-24 Isis Pharmaceuticals, Inc. Modulation of angiopoietin-like 3 expression
US20180087054A1 (en) * 2015-04-13 2018-03-29 Alnylam Pharmaceuticals, Inc. ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
CN111343994A (zh) * 2017-09-14 2020-06-26 箭头药业股份有限公司 用于抑制血管生成素-样3 (ANGPTL3)的表达的RNAi剂和组合物以及使用方法
WO2020099482A2 (fr) * 2018-11-13 2020-05-22 Lipigon Pharmaceuticals Ab Oligonucléotides d'angptl3 influençant la régulation du métabolisme des acides gras
WO2022068923A1 (fr) * 2020-09-30 2022-04-07 纳肽得(青岛)生物医药有限公司 Arnsi de l'angiopoïétine-like 3 (angptl3) et son utilisation
WO2022079222A1 (fr) * 2020-10-16 2022-04-21 Sanofi Nouvelles compositions d'arn et méthodes d'inhibition d'angptl3

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