WO2023134705A1 - 抑制angptl3表达的rna干扰剂及其用途 - Google Patents

抑制angptl3表达的rna干扰剂及其用途 Download PDF

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WO2023134705A1
WO2023134705A1 PCT/CN2023/071788 CN2023071788W WO2023134705A1 WO 2023134705 A1 WO2023134705 A1 WO 2023134705A1 CN 2023071788 W CN2023071788 W CN 2023071788W WO 2023134705 A1 WO2023134705 A1 WO 2023134705A1
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nucleotides
rna interference
antisense strand
modified
agent according
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French (fr)
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李冲
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上海金中锘美生物医药科技有限公司
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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
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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 present application relates to the technical field of biomedicine, in particular to an RNA interference agent for inhibiting the expression of ANGPTL3 and its use.
  • RNA interference refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. It is a mechanism left over from the process of biological evolution to regulate gene expression through RNA after transcription. Since the use of RNAi technology can specifically inhibit the expression of specific genes (dsRNA with a length of more than 30 bp can cause interferon toxicity), this technology has been widely used in the fields of exploring gene functions, treating infectious diseases and malignant tumors. RNAi is mediated by the RNA-induced silencing complex (RISC), a complex formed by the binding of siRNA to the Argonaute protein and Dicer enzyme.
  • RISC RNA-induced silencing complex
  • the small dsRNA intermediate molecule that is, the small RNA double strand
  • Dicer enzyme the small RNA double strand
  • these small RNA double strands are first melted to form a "functional" single strand, thus serving as a "guide” for the RISC complex "effect.
  • the guide strand can bind to a specific Argonaute protein to form an activated RISC complex.
  • RNA interfering agents such as siRNA and microRNA inhibit the gene translation of proteins by gene silencing, that is, by degrading mRNA molecules, thereby preventing the production of the corresponding proteins.
  • Angiopoietin-like 3 is an angiopoietin protein encoded by the human angiopoietin-like 3 gene, mainly expressed in the liver and used to regulate lipid metabolism.
  • ANGPTL3 inhibits the catalytic activity of lipoprotein lipase (LPL) and endothelial lipase (EL), resulting in increased plasma levels of triglycerides, high-density lipoprotein (HDL) and phospholipids.
  • ANGPTL3 is a 460 amino acid polypeptide consisting of a signal peptide, an N-terminal coiled-coil domain and a C-terminal fibrinogen (FBN)-like domain.
  • Dysregulation of lipid metabolism leads to elevated blood lipid levels (such as triglycerides and/or cholesterol), which are highly associated with hypertension, cardiovascular disease, diabetes, and other pathological conditions.
  • Hypertriglyceridemia is an instance of dysregulation of lipid metabolism cases, characterized by high levels of triglycerides in the blood.
  • Potent therapeutic agents targeting ANGPTL3 are useful in the treatment (including prophylactic treatment) of cardiometabolic diseases such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II Diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related disorders and disease.
  • cardiometabolic diseases such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II Diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia, and other metabolic-related disorders and disease.
  • RNAi agent capable of inhibiting the expression of ANGPTL3 in cells and subjects such as mammals (eg humans) and a composition comprising the interfering agent.
  • the human mRNA sequence is shown in Figure 1.
  • the ANGPTL3 RNA interference (RNAi) agent also known as RNAi trigger or trigger
  • the composition containing the ANGPTL3 RNAi agent can be used to selectively and effectively inhibit the expression of the ANGPTL3 gene.
  • Another object of the present invention is to provide the use of the RNAi agent or/and composition of the present invention, which is used to inhibit the expression of ANGPTL3 gene or/and to treat diseases related to the overexpression of ANGPTL3 gene, such as hyperlipidemia or hyperglycerol Hyperlipidemia and related diseases.
  • the first aspect of the present application provides an RNA interference (RNAi) agent that inhibits the expression of ANGPTL3, comprising a first nucleotide sequence, and the first nucleotide sequence includes any of SEQ ID NO:1 to SEQ ID NO:184 At least 12 consecutive nucleotides in a nucleotide sequence or its complementary nucleotide sequence, or a sequence that differs from said at least 12 consecutive nucleotides by no more than 3 nucleotides, said nucleotide The acid is in a modified or unmodified state.
  • RNAi RNA interference
  • the first nucleotide sequence is a single-stranded oligonucleotide or a double-stranded oligonucleotide.
  • one or more nucleotides in said first nucleotide sequence are modified to form modified nucleotides.
  • the first nucleotide sequence includes any one of the modified sense strand nucleotide sequences listed in Table 2 or a sequence that differs therefrom by no more than 3 nucleotides.
  • the first nucleotide sequence is a sense strand or an antisense strand.
  • the first nucleotide sequence includes a double-stranded structure of an antisense strand and a sense strand.
  • the sense strand has a length of 19-23 nucleotides
  • the antisense strand has a length of 19-26 nucleotides
  • the sense strand has a length of 19 nucleotides and the The length of the antisense strand is 21 nucleotides, or the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides, or the length of the sense strand is 23 nucleotides and the length of the antisense strand is 25; more preferably, the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.
  • the antisense strand nucleotide sequence is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:39, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:117, SEQ ID NO:135, SEQ ID NO:141, SEQ ID NO: 143, one of SEQ ID NO:151, SEQ ID NO:165 and SEQ ID NO:167.
  • the positive-sense strand nucleotide sequence is selected from: SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:112, SEQ ID NO:118, SEQ ID One of NO:136, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:166, and SEQ ID NO:168.
  • an overhang with 1 to 4 unpaired nucleotides is also included, preferably, the overhang is 2 or 3 unpaired nucleotides.
  • said overhang is located at the 3' end of said antisense strand.
  • the sense and/or antisense strands independently comprise one or more modified nucleotides.
  • the modified nucleotides are independently selected from: 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl base-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxynucleotide, 3'-deoxy-thymidine nucleotide, 2'-O-alkyl modified nucleotide, deoxyribonucleotide, 2'-deoxy-2'-fluoro modified core At least one of nucleotides, locked nucleotides, non-basic nucleotides, morpholino nucleotides, phosphoramidites, bicyclic nucleic acids, heterologous nucleotides, EVP,
  • the modified nucleotides are 2'-methoxy-modified nucleotides or 2'-fluoro-modified nucleotides.
  • part or all of the nucleotides at positions 7, 9, 10 and 11 of the sense strand are independently 2'-fluoro-modified nucleotides or 2'-deoxy-modified nucleotides;
  • the nucleotides at the 2nd, 4th, 5th, 6th, 8th, 9th, 10th, 12th, 14th and 16th positions of the antisense strand are partially or completely independent of each other
  • Ground is a 2'-fluoro-modified nucleotide
  • the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides;
  • the nucleotides at positions 2, 5, 6, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides;
  • the nucleotides at positions 2, 5, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide;
  • the nucleotides at positions 2, 4, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide;
  • the nucleotides at positions 2, 6, 12, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides;
  • the nucleotides at positions 2, 6, 10, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides;
  • the nucleotides at positions 2, 8, 9, 14 and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides;
  • the nucleotides at positions 2, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.
  • the sense strand and/or the antisense strand independently comprise one or more phosphorothioate linkages; preferably, the sense strand comprises two between the terminal nucleotides at the 3' and 5' ends Contiguous phosphorothioate linkages, alternatively, the antisense strand comprises two contiguous phosphorothioate linkages between the terminal nucleotides at the 3' and 5' ends.
  • the antisense strand includes any one of the modified antisense strand nucleotide sequences in Table 2 or a sequence that differs therefrom by no more than 3 nucleotides.
  • the sense strand and the antisense strand have the following characteristics: the sense strand comprises or consists of the modified sense strand sequence listed in Table 2, and the antisense strand comprises the modified sense strand sequence listed in Table 2 The antisense strand sequence of or consists of it.
  • the RNA interfering agent further comprises a ligand.
  • the ligand includes a targeting group.
  • the targeting group comprises an asialoglycoprotein receptor ligand.
  • the ligand comprises N-acetyl-galactosamine, and the ligand is attached to the 3' end or the 5' end of the sense strand of the siRNA.
  • said asialoglycoprotein receptor ligand comprises a galactose cluster.
  • said ligand is for conjugation to said sense and/or antisense strand, preferably for conjugation to the sense strand.
  • the ANGPTL3 RNA interfering agent comprises the following structure:
  • X is a nucleotide sequence
  • Z is the first linker part of the nucleotide sequence, wherein Z and X can be connected directly or through a chemical group, and the general formula of Z is as shown in (Z-1), One end of the O base is connected to the nucleotide sequence:
  • R 1 is O, S, NR 3 or CR 3 R 4 , wherein R 3 and R 4 are each independently hydrogen, halogen, substituted or unsubstituted Substituted aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl;
  • R 2 is -O-, -S-, -NH-, -CH 2 -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH 2 NH-, -CH 2 O-, -NH-C(O)-CH 2 -, -C(O)-CH 2 -NH-, or -NH(CO) NH-, wherein the -CH 2 - can also be optionally substituted by a substituent selected from halogen, alkyl, alkoxy and alkylamino; the a 1 and a 2 are the same or different, and An integer selected from 0 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
  • the nucleotide sequence represented by X is the above-mentioned first nucleotide sequence.
  • said first linker moiety Z is connected to a second linker moiety L1 , said second linker part L1 is connected to a branch point group E.
  • the branch point group E is connected to a targeting combination, and the a is an integer selected from 0 to 10, preferably an integer from 1 to 5; wherein the targeting combination includes a 1:1 ratio The tethering portion L 2 and the targeting portion T.
  • the structural formula of the ANGPTL3 RNA interference agent can be expressed as:
  • the ANGPTL3 RNA interfering agent has the following structure:
  • L3 includes a third linker part
  • L4 is a targeting part
  • L3 and L4 can be the same or different, preferably, L3 and L4 are respectively the following structures:
  • X is a nucleotide sequence, which can be a sense strand or an antisense strand
  • Y is O or S
  • b, c, d and e are respectively selected from integers from 0 to 10, and b and e are not 0 at the same time.
  • b is 0, and e is an integer from 1 to 6.
  • b is 0, d is 2, and e is 1.
  • the present application also provides the use of the aforementioned ANGPTL3 RNA interference agent in the preparation of medicines, wherein the medicines are used to reduce the expression of ANGPTL3 mRNA or protein in mammals, or to prevent and/or treat metabolic disorders or interact with ANGPTL3 overexpression is associated with a disease or disorder, or reduces the risk of a disease or disorder.
  • the metabolic disorder is a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia or high low density lipoprotein (LDL).
  • lipid metabolism such as hyperlipidemia or hypertriglyceridemia or high low density lipoprotein (LDL).
  • the disease is a cardiometabolic disease.
  • the cardiometabolic disease is hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary Arterial disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia or statin-resistant hypercholesterolemia, liver-derived disease, inflammation, cardiovascular and cerebrovascular, myocardial Infarction, or metabolic disease;
  • the cardiometabolic diseases include hyperlipidemia, stroke, atherosclerosis, thrombosis, coronary heart disease, cardiac apoplexy, cerebral apoplexy or aortic valve stenosis.
  • the medicament includes a pharmaceutically acceptable excipient, preferably, the pharmaceutically acceptable excipient is PBS buffer or physiological saline.
  • the medicament is a composition further comprising one or more additional therapeutic agents.
  • the therapeutic agent is selected from statins, PCSK9 RNAi inhibitors, PCSK9 antibody inhibitors, and PCSK9 small molecule inhibitors.
  • the present application provides a method for reducing ANGPTL3 mRNA or protein expression in cells or tissues, comprising: contacting cells or tissues with an effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition, and Maintaining the contact state for a period of time makes the contact time sufficient for ANGPTL3 mRNA degradation, thereby achieving RNAi inhibition of ANGPTL3 gene expression in cells.
  • the cells are hepatocytes.
  • tissue is liver tissue.
  • the present application provides a method for reducing ANGPTL3 mRNA or protein expression in a subject, which includes: administering an effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition to a subject in need thereof .
  • the subject is a human subject suffering from a lipid metabolism disorder (eg, hypertriglyceridemia and/or hyperlipidemia).
  • a lipid metabolism disorder eg, hypertriglyceridemia and/or hyperlipidemia.
  • the present application provides a method for preventing or treating a disease or condition, the method comprising administering to a target subject An effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition is administered.
  • said compound or said pharmaceutical composition is administered to a subject by subcutaneous, intravenous, oral, rectal or intraperitoneal administration routes.
  • the present invention provides a kit for performing the above method.
  • the invention provides a kit for inhibiting ANGPTL3 gene expression in a cell, comprising an RNAi agent as described above and optionally a means for administering the RNAi agent to a subject by administering This is accomplished by contacting the cell with an effective amount of the double-stranded RNAi agent.
  • Figure 1 is the specific sequence of human ANGPTL3 mRNA.
  • Fig. 2 has shown the detection result of embodiment 6.
  • Fig. 3 has shown the detection result of embodiment 7
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs) of any length, which can be Double-stranded and single-stranded molecules.
  • polynucleotides genes or gene fragments (such as probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, nuclear Enzymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, siRNA, miRNA, shRNA, RNAi reagents and primers.
  • Polynucleotides may be modified or substituted at one or more bases, sugars and/or phosphates with any of the various modifications or substitutions known in the art.
  • Polynucleotides can contain modified nucleosides acids, such as methylated nucleotides and nucleotide analogs. In addition, modifications to the nucleotide structure can be made either before or after polymer assembly. Polynucleotides can be modified after polymerization, for example by conjugation with labeling components. Unless otherwise stated or required, any embodiment of the present application that is a polynucleotide includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • target nucleic acid or “target sequence” generally means a nucleic acid molecule to which an antisense compound is intended to hybridize to produce a desired effect (eg, antisense activity).
  • antisense compounds include antisense oligonucleotides that have sufficient complementarity to their target nucleic acid to allow hybridization under physiological conditions.
  • a “target nucleic acid” includes, but is not limited to, DNA or RNA encoding mammalian ANGPTL3, such as human ANGPTL3 mRNA.
  • oligonucleotide generally refers to a oligonucleotide composed of multiple nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) via a phosphate diphosphate Ester linkages (or related structural variants or synthetic analogues thereof) link the constituent polymers.
  • oligonucleotide generally refers to polymers of nucleotides in which nucleotide residues and linkages between them are naturally occurring, it is to be understood that the term also includes within the scope of the term various analogs, including But not limited to: peptide nucleic acid (PNA), phosphoramidate, phosphorothioate, methylphosphonate, 2-O-methyl ribonucleic acid, etc. The exact size of the molecule can depend on the particular application.
  • PNA peptide nucleic acid
  • phosphoramidate phosphoramidate
  • phosphorothioate phosphorothioate
  • methylphosphonate 2-O-methyl ribonucleic acid
  • Oligonucleotides are generally shorter in length, usually about 10-30 nucleotide residues, but the term can refer to molecules of any length, although the terms "polynucleotide” or “nucleic acid” are generally used for larger Oligonucleotides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • modified oligonucleotide generally means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • modified nucleoside generally means a nucleoside comprising at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside.
  • Modified nucleosides comprise modified sugar moieties and/or modified nucleobases.
  • nucleobase generally means a heterocyclic pyrimidine or purine compound, which is a component of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleotides may include modified nucleotides or nucleotide mimetics, abasic sites (Ab or X), or surrogate replacement moieties. As used herein, “nucleobase sequence” generally means the sequence of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
  • unmodified nucleobase or “naturally occurring nucleobase” generally means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G); and The pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U).
  • Modified nucleobase generally means any nucleobase that is not a naturally occurring nucleobase.
  • sugar moiety generally means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • naturally occurring sugar moiety generally means a ribofuranosyl group as found in naturally occurring RNA or a deoxyribofuranosyl group as found in naturally occurring DNA.
  • Modified sugar moiety means a substituted sugar moiety or sugar substitute.
  • internucleoside linkage generally means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • sky naturally occurring internucleoside linkage
  • modified internucleoside linkage is meant any internucleoside linkage other than the naturally occurring internucleoside linkage. couplet.
  • antisense oligonucleotide refers to a single-stranded oligonucleotide molecule having a core complementary to a corresponding fragment of a target nucleic acid (for example, a target genomic sequence, mRNA precursor, or mRNA molecule). base sequence.
  • the antisense oligonucleotides are 12 to 30 nucleobases in length.
  • an antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence that is complementary to the sequence of a target nucleic acid (eg, ANGPTL3 polynucleotide).
  • the term "antisense strand” generally refers to the strand of an RNAi agent (eg, dsRNA) that includes a region that is substantially complementary to a target sequence.
  • region of complementarity generally refers to the region of the antisense strand that is substantially complementary to a sequence as defined herein (eg, a target sequence).
  • the mismatch can be in an internal or terminal region of the molecule.
  • the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3 or 2 nucleotides of the 5' end and/or the 3' end.
  • the term "sense strand” generally refers to a strand of an RNAi agent that includes a region that is substantially complementary to a region that is the term antisense strand as defined herein.
  • the "just" chain is sometimes called the "sense” chain.
  • the antisense strand targets the desired mRNA, while the sense strand targets a different target. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by the use of modifications on the sense strand or by the use of 5' caps.
  • the term "complementary" when used to describe a first nucleotide sequence (such as the sense strand of an RNAi agent or ANGPTL3 mRNA) with respect to a second nucleotide sequence (such as the antisense strand of an RNAi agent) means An oligonucleotide or polynucleotide comprising a first nucleotide sequence hybridizes (forms base pair hydrogen bonds) to an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions and forms a double strand ability to form bulk or double helix structures.
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, provided the above The need was fulfilled with regard to their ability to hybridize. "Complementary” does not necessarily have nucleobase complementarity at every nucleoside. Instead, some mismatches can be tolerated.
  • the term “fully complementary” generally means that all (100%) of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide .
  • the contiguous sequence may comprise all or part of the first or second nucleotide sequence.
  • “partially complementary” generally means that in a hybridized base sequence, at least about 70% of the bases in the contiguous sequence of the first polynucleotide will be identical to those in the contiguous sequence of the second polynucleotide. The same number of bases hybridize.
  • substantially complementary generally means that in hybridized base sequence pairs, at least about 90% of the bases in the contiguous sequence of the first polynucleotide will be identical to the contiguous sequence of the second polynucleotide. Hybridization of the same number of bases.
  • the terms “complementary”, “fully complementary” and “substantially complementary” as used in the present application may be between the sense strand and the antisense strand of the RNAi agent or between the antisense strand of the RNAi agent and the sequence of ANGPTL3 mRNA Used for base matching. sequence Identity or complementarity is independent of modification. For purposes of determining identity or complementarity, for example, A and Af are complementary to U (or T) and identical to A.
  • the term "ligand” generally refers to any compound or molecule capable of covalently or otherwise chemically binding to a biologically active substance such as an oligonucleotide.
  • the ligand is capable of interacting directly or indirectly with another compound, such as a receptor, which may be present on the cell surface, or alternatively may be intracellular and/or Or intercellular receptors, the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding.
  • the terms “induce”, “inhibit”, “enhance”, “elevate”, “increase”, “decrease”, “decrease”, etc. generally denote a quantitative difference between two states.
  • an amount effective to inhibit the activity or expression of ANGPTL3 means that the level of activity or expression of ANGPTL3 in a treated sample will be lower than the level of activity or expression of ANGPTL3 in an untreated sample.
  • the terms apply, for example, to expression levels and activity levels.
  • the terms “reduce” and “decrease” are used interchangeably and generally mean any change from the original.
  • “Reduce” and “decrease” are relative terms that require a comparison between before and after measurement.
  • “Reduce” and “decrease” include complete depletion.
  • the term “reduce” refers to a reduction in the expression level/amount of a gene or biomarker in a test sample, wherein the reduction refers to at least about 0.9 of the expression level/amount of the corresponding gene or biomarker times to 0.01 times.
  • expression generally means the process by which a gene ultimately produces a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modifications (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.
  • the term "pharmaceutically acceptable” generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations will generally contain salts, excipients, buffers, preservatives, compatible carriers and, optionally, other therapeutic agents.
  • the salt should be a pharmaceutically acceptable salt, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and they cannot be excluded from the scope of the present application.
  • Such pharmacologically and pharmaceutically acceptable salts include those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, Malonic acid, succinic acid, etc.
  • Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.
  • prevention and/or treatment includes not only preventing and/or treating a disease, but also generally preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing down one or more symptoms associated with a disease onset, reduction and/or alleviation of one or more symptoms associated with the disease, reduction in the severity of the disease and/or any symptoms associated therewith, and/or or the duration and/or prevention of a further increase in the severity of the disease and/or any symptoms associated therewith, the prevention, reduction or reversal of any physiological impairment caused by the disease, and any pharmacological effects generally beneficial to the patient being treated.
  • RNAi agent or pharmaceutical composition of the present application need not achieve a complete cure or eradicate any symptom or manifestation of a disease to be a viable therapeutic agent.
  • drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic agent.
  • a treatment administered prophylactically need not be fully effective in preventing the onset of the disorder to constitute a viable prophylactic. Simply reducing the effects of disease in a subject (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing disease occurrence or The possibility of deterioration is enough.
  • disease or “condition” are used interchangeably and generally refer to any deviation from the normal state of a subject, such as any change in the state of the body or certain organs that prevents or disrupts the performance of function , and/or cause symptoms such as malaise, dysfunction, suffering or even death in those who are sick or come into contact with it.
  • a disease or condition may also be called a disorder, ailing, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.
  • the term "administration" generally refers to introducing the pharmaceutical formulation of the present application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug may be used. Such administration may include, without limitation, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous or oral.
  • the daily dose may be divided into one, two or more doses of suitable form to be administered at one, two or more times during a certain period of time.
  • the term "contacting" generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time. Exposure can occur in vivo, ex vivo or in vitro. In some embodiments, it may refer to directly contacting a cell or tissue with an RNAi agent or composition of the present application. In other embodiments, the term refers to indirect contact of an RNAi agent or composition of the present application with a cell or tissue.
  • the methods of the present application include wherein the subject is exposed to the RNAi agent or composition of the present application, and then the RNAi agent or composition passes through diffusion or any other active or passive transport process known in the art (the process by which the compound is transported in vivo). Circulation) A method of contacting cells or tissues.
  • the term “effective amount” or “effective dose” generally refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is typically one that, when used alone or in combination with another therapeutic agent, promotes regression of disease (this is achieved by a reduction in the severity of symptoms of the disease, frequency of asymptomatic periods of the disease) any amount of drug that is evidenced by an increase in the degree and duration of the disease, or by the prevention of impairment or disability due to the presence of a disease.
  • a “prophylactically effective amount” or “prophylactically effective dose” of a drug generally refers to the amount of the drug that, alone or in combination with another therapeutic agent, inhibits the development or recurrence of the disease when administered to a subject at risk of disease development or disease recurrence .
  • An “effective amount” refers to the amount of an RNAi agent that produces a desired pharmacological, therapeutic or prophylactic result.
  • the term "subject” generally refers to a human or non-human animal (including mammals), such as a human, a non-human primate (ape, or , gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects include animal disease models.
  • RNAi agent used to inhibit the expression of the ANGPTL3 gene described in the present application is delivered to cells expressing the ANGPTL3 gene in vitro and/or through the biological process of RNA interference (RNAi). Or inhibit or knock down the expression of ANGPTL3 in vivo.
  • RNAi agents include, but are not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA).
  • the application provides a kind of ANGPTL3 RNAi agent, it can comprise the first nucleotide sequence targeting the nucleic acid molecule of coding ANGPTL3, wherein said first nucleotide sequence comprises SEQ ID NO:1 to SEQ ID NO At least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 or at least 23 contiguous nucleotides of :184.
  • the nucleotide sequence can be in a modified state (and ligand added after modification) and an original state without modification.
  • the first nucleotide sequence consists of 12 to 30 nucleotides. In certain embodiments, said first nucleotide sequence may be 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
  • the first nucleotide sequence comprises a nucleotide sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 184 or differs therefrom by no more than 3 nucleosides acid sequence.
  • the derived RNAi agent can still have reduced
  • the inhibitory activity of ANGPTL3 expression is not less than 20% inhibitory effect compared with the nucleotide sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 184 as the source of the derivative.
  • the antisense strand nucleotide sequence is selected from: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:23, SEQ ID NO:39, SEQ ID NO:49, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO : 117, SEQ ID NO: 135, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 151, SEQ ID NO: 165 and SEQ ID NO: 167.
  • the sense strand nucleotide sequence is selected from: SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:112, SEQ ID NO: 118, one of SEQ ID NO:136, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:166, and SEQ ID NO:168.
  • the first nucleotide sequence is a single-stranded oligonucleotide or a double-stranded oligonucleotide.
  • the first nucleotide sequence can be an antisense oligonucleotide.
  • the oligonucleotide is siRNA.
  • the first nucleotide sequence may comprise only the antisense strand.
  • the first nucleotide sequence may comprise a sense strand and an antisense strand.
  • the sense and antisense strands of the ANGPTL3 RNAi agent are independently 17 to 30 nucleotides in length. In certain embodiments, the sense and antisense strands are independently 17 to 26 nucleotides in length. In certain embodiments, the sense and antisense strands are 19-26 nucleotides in length. The sense and antisense strands can be the same length or they can be different lengths. In certain embodiments, the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length. In certain embodiments, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.
  • the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long. In certain embodiments, the sense strand is 23 nucleotides long and the antisense strand is 25 nucleotides long.
  • the antisense strand sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to a nucleotide sequence present in ANGPTL3 mRNA (sometimes referred to as the target sequence).
  • the sense strand sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary to the sequence in the antisense strand, and thus the sense strand sequence is perfectly identical or at least 90% identical to the nucleotide sequence (target sequence) present in the ANGPTL3 mRNA %same.
  • the sense and antisense strands of the ANGPTL3 RNAi agent anneal to form a duplex.
  • the sense and antisense strands of the ANGPTL3 RNAi agent are partially, substantially or fully complementary to each other.
  • the sense strand core sequence is at least 90% complementary or 100% complementary to the antisense core sequence.
  • the sense strand core sequence contains at least 90% or 100% of the corresponding 17, 18, 19, 20, or 21 nucleotide sequence of the antisense strand core sequence
  • a complementary sequence of at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides i.e., the sense strand and antisense core sequence of the ANGPTL3 RNAi agent have at least 90% base pairing or 100% base pairing paired regions of at least 17, at least 18, at least 19, at least 20 or at least 21 nucleotides).
  • the sense and/or antisense strands may optionally and independently contain additional 1, 2, 3, 4, 5 or 6 nucleotides (extension) (overhang). In certain embodiments, the sense and/or antisense strands may optionally and independently contain additional 1 , 2, 3 or 4 nucleotides (extension). In certain embodiments, the sense and/or antisense strands may optionally and independently contain an additional 2 or 3 nucleosides at the 3' end, the 5' end, or both the 3' and 5' ends of the core sequence acid (extension). In certain embodiments, the antisense strand independently contains an additional 2 or 3 nucleotides (extension) at the 3' end of the core sequence.
  • the one or more antisense strand extension nucleotides include uracil or thymidine nucleotides or nucleotides that are complementary to the corresponding ANGPTL3 mRNA sequence.
  • the sense strand comprises any one of the nucleotide sequences provided in Table 1 or a sequence different from it by 1-4 nucleotides.
  • the antisense strand includes a nucleotide sequence that is at least partially complementary to the sense strand.
  • the antisense strand includes a region of complementarity that differs from any of the antisense strand sequences listed in Tables 1-3 by at least 15 consecutive nucleosides differ by no more than 3 nucleotides in the acid sequence.
  • sequences of the antisense strand and the sense strand are as shown in Table 1.
  • the ANGPTL3 RNAi agents described herein can be formed by annealing the antisense strand to the sense strand.
  • the sense strand containing the sequences listed in Table 1 can be hybridized to any antisense strand as long as the two sequences are at least 90% complementary over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence sex.
  • the ANGPTL3 RNAi agents of the present application include unmodified nucleic acids as well as nucleic acids modified for enhanced efficacy, and multimers of nucleoside substitutes.
  • Unmodified nucleic acid means that the sugar, base and phosphate structure components are the same or substantially the same as the natural components, preferably the natural components in the human body.
  • the prior art considers rare or unusual but naturally occurring RNAs to be modified RNAs, see Limbach et al. (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual modified RNAs are often referred to as modified RNAs (apparently because they are the result of post-transcriptional modifications), and are included in this application as unmodified RNAs.
  • RNA as used in this application refers to one or more components of the nucleic acid in which the components of the sugar, base and phosphate structures are different from the natural components, preferably different from the natural components produced by the human body .
  • Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate construct that places the bases in the correct spatial relationship for hybridization and hybridization seen with the ribose phosphate backbone basically similar, e.g. non- An analogue of a charged ribose phosphate backbone.
  • the ANGPTL3 RNAi agent contains one or more modified nucleotides.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the nucleotides in the ANGPTL3 RNAi agent are modified.
  • the sense strand and/or the antisense strand independently comprise one or more modified nucleotides.
  • RNAi agents may comprise non-naturally occurring bases, or non-naturally occurring sugars, such as non-saccharide ring carrier molecules, typical properties of non-naturally occurring sugars for RNAi agents.
  • RNAi agents may include linkages between nucleotides (eg, chiral phosphorothioate linkages) for increased nuclease resistance.
  • RNAi agents may additionally, or alternatively, contain ribose analogs to increase nuclease resistance.
  • Modified nucleotides include, but are not limited to, deoxynucleotides, nucleotide mimetics, non-basic nucleotides (denoted in this application as X, Ab), 2'-modified nucleotides, 3' To 3 linked (reverse) nucleotides (denoted in this application as invdN, invN, invn, invX, invAb, nucleotides containing unnatural bases, bridging nucleotides, peptide nucleic acid (PNA), 2 ', 3'-broken nucleotide mimetic (unlocked nucleobase analog, denoted NUNA or NUNA in this application), locked nucleotide (denoted NLNA or NLNA in this application), 3'-O - Methoxy (2' internucleoside linked) nucleotides (denoted 3'-OMen in this application), 2'-F-arabinonucleotides (denoted Nf
  • 2'-modified nucleotides include, but are not limited to, 2'-O-methyl modified nucleosides acid (represented in this application as a lowercase letter n in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotide (represented as Nf in this application, also represented as 2'- Fluorine nucleotides), 2'-deoxynucleotides (denoted as dN in this application), 2'-methoxyethyl (2'-O-2-methoxyethyl) modified nucleotides ( Denoted in this application as NM or 2'-MOE), 2'-O-allyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-amino nucleotides and 2'- Alkyl nucleotides.
  • 2'-O-methyl modified nucleosides acid represented in this application as a lowercase letter n in the nucleotide sequence
  • more than one modification can be incorporated in a single ANGPTL3 RNAi agent or even in a single nucleotide thereof.
  • the sense and antisense strands of the ANGPTL3 RNAi agent can be synthesized and/or modified by methods known in the art.
  • a modification at one nucleotide is independent of a modification at another nucleotide.
  • the modified nucleotides are 2'-methoxy-modified nucleotides or 2'-fluoro-modified nucleotides.
  • nucleotides at positions 7, 9, 10, and 11 of the sense strand are independently 2'-fluoro-modified Nucleotides or 2'-deoxy-modified nucleotides;
  • nucleotides at positions 2, 4, 5, 6, 8, 9, 10, 12, 14, and 16 of the antisense strand in the direction from the 5' end to the 3' end ground each independently being a 2'-fluoro-modified nucleotide
  • the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are each independently 2'-fluoro-modified in the direction from the 5' end to the 3' end Nucleotide;
  • the nucleotides at positions 2, 5, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid;
  • the nucleotides at positions 2, 5, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide
  • the nucleotides at positions 2, 4, 6, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid;
  • the nucleotides at positions 2, 6, 12, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid;
  • the nucleotides at positions 2, 6, 10, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid;
  • the nucleotides at positions 2, 8, 9, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleoside according to the direction from the 5' end to the 3' end acid;
  • the nucleotides at positions 2, 14 and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide in the direction from the 5' end to the 3' end.
  • the sense strand and/or the antisense strand independently comprise one or more phosphorothioate linkages; preferably, the sense strand is between the terminal nucleotides at the 3' and 5' ends Alternatively, the antisense strand contains two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends.
  • the ANGPTL3 RNAi agent further comprises a ligand.
  • the ligand alters the distribution, targeting, or lifetime of the RNAi agent to which it binds. In certain embodiments, the ligand enhances the affinity of the selected target compared to a species lacking the ligand.
  • the selected target is, for example, a molecule, a cell or a class of cells and a cavity such as a cell or an organ cavity, a tissue, an organ or a region of the body.
  • the ligand can promote the properties of transport, hybridization and specificity, and can promote the nuclease resistance of the obtained natural or modified oligoribonucleotides, or promote Nuclease resistance of monomeric and/or polymeric molecules of combinations of natural or modified ribonucleotides is described.
  • ligands may also include targeting groups, eg, cell or tissue targeting agents such as lectins, glycoproteins, lipids or proteins, eg antibodies that bind to a specific cell such as hepatocytes or jejunum cells.
  • targeting groups eg, cell or tissue targeting agents such as lectins, glycoproteins, lipids or proteins, eg antibodies that bind to a specific cell such as hepatocytes or jejunum cells.
  • targeting group Adrenaline, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, multivalent Mannose, polyvalent trehalose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, cholic acid, Folic acid, vitamin B12, or biotin.
  • Adrenaline melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, multivalent Mannose, polyvalent trehalose, glycosylated polyamino acid, polyvalent galactos
  • the ligand includes a targeting group.
  • the targeting group can be monovalent, divalent, trivalent, tetravalent, or have a higher valence.
  • Representative targeting groups include compounds with affinity for cell surface molecules, cellular receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics with affinity for somatic cell surface molecules.
  • the ligand comprises N-acetyl-galactosamine.
  • the targeting group is capable of targeting hepatocytes or the liver.
  • the targeting group comprises an asialoglycoprotein receptor ligand.
  • the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives or galactose clusters.
  • galactose derivatives includes both galactose, and galactose derivatives having an affinity (equal to or greater than that of galactose) for the asialoglycoprotein receptor.
  • Galactose derivatives include: galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-Iso-butyrylgalactosyl-amine.
  • Galactose derivatives have been used in vivo to target molecules to hepatocytes by binding to the asialoglycoprotein receptor (ASGPr) on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr promotes cell-specific targeting to hepatocytes and endocytosis of the molecule into hepatocytes.
  • the galactose cluster can be attached to the 3' or 5' end of the RNAi polynucleotide using methods known in the art.
  • the ligand is conjugated to said sense and/or antisense strand.
  • the ligand may be conjugated to the 5' and/or 3' ends of the sense and/or antisense strands.
  • a delivery vehicle can be used to deliver an ANGPTL3 RNAi agent to a cell or tissue.
  • the delivery vehicle is a compound that improves delivery of the ANGPTL3 RNAi agent to cells or tissues.
  • Delivery vehicles may comprise or consist of polymers, such as amphiphilic polymers, reversibly modified polymers or peptides, or reversibly modified membrane active polyamines.
  • ANGPTL3 RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art.
  • ANGPTL3 RNAi agents can also be chemically conjugated to targeting groups, lipids (including cholesterol and cholesteryl derivatives), nanoparticles, polymers, or other delivery systems available in the art.
  • a delivery vehicle may be used. Delivery vehicles can be, but are not limited to: polymers (such as amphiphilic polymers, membrane active polymers), ligands, and the like.
  • the ANGPTL3 RNAi agent is linked to a Targeting ligands for protein ligands.
  • an ANGPTL3 RNAi agent can be linked to a targeting ligand comprising or consisting of a galactose cluster.
  • siRNA conjugate which has the following general structure
  • X is a nucleotide sequence, which can be the sense strand or antisense strand of siRNA, and Z is the first linker part of the nucleotide sequence, wherein Z and X can be connected directly or through a chemical group, and the general The formula is shown in (Z-1), wherein one end of the O group is connected to the nucleotide sequence:
  • R 1 is O, S, NR 3 or CR 3 R 4 , wherein R 3 and R 4 are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted Substituted heteroaryl, or substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl;
  • R 2 is -O-, -S-, -NH-, -CH 2 -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH 2 NH-, -CH 2 O-, -NH-C(O)-CH 2 -, -C(O)-CH 2 -NH-, or -NH(CO) NH-, wherein the -CH 2 - can also be optionally substituted by a substituent selected from halogen, alkyl, alkoxy and alkylamino; the a 1 and a 2 are the same or different, and An integer selected from 0 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 5.
  • the nucleotide sequence represented by X is the above-mentioned first nucleotide sequence.
  • said first linker moiety Z is connected to a second linker moiety L1 , said second linker part L1 is connected to a branch point group E.
  • the branch point group E is connected to a targeting combination, and the a is an integer selected from 0 to 10, preferably an integer from 1 to 5; wherein the targeting combination includes a 1:1 ratio The tethering portion L 2 and the targeting portion T.
  • the structural formula of the ANGPTL3 RNA interfering agent can be expressed as:
  • L 3 includes a third linker moiety
  • L 4 is a targeting moiety
  • T in general formula (I) and L 3 and L 4 in general formula (II) can be the same or different, preferably, L 3 and L 4 are the following structures respectively:
  • X is a nucleotide sequence, which can be a sense strand or an antisense strand
  • Y is O or S
  • the second linker part L1 has the following structure:
  • f, g, h and i are integers from 1 to 20, preferably integers from 1 to 10, respectively.
  • said tethering portion L2 has the following structural formula:
  • j, k, l, m, n and o are integers from 1 to 20, preferably integers from 1 to 10, respectively.
  • Another aspect of the present invention also provides an ANGPTL3 RNA interference agent, comprising: a sense strand and an antisense strand, and the above-mentioned siRNA conjugate.
  • Another object of the embodiments of the present invention is to provide a composition comprising the above-mentioned RNA interference agent.
  • siRNA conjugates provided in the embodiments of the present invention can improve the efficiency of targeting moieties binding to cells or cell receptors, increase The effect of adding RNA interference.
  • R 2 is -NH-, at this time compound Z is:
  • R 2 is -C(O)-, at this time compound Z is
  • L 1 is the following structure:
  • the compound E is selected from one of the following five structures:
  • compound L in general formula (I) 2 is
  • j, k, l, m, n and o are integers from 1 to 20 respectively;
  • L2 has the following structural formula:
  • the targeting ligand described in general formula (I) (excluding part X) has the following structure:
  • T in the targeting ligand is selected from N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N - one of n-butyryl galactosamine and N-isobutyryl-galactosamine, preferably N-acetyl-galactosamine, the structural formula is as follows:
  • the targeting ligand in the general formula (I) is connected to the end of the siRNA through a phosphate group, a phosphorothioate group or a phosphonic acid group.
  • the targeting ligand has the following structure:
  • H—O— L in the targeting ligand is:
  • R 2 are each independently -O-, -S-, -NH-, -CH 2 -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O )-, -C(O)NH-, -CH 2 NH-, -CH 2 O-, -NH-C(O)-CH 2 -, -C(O)-CH 2 -NH-, -NH( CO)NH-, the -CH 2 - can also be optionally substituted by halogen or alkyl, wherein the alkyl is optionally further substituted by one selected from hydroxyl, amino, halogen, alkoxy and alkylamino base substitution);
  • the p, q, r, s, t and u are each independently selected from an integer of 0 to 20, preferably an integer of 1 to 10.
  • H—O—L 3 has the following structure:
  • H—O— L in the targeting ligand is:
  • H—O—L 4 has the following structure:
  • general formula (II) has the following structure:
  • Y is O or S.
  • the siRNA conjugate has the following structure:
  • Y is O or S.
  • the ligand comprises N-acetyl-galactosamine, and the ligand is linked to the 3' end or 5' end of the sense strand of the siRNA.
  • the ligand comprises one or more structures selected from:
  • the application provides a pharmaceutical composition, which comprises the aforementioned ANGPTL3 RNAi agent and optionally a pharmaceutically acceptable excipient.
  • the ANGPTL3 RNAi agent is useful for inhibiting the expression of ANGPTL3, eg, in a cell, population of cells, or tissue in a subject.
  • the ANGPTL3 RNAi agent is used to formulate a composition, i.e., a pharmaceutical composition or medicament, for administration to a subject.
  • a pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one such ANGPTL3 RNAi agent and one or more pharmaceutically acceptable excipients.
  • a pharmaceutically acceptable excipient is a substance other than the active pharmaceutical ingredient (API, therapeutic product, such as an ANGPTL3 RNAi agent) that has undergone appropriate safety evaluation and is intentionally included in the drug delivery system.
  • An excipient does not exert or is not intended to exert a therapeutic effect at the intended dosage.
  • Excipients can be used to a) aid in the handling of the drug delivery system during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API, c) aid in product identification and/or d) Any other attribute that enhances the overall safety, efficacy of the delivered API during storage or use.
  • Pharmaceutically acceptable excipients may or may not be inert substances.
  • Excipients include: absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, binders, buffers, vehicles, coatings, pigments, delivery enhancers, delivery polymers, dextran Sugar, dextrose, diluent, disintegrant, emulsifier, bulking agent, filler, flavoring agent, glidant, humectant, lubricant, oil, polymer, preservative, brine, salt, solvent, Sugars, suspending agents, sustained release bases, sweetening agents, thickening agents, tonicity agents, vehicles, hydrophobic agents and wetting agents.
  • the pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions.
  • additional components include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (eg, antihistamines, diphenhydramine, etc.).
  • antipruritics e.g., antipruritics, astringents, local anesthetics, or anti-inflammatory agents (eg, antihistamines, diphenhydramine, etc.).
  • a cell, tissue or isolated organ expressing or comprising an RNAi agent as defined herein may be used as a "pharmaceutical composition”.
  • the ANGPTL3 RNAi agent is combined with one or more additional therapeutic agents or treatments including, but not limited to: statins, PCSK9 RNAi inhibitors, PCSK9 antibody inhibition agent and Small molecule inhibitors of PCSK9.
  • additional therapeutic agents or treatments including, but not limited to: statins, PCSK9 RNAi inhibitors, PCSK9 antibody inhibition agent and Small molecule inhibitors of PCSK9.
  • the RNAi agents and pharmaceutical compositions comprising the ANGPTL3 RNAi agents disclosed herein can be packaged or included in a kit, container, package or dispenser.
  • the ANGPTL3 RNAi agent and the pharmaceutical composition comprising the ANGPTL3 RNAi agent can be packaged in a prefilled syringe or vial.
  • the pharmaceutical composition of the present application can be used to inhibit the expression of ANGPTL3 gene in cells, tissues or organisms.
  • the pharmaceutical composition is used to treat a subject suffering from a disease, disorder or condition that would benefit from reduction or inhibition of ANGPTL3 expression.
  • the pharmaceutical composition is used to treat a subject at risk of developing a disease, disorder or condition that would benefit from reduction or inhibition of ANGPTL3 expression.
  • ANGPTL3 expression diseases, disorders or conditions that would benefit from reduction or inhibition of ANGPTL3 expression include, but are not limited to: hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II diabetes , cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia or statin-resistant hypercholesterolemia, hepatic disease, inflammation , cardiovascular and cerebrovascular diseases, myocardial infarction, or metabolic diseases.
  • the subject is a mammal, including but not limited to a human.
  • Cells, tissues and non-human organisms comprising at least one ANGPTL3 RNAi agent described herein are contemplated.
  • the cell, tissue or non-human organism is prepared by delivering the ANGPTL3 RNAi agent to the cell, tissue or non-human organism by any means available in the art.
  • the cells are mammalian cells, including but not limited to human cells.
  • Cells, tissues, or non-human organisms can be used in research or as research tools (such as drug testing or diagnostics).
  • the application provides the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition in the preparation of medicines, wherein the medicines are used to reduce ANGPTL3 mRNA or protein expression in mammals, or to prevent and/or treat metabolic disorders. Dysregulation or a disease or disorder associated with overexpression of ANGPTL3, or reduced risk of a disease or disorder.
  • the metabolic disorder is a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia or high low density lipoprotein (LDL).
  • lipid metabolism such as hyperlipidemia or hypertriglyceridemia or high low density lipoprotein (LDL).
  • the disease is a cardiometabolic disease.
  • the cardiometabolic disease is hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary Arterial disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia or statin-resistant hypercholesterolemia, liver-derived disease, inflammation, cardiovascular and cerebrovascular, myocardial Infarction, or metabolic disease;
  • the cardiometabolic diseases include hyperlipidemia, stroke, atherosclerosis, thrombosis, coronary heart disease, cardiac apoplexy, cerebral apoplexy or aortic valve stenosis.
  • the present application provides a method for preventing and/or treating a disease or a disease, which involves contacting cells or tissues with an effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition, and maintaining the contact state for a period of time so that the The exposure time is sufficient for the degradation of ANGPTL3 mRNA, so that RNAi inhibits the expression of ANGPTL3 gene in cells.
  • the ANGPTL3 RNAi agents described herein are useful for treating diseases or conditions that would benefit from reducing ANGPTL3 expression.
  • diseases or conditions include, but are not limited to: hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease , nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous and heterozygous familial hypercholesterolemia or statin drug-resistant hypercholesterolemia, liver-derived disease, inflammation, cardiovascular and cerebrovascular, myocardial infarction, or metabolic disease.
  • the method comprises administering a composition, such as a pharmaceutical composition comprising an ANGPTL3 RNAi agent described herein, to the target mammal.
  • a therapeutically effective amount of one or more of the ANGPTL3 RNAi agents is administered to the subject, thereby inhibiting expression of ANGPTL3 in the subject (e.g., an amount effective to inhibit expression of ANGPTL3 in the subject ).
  • the method further comprises the step of administering a second therapeutic agent or treatment.
  • the second therapeutic agent is another ANGPTL3 RNAi agent (e.g., an ANGPTL3 RNAi agent that targets a different sequence within an ANGPTL3 target).
  • the second therapeutic agent may be selected from the group consisting of statins, PCSK9 RNAi inhibitors, PCSK9 antibody inhibitors, and PCSK9 small molecule inhibitors.
  • the route of administration is the route by which the RNAi agent comes into contact with the body.
  • routes of administration is the route by which the RNAi agent comes into contact with the body.
  • methods of administering drugs and nucleic acids for treating subjects are well known in the art and can be applied to the administration of the compositions described herein.
  • the compounds described herein may be administered via any suitable route in formulations suitably tailored for a particular route.
  • the compounds described herein may be administered by injection, eg, intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally.
  • an ANGPTL3 RNAi agent or composition described herein can be delivered to a cell, cell population, tissue, or subject using a first nucleotide sequence delivery technique known in the art.
  • a first nucleotide sequence delivery technique known in the art.
  • any suitable method recognized in the art for delivering nucleic acid molecules can be applied to the ANGPTL3 RNAi agents described herein.
  • delivery can be by local administration (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (such as intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical (including buccal and sublingual) administration.
  • local administration e.g., direct injection, implantation, or topical administering
  • systemic administration or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (such as intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical (including buccal and sublingual) administration.
  • intracranial such as intraventricular, intraparenchymal, and intrathecal
  • nasal, rectal or topical (including
  • ANGPTL3 RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. RNAi agents can also be chemically conjugated to targeting moieties, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art .
  • the ANGPTL3 RNAi agent can be conjugated to a delivery polymer.
  • the delivery polymer is a reversibly masked/modified amphiphilic membrane-active polyamine.
  • the present application provides a method for reducing ANGPTL3 mRNA or protein expression in cells or tissues, which includes: contacting cells or tissues with an effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition.
  • the cells are hepatocytes. In certain embodiments, wherein the tissue is liver tissue.
  • the present application provides a method for reducing ANGPTL3 mRNA or protein expression in a subject, which includes: administering an effective amount of the aforementioned ANGPTL3 RNAi agent or the aforementioned pharmaceutical composition to the target subject.
  • the term “silencing”, “reducing”, “inhibiting”, “down-regulating” or “knocking down gene expression” when referring to the ANGPTL3 gene means that when a cell, cell population or tissue is treated with said ANGPTL3 RNAi
  • the expression of the gene (such as by the level of RNA transcribed from the gene or from the mRNA in the cell, cell population, or tissue in which the ANGPTL3 gene is transcribed) is compared to the same cell, cell population, or tissue before administration of the ANGPTL3 RNAi agent.
  • the gene expression level and/or mRNA level of ANGPTL3 in a subject administered the ANGPTL3 RNAi agent is relative to a subject prior to administration of the ANGPTL3 RNAi agent or a subject not receiving the ANGPTL3 RNAi agent A reduction of at least about 5% or more, such as 5% to 98%.
  • Gene expression levels and/or mRNA levels in a subject can be reduced in cells, cell populations and/or tissues of the subject.
  • the protein level of ANGPTL3 in the subject administered the ANGPTL3 RNAi agent is reduced by more than 5% relative to a subject before administration of the ANGPTL3 RNAi agent or a subject not receiving the ANGPTL3 RNAi agent, e.g. 5% to 98%.
  • Protein levels in a subject can be reduced in cells, cell populations, tissues, blood and/or other fluids of the subject. Reduction in gene expression, mRNA or protein levels can be assessed by any method known in the art. Reduction or reduction of ANGPTL3 mRNA levels and/or protein levels is collectively referred to herein as reduction or reduction of ANGPTL3, or inhibition or reduction of ANGPTL3 expression.
  • introducing into a cell when referring to an ANGPTL3 RNAi agent means that the ANGPTL3 RNAi agent is functionally delivered into the cell.
  • Functional delivery means that the RNAi agent is delivered to the cell and has the desired biological activity (eg, sequence-specific inhibition of gene expression).
  • Embodiment 1 compound GENO-Gal-6 is synthesized
  • Int-6-1 (2.1g, 2.35mmol) was dissolved in DCM (30mL), Tetrazole (33mg, 0.47mmol), NMI (77.2mg, 0.94mmol) and 2g molecular sieves were added, and the reaction solution was replaced with argon three times. Stir at room temperature for 20 min, then dissolve phosphorus reagent (920.81 mg, 3.06 mmol) in a small amount of dichloromethane and add to the reaction solution, and stir at room temperature for 1 hour.
  • Gal-5-1 (200 mg, 0.26 mmol) was dissolved in 5 mL of ethyl acetate, 50 mg of palladium carbon and triethylamine (0.11 mL, 0.78 mmol) were added, and the reaction solution was stirred under hydrogen (15 psi)) at room temperature for 16 After 1 hour, the reaction solution was concentrated by filtration to obtain Gal-5-2 (150 mg).
  • Gal-5-4 (220mg, 0.086mmol) was suspended in 4ml CH 3 CN and 2mL DMF, DIEA (0.035mL, 0.216mmol) and HBTU (49.07mg, 0.129mmol) were added dropwise, stirred at room temperature for 5 minutes, and added to the reaction
  • Add aminomethyl resin 99.51 mg, 100-200 mesh, amino load 250 umol/g, react on a shaking table at 25°C, rotate at 220 rpm, react for 16 hours and filter, and filter the cake with DCM for 3 times , 30ml each time, acetonitrile rinse 3 times, 30ml each time, 30ml n-hexane rinse 3 times, vacuum oil pump drying 2h, then add mixed reagents (CapB1, 4-dimethylaminopyridine, N-methylimidazole and acetonitrile, 11.2mL/12.4mg/0.50mL/4.32mL) for capping reaction.
  • CapB1 4-di
  • the nucleotide monomers are linked to each other by 5'-3'-phosphodiester linkages, including phosphorothioate linkages and phosphodiester linkages.
  • the oligoribonucleotides are cleaved from the solid support, and soaked in a 3:1 solution of 28% ammonia water and ethanol at 50° C. for 15 hours. Then centrifuged, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, purified by C18 reverse chromatography, the mobile phase was 0.1M TEAA and acetonitrile, and 3% trifluoroacetic acid solution was used to remove DMTr.
  • the target first nucleotide sequence was collected and lyophilized, and identified as the target product by LC-MS, and then quantified by UV (260nm).
  • the obtained single-stranded first nucleotide sequence is annealed according to the equimolar ratio and complementary pairing, and finally the obtained double-stranded siRNA is dissolved in 1 ⁇ PBS or sterile water, and adjusted to the concentration required for the experiment for later use.
  • the lowercase letter m indicates that the nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide
  • the lowercase letter f indicates that the nucleotide adjacent to the left side of the letter f is a fluorine-modified nucleus nucleotide
  • the nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group.
  • Gal-6 is the abbreviation of the target product Geno-Gal-6 prepared in Example 1;
  • the human ANGPTL3 mRNA sequence (Accession No. NM_014495.4) or fragments thereof were subcloned from a commercially available mammalian expression vector (Origene) into a commercially available reporter-based screening plasmid psiCHECK2 (Promega) to generate Renilla firefly Luciferase/ANGPTL3 fusion mRNA.
  • Huh7 cells (a human hepatocellular carcinoma line) were plated at approximately 10,000 cells/well in a 96-well format.
  • Each ANGPTL3 RNAi agent (modified and unmodified, from Table 1 and Table 2, respectively) was mixed at two concentrations (1 nM and 0.1 nM or 1 nM and 0.01 nM)) with 25 ng psiCHECK2-ANGPTL3 plasmid DNA per well and 0.2 ⁇ L LipoFectamine 2000 together for co-transfection. Renilla luciferase levels normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid were measured by using a dual luciferase reporter assay (purchased from Promega, Madison, WI) To determine gene knockdown (Table 4).
  • Example 4 In vitro screening of non-conjugated ANGPTL3 RNAi agents by inhibiting ANGPTL3 mRNA expression in human huh7 and Hep3B cells
  • RNA isolation was carried out using RNeasy 96 kit (purchased from Qiagen, 74182). Using Novizan cDNA reverse transcription kit and qPCR kit, cDNA synthesis and real-time PCR detection were performed (results are shown in Table 5).
  • Example 3 The same transfection conditions as in Example 3 were used to generate dose responses of 6 concentrations in Huh7 cells, wherein the concentration range of ANGPTL3 RNAi agent (modified) was 8pM-8nM (see Table 6 for test results).
  • mice Female C57 BL/6 mice were used. After adapting to the environment and facilities, the mice were divided into equal groups according to body weight, with 3 mice in each group. On the day of administration (the 0th day), the mice were subcutaneously injected with ANGPTL3 RNAi solution, the mice in the control group were given PBS solution, and all mice were subcutaneously injected once at the back of the neck. The administration dose was 1 mg/Kg, and the administration volume 5ml/kg.
  • the knockdown of mANGPTL3 expression was evaluated by detecting the protein level of mANGPTL3 in mouse serum by ELISA method (mANGPTL3, R&D).
  • mANGPTL3, R&D ELISA method
  • each animal's mANGPTL3 level at a given time point was divided by the pretreatment level in this animal (Day-3) to determine the ratio of "normalized to pretreatment” expression;
  • the "normalized to pretreatment” ratio for each animal was divided by the average “normalized to pretreatment” ratio for all mice in the control group to normalize expression at a specific time point to the control group. This normalizes the expression at each time point to the expression of the control group, and the specific detection results after normalization are shown in FIG. 2 .
  • ANGPTL3 RNAi agents were administered to C57 BL/6 mice as described above. Each mouse received a single subcutaneous (SC) dose of 1 mg/Kg ANGPTL3 RNAi agent solution, and mANGPTL3 protein levels in serum were monitored for up to 28 days. Knockdown levels and duration of response are shown in Table 7. As can be seen from Table 7, when using the 5th day, 9 ANGPTL3 RNAi agents 1 mg/Kg administered showed a knockdown greater than 40%, and 7 ANGPTL3 RNAi agents 1 mg/Kg administered showed a knockdown greater than 50%.
  • mice Female hANGPTL3 C57 BL/6 transgenic mice were used. After adapting to the environment and facilities, the serum of the mice was collected three days before the administration, and the mice were divided into 18 groups according to the level of hANGPTL3 in the serum, with 3 mice in each group. On the day of administration (day 0), hANGPTL3 C57 BL/6 transgenic mice were subcutaneously injected with ANGPTL3 RNAi solution, and mice in the control group were given PBS solution. All mice were subcutaneously injected once at the back of the neck, with a dose of 1 mg/Kg and a volume of 5 ml/kg.
  • Knockdown of hANGPTL3 expression was evaluated by detecting hANGPTL3 protein level in mouse serum by ELISA method (hANGPTL3, Abcam).
  • hANGPTL3 level at a given time point was divided by the pretreatment level in this animal (Day0) to determine the ratio of "normalized to pretreatment” expression;
  • Expression at a particular time point was normalized to the control group by dividing the "normalized to pretreatment” ratio of the animal by the mean “normalized to pretreatment” ratio of all mice in the control group. This normalizes the expression at each time point to the expression of the control group, and the specific detection results after normalization are shown in FIG. 3 .
  • L96-conjugated ANGPTL3 RNAi agents were administered to hANGPTL3 C57 BL/6 transgenic mice as described above. Each mouse received a single subcutaneous (SC) dose of 1 mg/Kg ANGPTL3 RNAi agent solution, and hANGPTL3 protein levels in serum were monitored for up to 35 days. Knockdown levels and duration of response are shown in Table 8. As can be seen from Table 8, on the 5th day of administration, 8 ANGPTL3 RNAi agents 1 mg/Kg administered showed greater than 50% knockdown, and 2 ANGPTL3 RNAi agents 1 mg/Kg administered showed greater than 70% knockdown.
  • Example 8 In vivo activity evaluation (multiple doses) of ANGPTL3 RNA interfering agent
  • mice Female hANGPTL3 C57 BL/6 transgenic mice were used. After adapting to the environment and facilities, the serum of the mice was collected 3 days before administration, and the mice were divided into 12 groups according to the level of hANGPTL3 in the serum, with 3 mice in each group. On the day of administration (day 0), hANGPTL3 C57 BL/6 transgenic mice were subcutaneously injected with ANGPTL3 RNAi solution, and mice in the control group were given PBS solution. All mice were subcutaneously injected once at the back of the neck, the dose was 1 mg/Kg or 3 mg/Kg, and the volume of administration was 5 ml/kg.
  • Knockdown of hANGPTL3 expression was evaluated by detecting hANGPTL3 protein level in mouse serum by using ELISA method (hANGPTL3, Abcam).
  • hANGPTL3, Abcam ELISA method
  • each animal's hANGPTL3 level at a given time point was divided by the pretreatment level in this animal (Day0) to determine the ratio of "normalized to pretreatment”expression;
  • Expression at a particular time point was normalized to the control group by dividing the "normalized to pretreatment” ratio of the animal by the mean “normalized to pretreatment” ratio of all mice in the control group.
  • Gal6-conjugated ANGPTL3 RNAi agents were administered to hANGPTL3 C57BL/6 transgenic mice as described above. Each mouse received a single subcutaneous (SC) dose of 1 mg/Kg or 3 mg/Kg ANGPTL3 RNAi agent solution, and serum hANGPTL3 protein levels were monitored for up to 28 days. Knockdown levels and duration of response are shown in Table 9.
  • ANGPTL3 RNAi agent Geno-2-167M, Geno-2-168M, Geno-2-169M and Geno-2-171M all showed greater than 1mg/Kg and 3mg/Kg 50% knockdown, where Geno-2-167M 3mg/Kg all showed greater than 80% knockdown, Geno-2-168M, Geno-2-170M and Geno-2-171M 3mg/Kg showed greater than 70% knockdown; when administered on the 14th day, ANGPTL3 RNAi agents Geno-2-167M, Geno-2-168M, Geno-2-170M and Geno-2-171M 3mg all showed greater than 80% knockdown; On day 21, ANGPTL3 RNAi agents Geno-2-168M and Geno-2-171M 3mg/Kg showed greater than 90% knockdown; on day 28 of administration, ANGPTL3 RNAi agent 3mg/Kg still showed greater than 60% knockdown, Among them, Geno-2-168M, Geno-2-170M and Geno

Abstract

本申请涉及一种抑制ANGPTL3表达的RNA干扰(RNAi)剂，包含靶向编码ANGPTL3的核酸分子的核苷酸，其中所述第一核苷酸序列包含SEQ ID NO:1至SEQ ID NO:184中的任一个核苷酸序列中的至少12个连续核苷酸。本申请还涉及包含配体的RNA干扰剂，配体可作为递送媒介物体内递送所述RNA干扰剂至肝脏细胞。本申请还涉及包含一种或多种RNA干扰剂的药物组合物，使用这种基于核酸的药物组合物能够降低ANGPTL3蛋白的表达水平并治疗或预防心脏代谢疾病。

Description

抑制ANGPTL3表达的RNA干扰剂及其用途
交叉引用
本申请要求2022年01月11日递交的，第202210026997.2号，名称为抑制ANGPTL3表达的RNA干扰剂及组合物的中国专利申请，和2022年01月20日递交的，第202210068123.3号，名称为siRNA缀合物及其用途中国专利申请的优先权，他们通过引用全文结合至本申请中。
技术领域
本申请涉及生物医药技术领域，具体的涉及一种抑制ANGPTL3表达的RNA干扰剂及其用途。
背景技术
RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。它是生物进化过程中遗留下来的一种在转录后通过RNA调控基因表达的机制。由于使用RNAi技术可以特异性抑制特定基因的表达(其中长度超过30bp的dsRNA会引起干扰素毒性)，所以该技术已被广泛用于探索基因功能、治疗传染性疾病及恶性肿瘤的领域。RNAi由RNA诱导的沉默复合物(RISC)介导，RISC是一种由siRNA与Argonaute蛋白和Dicer酶结合形成的复合物。具体工作时，先由Dicer酶剪切得到小dsRNA中间分子即小RNA双链，然后这些小RNA双链先解链，形成“有功能的”单链，由此为RISC复合体起到“向导”作用。对于每一个小dsRNA分子来说，只有一条链——即向导链才能与特定的Argonaute蛋白结合，形成活化的RISC复合体。RNA干扰剂诸如siRNA和微小RNA是通过基因沉默，即通过使mRNA分子降解来抑制蛋白质的基因翻译，由此阻止对应蛋白质的生成。
血管生成素样3(ANGPTL3)是由人类血管生成素-样3基因编码的血管生成素蛋白，主要在肝脏中表达并用于调节脂质新陈代谢。ANGPTL3抑制脂蛋白脂肪酶(LPL)和内皮脂肪酶(EL)的催化活性，从而导致甘油三酯、高密度脂蛋白(HDL)和磷脂的血浆水平增加。ANGPTL3是460个氨基酸组成的多肽，其由信号肽、N-末端卷曲螺旋结构域和C-末端纤维蛋白原(FBN)-样结构域组成。
脂质代谢的失调会导致血脂水平(例如甘油三酯和/或胆固醇)的升高,这与高血压、心血管疾病、糖尿病以及其他病理学病症高度相关。高甘油三酯血症是脂质代谢作用失调的一个实 例，其特征为血液中含有高水平的甘油三酯。靶向ANGPTL3的有效治疗剂可用于治疗(包括预防性治疗)心脏代谢疾病，例如高甘油三酯血症、肥胖、高血脂症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症、他汀类药物抗性高胆固醇血症和其它代谢相关病症和疾病。
发明内容
本发明的目的之一在于提供一种能够抑制细胞、受试者体内如哺乳动物(例如人)中ANGPTL3表达的RNAi剂及包括该干扰剂的组合物。人类的mRNA序列如图1所示。本发明提供的ANGPTL3 RNA干扰(RNAi)剂(也被称为RNAi触发剂或触发剂)和含有ANGPTL3 RNAi剂的组合物能用于选择性且有效抑制ANGPTL3基因的表达。
本发明的另一个目的在于提供本发明的RNAi剂或/和组合物的用途，其用于抑制ANGPTL3基因的表达或/和用于治疗ANGPTL3基因过量表达相关的疾病，比如高血脂或高甘油三酯血症及相关的疾病。
本申请第一方面提供一种抑制ANGPTL3表达的RNA干扰(RNAi)剂，包含第一核苷酸序列，所述第一核苷酸序列包括SEQ ID NO:1至SEQ ID NO:184中的任一个核苷酸序列或者与之互补的核苷酸序列中的至少12个连续核苷酸，或者与所述至少12个连续核苷酸相差不超过3个核苷酸的序列，所述核苷酸为经修饰或者未被修饰的状态。
优选地，所述第一核苷酸序列为单链寡核苷酸或双链寡核苷酸。
优选地，所述第一核苷酸序列中的一个或多个核苷酸被修饰以形成经修饰的核苷酸。
优选地，所述第一核苷酸序列包括表2中列举的任意一个经修饰的正义链核苷酸序列或与之相差不超过3个核苷酸的序列。
优选地，所述第一核苷酸序列为正义链或反义链。
优选地，所述第一核苷酸序列包括反义链和正义链的双链结构。
优选地，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸；优选地，所述正义链的长度为19个核苷酸且所述反义链的长度为21个核苷酸，或所述正义链的长度为21个核苷酸且所述反义链的长度为23个核苷酸，或所述正义链的长度为23个核苷酸且所述反义链的长度为25个；更优选地，所述正义链的长度为21个核苷酸且所述反义链的长度为23个核苷酸。
优选地，所述反义链核苷酸序列选自：SEQ ID NO:1，SEQ ID NO:5，SEQ ID NO:23，SEQ ID NO:39，SEQ ID NO:49，SEQ ID NO:51，SEQ ID NO:53，SEQ ID NO:59，SEQ ID  NO:67，SEQ ID NO:73，SEQ ID NO:89，SEQ ID NO:101，SEQ ID NO:111，SEQ ID NO:117，SEQ ID NO:135，SEQ ID NO:141，SEQ ID NO:143，SEQ ID NO:151，SEQ ID NO:165和SEQ ID NO:167中的一种。
优选地，所述正义链核苷酸序列选自：SEQ ID NO:2，SEQ ID NO:6，SEQ ID NO:24，SEQ ID NO:40，SEQ ID NO:50，SEQ ID NO:52，SEQ ID NO:54，SEQ ID NO:60，SEQ ID NO:68，SEQ ID NO:74，SEQ ID NO:90，SEQ ID NO:102，SEQ ID NO:112，SEQ ID NO:118，SEQ ID NO:136，SEQ ID NO:142，SEQ ID NO:144，SEQ ID NO:152，SEQ ID NO:166，和SEQ ID NO:168中的一种。
优选地，还包含具有1至4个未配对核苷酸的突出端，优选地，所述突出端为2个或3个未配对核苷酸。
优选地，所述突出端位于所述反义链的3'端。
优选地，所述正义链和/或反义链独立地包含一个或多个经修饰的核苷酸。
优选地，所述修饰的核苷酸相互独立地选自：2'-O-甲基修饰核苷酸、2'-O-甲氧基乙基修饰核苷酸、2'-O-烯丙基修饰核苷酸、2'-烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧核苷酸、3'-脱氧-胸腺嘧啶核苷酸、、2'-O-烷基修饰核苷酸、脱氧核糖核苷酸、2'-脱氧-2'-氟修饰的核苷酸、锁定核苷酸、非碱基核苷酸、吗啉代核苷酸、磷酰胺酯、双环核酸、异源核苷酸、EVP、LNA、GNA和UNA中的至少一种；
优选地，所述修饰的核苷酸为2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸。
优选地，按照5'末端到3'末端的方向，正义链的第7、9、10和11位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸或2'-脱氧-修饰的核苷酸；
优选地，按照5'末端到3'末端的方向，反义链的第2、4、5、6、8、9、10、12、14和16位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸；
优选地，按照5'末端到3'末端的方向，反义链的第2、6、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、5、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、5、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、4、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、6、12、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、6、10、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
还更优选地，按照5'末端到3'末端的方向，反义链的第2、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
更优选地，按照5'末端到3'末端的方向，反义链的第2、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
优选地，所述正义链和/或反义链独立地包含一个或多个硫代磷酸酯键；优选地，所述正义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键，或者，所述反义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键。
在某些实施方案中，所述反义链包括表2中任意一个经修饰的反义链核苷酸序列或与其相差不超过3个核苷酸的序列。
进一步优选地，所述正义链和反义链具有以下特征：所述正义链包含表2中列举的经修饰的正义链序列或由其组成，所述反义链包含表2中列举的经修饰的反义链序列或由其组成。
优选地，所述RNA干扰剂还包含配体。
优选地，所述配体包括靶向基团。
优选地，所述靶向基团包括脱唾液酸糖蛋白受体配体。
优选地，所述配体包含N-乙酰基-半乳糖胺，且所述配体连接至siRNA的正义链3'末端或5'末端。
优选地，所述脱唾液酸糖蛋白受体配体包含半乳糖簇。
优选地，所述配体用于缀合至所述正义链和/或反义链，优选地，用于缀合至正义链。
在一个实施方式中，所述ANGPTL3 RNA干扰剂包括如下结构：
Z-X  (I)，
其中X为核苷酸序列，Z为核苷酸序列的第一接头部分，其中Z与X可以直接连接，也可以通过化学基团连接，且Z的通式如(Z-1)所示，其中O基一端与核苷酸序列连接：
其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取 代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、或者取代或未取代的杂环或取代或未取代的环烷基；
其中R2为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-还可以任选被选自卤素、烷基、烷氧基和烷氨基中的一种取代基取代；所述a1和a2相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数，进一步优选为1到5的整数。
优选地，X代表的核苷酸序列为上述第一核苷酸序列。
优选地，所述第一接头部分Z与第二接头部分L1连接，所述第二接头部分L1与分支点基团E连接。
优选地，所述分支点基团E与a个靶向组合体连接，所述a为选自0到10的整数，优选1到5的整数；其中所述靶向组合体包括1：1比例的栓系部分L2和靶向部分T。
具体地，所述所述ANGPTL3 RNA干扰剂结构式可以表示为：
一个实施方案中，所述ANGPTL3 RNA干扰剂具有如下结构：
通式(II)中：
L3包括第三接头部分，L4为靶向部分，具体地，L3和L4可以相同或者不同，优选地，L3和L4分别为以下结构：
X为核苷酸序列，可以为正义链或反义链，
Y是O或S，
其中b,c,d和e分别选自0到10的整数，且b和e不同时为0。在一个实施方案中，b 为0，e为1至6的整数。一个优选实施例中，b为0，d为2，e为1。
另一方面，本申请还提供了前述的ANGPTL3 RNA干扰剂在制备药物中的用途，其中所述药物用于减少哺乳动物中ANGPTL3 mRNA或蛋白质表达，或者用于预防和/或治疗代谢失调或者与ANGPTL3过量表达相关疾病或病症、或者降低疾病或病症的风险。
在一个实施方案中，所述代谢失调为脂代谢作用失调，例如高血脂或高甘油三酯血症或高低密度脂蛋白(LDL)。
在另一个实施方案中，所述疾病是心脏代谢疾病。
在另一个实施方案中，所述心脏代谢疾病是高甘油三酯血症、肥胖、高脂血症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症或他汀类药物抗性高胆固醇血症，肝源性疾病，炎症、心脑血管、心肌梗塞、或代谢疾病；
优选地，所述心血管代谢疾病包括高脂血症、中风、动脉粥样硬化、血栓形成、冠心病，心卒中，脑卒中或主动脉瓣狭窄。
在某些实施方案中，所述药物包括药学上可接受的赋形剂，优选地，所述药学上可接受的赋形剂为PBS缓冲液或生理盐水。
在某些实施方案中，所述药物为组合物，还包含一种或多种另外的治疗剂。
在某些实施方案中，所述治疗剂选自他汀类药物、PCSK9 RNAi抑制剂、PCSK9抗体抑制剂和PCSK9小分子抑制剂。
另一方面，本申请提供一种用于减少细胞或组织中ANGPTL3 mRNA或蛋白质表达的方法，其包括：使细胞或组织与有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物的接触，并维持接触状态一段时间使得该接触时间足够ANGPTL3 mRNA降解，从而实现RNAi抑制细胞中ANGPTL3基因表达。
在某些实施方案中，其中所述细胞是肝细胞。
在某些实施方案中，其中所述组织是肝脏组织。
在某些实施方案中，其中所述细胞和组织是离体的。
在某些实施方案中，其中所述细胞和组织位于受试者体内。
另一方面，本申请提供一种用于减少受试者体内ANGPTL3 mRNA或蛋白质表达的方法，其包括：向有此需要的受试者施用有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物。
在某些实施方案中，所述受试者为患有脂代谢紊乱(例如高甘油三酯血症或/和高血脂症)的人类受试者。
另一方面，本申请提供一种预防或治疗疾病或病症的方法，所述方法包括向目标受试者 施用有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物。
在某些实施方案中，其中所述化合物或所述药物组合物以皮下方式、静脉内方式、口服、经直肠或腹膜内施加途径向受试者施用。
另一方面，本发明提供一种用于执行上述的方法的试剂盒。
在某些实施方案中，本发明提供用于抑制细胞中ANGPTL3基因表达的试剂盒，包括上述的RNAi剂和任选地用于将该RNAi剂给予受试者的工具，所述抑制是通过施用有效量的双链RNAi剂接触细胞来实现的。
有益效果：实验证明，本申请的ANGPTL3 RNAi剂，能使得受试者的甘油三酯水平、胆固醇水平和/或低密度脂蛋白水平得以降低，在离体实验和活体(在体)实验(小鼠体内)中，均可以显著抑制ANGPTL3蛋白及对应的mRNA的表达。其中，在小鼠实验中，对于ANGPTL3蛋白的表达，高剂量下最高可以实现大于90％的敲低抑制，使用方面，可以定向发挥作用。
附图说明
图1为人类ANGPTL3的mRNA的具体序列。
图2显示了实施例6的检测结果。
图3显示了实施例7的检测结果
图4A和4B显示了实施例8中两个不同浓度抑制剂的检测结果。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图、实施例及化学反应式，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
术语定义
在本申请中，术语“核酸”和“多核苷酸”可互换使用，并指任何长度的核苷酸(脱氧核糖核苷酸或者核糖核苷酸或者其类似物)的聚合形式，可以是双链和单链分子。以下是多核苷酸的非限制性实例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、分支的多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、siRNA、miRNA、shRNA、RNAi试剂和引物。多核苷酸可以在一个或多个碱基、糖和/或磷酸酯处以本领域已知的任何各种修饰或取代进行修饰或取代。多核苷酸可以包含修饰的核苷 酸，例如甲基化的核苷酸和核苷酸类似物。此外，可以在聚合物组装之前或之后对核苷酸结构进行修饰。多聚核苷酸可以在聚合后修饰，例如通过与标记组分偶联。除非另有说明或要求，否则本申请作为多核苷酸的任何实施方案包括双链形式和已知或据预测构成双链形式的两种互补单链形式中的每一种。
在本申请中，“靶核酸”或“靶序列”通常意指反义化合物意图与其杂交以产生希望的效应(如反义活性)的核酸分子。示例性的反义化合物包括反义寡核苷酸，反义寡核苷酸与其靶核酸具有足够的互补性以允许在生理条件下杂交。在某些实施方案中，“靶核酸”包括但不限于编码哺乳动物ANGPTL3的DNA或RNA，诸如人ANGPTL3 mRNA。
在本申请中，术语“寡核苷酸”通常是指由多个核苷酸残基(脱氧核糖核苷酸或核糖核苷酸，或其相关的结构变体或合成类似物)通过磷酸二酯键(或其相关的结构变体或合成类似物)连接组成的聚合物。因此，虽然术语“寡核苷酸”一般指其中核苷酸残基和它们之间的连接是天然产生的核苷酸聚合物，但应理解，该术语的范围也包括各种类似物，包括但不限于：肽核酸(PNA)、氨基磷酸酯、硫代磷酸酯、甲基膦酸酯、2-O-甲基核糖核酸等。该分子的确切大小可取决于具体应用。寡核苷酸一般长度较短，通常约有10-30个核苷酸残基，但该术语也可指任何长度的分子，尽管术语“多核苷酸”或“核酸”一般用于较大的寡核苷酸。
在某些实施方案中，寡核苷酸包含一个或多个未修饰的核糖核苷(RNA)和/或未修饰的脱氧核糖核苷(DNA)和/或一个或多个修饰核苷。术语“修饰寡核苷酸”通常意指包含至少一个修饰核苷和/或至少一个修饰的核苷间键联的寡核苷酸。
在本申请中，术语“修饰的核苷”通常意指与天然存在的RNA或DNA核苷相比包含至少一个化学修饰的核苷。修饰的核苷包含修饰的糖部分和/或修饰的核碱基。
在本申请中，术语“核碱基”通常意指杂环嘧啶或嘌呤化合物，它是所有核酸的组分且包括腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U)。核苷酸可包括经修饰的核苷酸或核苷酸模拟物、无碱基位点(Ab或X)或替代物替代部分。如本申请所使用，“核碱基序列”通常意指不依赖于任何糖、键联或核碱基修饰的连续核碱基的顺序。术语“未修饰的核碱基”或“天然存在的核碱基”通常意指RNA或DNA的天然存在的杂环核碱基：嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)；以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)(包括5-甲基C)和尿嘧啶(U)。“修饰的核碱基”通常意指并非天然存在的核碱基的任何核碱基。
在本申请中，术语“糖部分”通常意指核苷的天然存在的糖部分或修饰的糖部分。术语“天然存在的糖部分”通常意指如在天然存在的RNA中发现的呋喃核糖基或如在天然存在的DNA中发现的脱氧呋喃核糖基。“修饰的糖部分”意指取代的糖部分或糖替代物。
在本申请中，术语“核苷间键联”通常意指寡核苷酸中相邻核苷之间的共价键联。“天 然存在的核苷间键联”意指3’至5’磷酸二酯键联。“修饰的核苷间键联”意指除了天然存在的核苷间键联之外的任何核苷间键联。
在本申请中，术语“反义寡核苷酸”是指单链寡核苷酸分子，其具有与靶核酸(例如，目标基因组序列，mRNA前体，或mRNA分子)的相应片段互补的核碱基序列。在某些实施方案中，反义寡核苷酸的长度为12至30个核碱基。在某些实施方案中，反义寡核苷酸是具有与靶核酸(如ANGPTL3多核苷酸)序列互补的核苷酸序列的未经修饰或经修饰的核酸。
在本申请中，术语“反义链”通常是指RNAi剂(例如dsRNA)的包括与靶序列实质上互补的区域的链。在本文中使用时，术语“互补性区域”通常指反义链上与本申请定义的序列(例如靶序列)实质上互补的区域。当互补性区域与靶序列不完全互补时，错配可以在分子的内部或末端区域。通常，最被容许的错配在末端区域，例如，在5’末端和/或3’末端的5、4、3或2个核苷酸内。
在本申请中，术语“正义链”(S)通常是指RNAi剂的这样一条链，所述链包括与作为在此定义的术语反义链的区域基本互补的区域。“正义”链有时被称为“有义”链。借助它们的序列，反义链靶向所希望的mRNA，同时正义链靶向不同靶标。因此，如果反义链被掺入RISC中，则正确的靶标被靶向。正义链的掺入可以导致脱靶效应。这些脱靶效应可以通过在正义链上使用修饰或使用5’端帽加以限制。
在本申请中，术语“互补”当用于描述就第二核苷酸序列(如RNAi剂反义链)而言的第一核苷酸序列(如RNAi剂正义链或ANGPTL3 mRNA)时是指包含第一核苷酸序列的寡核苷酸或多核苷酸在一定条件下与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交(形成碱基对氢键)并形成双链体或双螺旋结构的能力。互补序列包括沃森-克里克碱基对(Watson-Crick base pairs)或非沃森-克里克碱基对，并且包括天然或经修饰的核苷酸或核苷酸模拟物，只要以上关于它们的杂交能力而言的需求得以实现。“互补”不必需在每个核苷上均具有核碱基互补性。相反，可以容忍一些错配。
在本申请中，术语“完全互补”通常意指第一多核苷酸的连续序列中的全部(100％)碱基将与第二多核苷酸的连续序列中的相同数目的碱基杂交。连续序列可以包含第一或第二核苷酸序列的全部或一部分。如本申请所用，“部分互补”通常意指在杂交的碱基序列对中，第一多核苷酸的连续序列中至少约70％的碱基将与第二多核苷酸的连续序列中相同数目的碱基杂交。如本申请所用，“基本上互补”通常意指在杂交的碱基序列对中，第一多核苷酸的连续序列中至少约90％的碱基将与第二多核苷酸的连续序列中相同数目的碱基杂交。如本申请所用的术语“互补”、“完全互补”和“基本上互补”可以就在RNAi剂的正义链与反义链之间或者在RNAi剂的反义链与ANGPTL3 mRNA的序列之间的碱基匹配而言使用。序列 同一性或互补性不依赖于修饰。为了测定同一性或互补性的目的，例如，A和Af与U(或T)互补并且与A同一。
在本申请中，术语“配体”通常是指能够共价地或以其它化学方式与生物活性物质(如寡核苷酸)结合的任何化合物或分子。在某些实施方案中，配体能够与另一种化合物例如受体直接或间接地相互作用，与配体相互作用的受体可以存在于细胞表面上，或可替代地可以是细胞内和/或细胞间受体，配体与受体的相互作用可以导致生化反应，或可以仅仅是物理相互作用或结合。
在本申请中，术语“诱导”、“抑制”、“加强”、“升高”、“增加”、“减少”、“降低”等通常表示两个状态之间的定量差异。例如，“有效抑制ANGPTL3的活性或表达的量”意指处理样品中ANGPTL3的活性或表达的水平将低于未处理样品中ANGPTL3活性或表达的水平。所述术语例如适用于表达水平和活性水平。术语“减少”和“降低”可互换使用并且通常表示小于原来的任何变化。“减少”和“降低”是相对的术语，需要在测量前和测量后间进行比较。“减少”和“降低”包括完全耗竭。
在某些实施方案中，术语“降低”可以通过本领域已知标准方法(诸如本申请中描述的那些)检测的，基因、基因产物例如蛋白质或生物标志物在第一样品中的表达水平/量与相应基因、基因产品例如蛋白质或生物标志物在第二样品中的表达水平/量相比约5％至95％、或100％的总体降低。在某些实施方案中，术语“降低”指受试样品中基因或生物标志物的表达水平/量的降低，其中该降低是指相应基因或生物标志物的表达水平/量的至少约0.9倍至0.01倍。
在本申请中，术语“表达”通常意指基因最终产生蛋白质的过程。表达包括但不限于转录、转录后修饰(例如，剪接、聚腺苷酸化、添加5’-帽)以及翻译。
在本申请中，术语“药学上可接受的”通常是指不干扰活性成分生物学活性的有效性的一种或多种无毒物质。这类制剂通常可含有盐、赋形剂、缓冲剂、防腐剂、相容性载体和任选的其它治疗剂。用于医药时，盐应该是药学上可接受的盐，但可方便地使用非药学上可接受的盐来制备药学上可接受的盐，不能将它们排除在本申请范围以外。这类药理学和药学上可接受的盐包括由以下酸制备的盐：氢氯酸、氢溴酸、硫酸、硝酸、磷酸、马来酸、乙酸、水杨酸、柠檬酸、硼酸、甲酸、丙二酸、琥珀酸等。药学上可接受的盐也可制备成碱金属盐或碱土金属盐，如钠盐、钾盐或钙盐。
在本申请中，术语“预防和/或治疗”不仅包括预防和/或治疗疾病，还通常包括预防疾病的发作，减缓或逆转疾病的进展，预防或减缓与疾病相关的一种或多种症状的发作，减少和/或减轻与疾病相关的一种或多种症状，降低疾病和/或与其相关的任何症状的严重程度和/ 或持续时间和/或预防疾病和/或与其相关的任何症状的严重程度的进一步增加，预防、减少或逆转由疾病引起的任何生理损伤，以及通常对正在治疗的患者有益的任何药理学作用。本申请的RNAi剂或药物组合物形成可行的治疗剂不需要实现完全治愈或根除疾病的任何症状或表现。如在相关领域中所认识到的，用作治疗剂的药物可降低给定疾病状态的严重程度，但不需要消除疾病的每种表现才能被认为是有用治疗剂。类似地，预防性施用的治疗构成可行的预防剂不需要完全有效地预防病症的发作。简单地在受试者中减少疾病的影响(例如，通过减少其症状的数量或严重程度，或通过提高另一种治疗的有效性，或通过产生另一种有益效果)，或减少疾病发生或恶化的可能性就足够了。
在本申请中，术语“疾病”或“病症”可以互换使用，通常是指受试者与正常状态的任意偏离，例如身体或某些器官的状态的任何变化，妨碍或扰乱了功能的履行，和/或在患病或与其接触的人中引起症状例如不适、机能障碍、痛苦或甚至死亡。疾病或病症还可以称为失调(distemper)、不适(ailing)、小病(ailment)、疾病(malady)、紊乱(disorder)、疾病(sickness)、生病(illness)、身体不适(complaint)、inderdisposion或affectation。
本申请中，术语“施用”通常是指通过任意引入或递送途径将本申请药物制剂引入受试者的身体中。可以采用本领域技术人员已知的用于使细胞、器官或组织与所述药物接触的任何方法。所述施用可以包括而不限于静脉内、动脉内、鼻内、腹内、肌内、皮下或口服。每日剂量可以划分成一个、两个或更多个合适形式的剂量以在某个时间段期间的一个、两个或更多个时间施用。
在本申请中，术语“接触”通常是指两种两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。接触可以发生在体内(in vivo)、间接体内(ex vivo)或体外(in vitro)。在一些实施方案中，可以是指使本申请的RNAi剂或组合物直接接触细胞或组织。在另一些实施方案中，该术语是指使本申请的RNAi剂或组合物间接接触细胞或组织。例如，本申请的方法包括其中受试者接触本申请的RNAi剂或组合物，然后RNAi剂或组合物通过扩散或本领域已知的任何其他主动运输或被动运输过程(化合物通过该过程在体内循环)接触细胞或组织的方法。
在本申请中，术语“有效量”或“有效剂量”通常是指足以实现或至少部分实现所需效果的量。药物或治疗剂的“治疗有效量”或“治疗有效剂量”通常是当单独使用或与另一种治疗剂组合使用时促进疾病消退(这通过疾病症状严重程度的降低、疾病无症状期的频度和持续时间的增加、或者由于罹患疾病而引起的损害或残疾的预防来证明)的任何药物量。药物的“预防有效量”或“预防有效剂量”通常是指当单独或与另一种治疗剂组合给有疾病发展或疾病复发的风险的受试者施用时抑制疾病的发展或复发的药物量。在某些实施方案中， “有效量”是指产生预期药理学、治疗性或预防性结果的RNAi剂的量。
在本申请中，术语“受试者”通常是指需要诊断、预后、改善、预防和/或治疗疾病的人或非人动物(包括哺乳动物)，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型。
在本申请中，术语“包括”、“包含”、“具有”、“可以”、“含有”及其变体通常旨在是开放式过渡性短语、术语或词语，其不排除额外行为或结构的可能性。术语“由……组成”通常表示不能存在别的组分(或同样地，特征、整数、步骤、等)。除非上下文另有明确规定，未限定数量的名词也包括复数指示物。
当术语“约”用于指涉数值范围时，截值或特定数值用于指示所载明的数值可与该列举数值有多达10％的差异。因此，术语“约”可用于涵盖自特定值±10％或更少的变异、±5％或更少的变异、±1％或更少的变异、±0.5％或更少的变异、或±0.1％或更少的变异。
ANGPTL3 RNAi剂
本申请所述的用于抑制ANGPTL3基因的表达的RNAi剂(在本申请中被称为ANGPTL3 RNAi剂)在递送至表达ANGPTL3基因的细胞后，通过RNA干扰(RNAi)的生物过程在体外和/或体内抑制或敲低ANGPTL3的表达。如本申请所用，除非另外特别指示，否则ANGPTL3在适当情况下可能是指ANGPTL3基因、ANGPTL3 mRNA或ANGPTL3蛋白质。RNAi剂包括但不限于：短干扰RNA(siRNA)、双链RNA(dsRNA)、微RNA(miRNA)和短发夹RNA(shRNA)。
一方面，本申请提供了一种ANGPTL3 RNAi剂，其可以包含靶向编码ANGPTL3的核酸分子的第一核苷酸序列，其中所述第一核苷酸序列包含SEQ ID NO:1至SEQ ID NO:184的至少12、至少13、至少14、至少15、至少16、至少17、至少18、至少19、至少20、至少21、至少22或至少23个连续核苷酸。所述核苷酸序列可以为修饰状态(以及修饰后加上配体)和无修饰的原始状态。
在某些实施方案中，所述第一核苷酸序列由12至30个核苷酸组成。在某些实施方案中，所述的所述第一核苷酸序列的长度可以为17、18、19、20、21、22、23、24、25或26个核苷酸长。
在某些实施方案中，其中所述第一核苷酸序列包含选自以下序列的核苷酸序列：SEQ ID NO:1至SEQ ID NO:184中任意一个或与其相差不超过3个核苷酸的序列。例如，当不同于SEQ ID NO:1至SEQ ID NO:184中任意一个所示的核苷酸序列，发生最多一个、二个或三个核苷酸替代(例如，腺苷被尿嘧啶替代)、缺失或添加时，衍生的RNAi剂仍可以具有降低 ANGPTL3表达的抑制活性，与作为衍生物来源的SEQ ID NO:1至SEQ ID NO:184中任意一个所示的核苷酸序列相比，不低于20％的抑制作用。
在某些实施方案中，所述反义链核苷酸序列选自：SEQ ID NO:1，SEQ ID NO:5，SEQ ID NO:23，SEQ ID NO:39，SEQ ID NO:49，SEQ ID NO:51，SEQ ID NO:53，SEQ ID NO:59，SEQ ID NO:67，SEQ ID NO:73，SEQ ID NO:89，SEQ ID NO:101，SEQ ID NO:111，SEQ ID NO:117，SEQ ID NO:135，SEQ ID NO:141，SEQ ID NO:143，SEQ ID NO:151，SEQ ID NO:165和SEQ ID NO:167中的一种。
在某些实施方案中，所述正义链核苷酸序列选自：SEQ ID NO:2，SEQ ID NO:6，SEQ ID NO:24，SEQ ID NO:40，SEQ ID NO:50，SEQ ID NO:52，SEQ ID NO:54，SEQ ID NO:60，SEQ ID NO:68，SEQ ID NO:74，SEQ ID NO:90，SEQ ID NO:102，SEQ ID NO:112，SEQ ID NO:118，SEQ ID NO:136，SEQ ID NO:142，SEQ ID NO:144，SEQ ID NO:152，SEQ ID NO:166，和SEQ ID NO:168中的一种。
在某些实施方案中，其中所述第一核苷酸序列为单链寡核苷酸或双链寡核苷酸。
例如，所述第一核苷酸序列可以为反义寡核苷酸。又例如，所述寡核苷酸为siRNA。
例如，所述第一核苷酸序列可以仅包含反义链。又例如，所述第一核苷酸序列可以包含正义链与反义链。
在某些实施方案中，所述的ANGPTL3 RNAi剂正义链和反义链的长度独立地为17至30个核苷酸长。在某些实施方案中，正义链和反义链独立地为17至26个核苷酸长。在某些实施方案中，正义链和反义链为19-26个核苷酸长。正义链和反义链可为相同长度或它们可为不同长度。在某些实施方案中，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。在某些实施方案中，正义链为19个核苷酸长且反义链为21个核苷酸长。在某些实施方案中，正义链为21个核苷酸长且反义链为23个核苷酸长。在某些实施方案中，正义链为23个核苷酸长且反义链为25个核苷酸长。
在某些实施方案中，反义链序列与ANGPTL3 mRNA中存在的核苷酸序列(有时被称为靶序列)100％(完美)互补或至少90％(基本上)互补。正义链序列与反义链中的序列100％(完美)互补或至少90％(基本上)互补，并因此正义链序列与ANGPTL3 mRNA中存在的核苷酸序列(靶序列)完美相同或至少90％相同。
在某些实施方案中，所述ANGPTL3 RNAi剂的正义链和反义链退火以形成双链体。ANGPTL3 RNAi剂的正义链和反义链彼此部分、基本上或完全互补。在互补双链体区域内，正义链核心序列与反义核心序列至少90％互补或100％互补。在某些实施方案中，正义链核心序列含有与反义链核心序列的相应的17、18、19、20或21个核苷酸序列至少90％或100％ 互补的至少17、至少18、至少19、至少20或至少21个核苷酸的序列(即，ANGPTL3 RNAi剂的正义链和反义核心序列具有至少90％的碱基配对或100％的碱基配对的至少17、至少18、至少19、至少20或至少21个核苷酸的区域)。
在某些实施方案中，正义链和/或反义链可以在核心序列的3’末端、5’末端或3’和5’末端两处任选且独立地含有另外的1、2、3、4、5或6个核苷酸(延伸)(突出端)。在某些实施方案中，正义链和/或反义链可以在核心序列的3’末端、5’末端或3’和5’末端两处任选且独立地含有另外的1、2、3或4个核苷酸(延伸)。在某些实施方案中，正义链和/或反义链可以在核心序列的3’末端、5’末端或3’和5’末端两处任选且独立地含有另外的2或3个核苷酸(延伸)。在某些实施方案中，反义链在核心序列的3’末端独立地含有另外的2或3个核苷酸(延伸)。
在某些实施方案中，一个或多个反义链延伸核苷酸包括尿嘧啶或胸苷核苷酸或与相应的ANGPTL3 mRNA序列互补的核苷酸。
在某些实施方案中，其中所述正义链包含表1中提供的任何一个核苷酸的序列或者与之相差1-4个核苷酸的序列。
在某些实施方案中，其中所述反义链包括与所述正义链至少部分互补的核苷酸序列。
在某些实施方案中，所述反义链包括一个互补性区域，该互补性区域与表1-3中所列出的反义链序列中的任一个的区别在于至少15个连续的核苷酸序列中有不多于3个核苷酸的不同。
在某些实施方案中，其中所述反义链和正义链序列如表1所示。
本申请所述的ANGPTL3 RNAi剂可以通过使反义链与正义链退火形成。可使含有表1中列出的序列的正义链与任何反义链杂交，只要这两条序列在连续的16、17、18、19、20或21个核苷酸序列上具有至少90％互补性。
经修饰的核苷酸
本申请的ANGPTL3 RNAi试剂包括未修饰的核酸以及为提高功效而修饰的核酸，和核苷替代物的多聚体。未修饰核酸是指其中糖、碱基和磷酸酯结构成份与天然的成份一样或基本相同，优选为人体内的天然成份。现有技术将稀有的或不寻常但是天然发生的RNAs看作是修饰的RNAs，参见Limbach等，(1994)Nucleic Acids Res.22：2183-2196。这样的稀有或不寻常的修饰的RNAs通常被称为修饰的RNAs(很显然是因为它们是转录后修饰的结果)，并在本申请属于未修饰的RNA。在本申请所使用的修饰的RNA是指其中核酸的组分，即糖、碱基和磷酸酯结构的一或多个成份，与天然的成份不一样，优选为不同于产生自人体的天然成份。核苷替代物是这样一些分子，其中核糖磷酸酯主链被非核糖磷酸酯构造所替代，该构造使碱基处于正确的空间关系，以便杂交与所见到的与核糖磷酸酯主链的杂交基本相似，例如，非 带电的核糖磷酸酯主链的类似物。
在某些实施方案中，ANGPTL3 RNAi剂含有一个或多个经修饰的核苷酸。
在某些实施方案中，ANGPTL3 RNAi剂中至少50％、至少60％、至少70％、至少80％、至少90％、至少95％或100％的核苷酸经修饰。
在某些实施方案中，其中所述正义链和/或反义链独立地包含一种或多种经修饰的核苷酸。
对RNAi剂进行修饰的两个主要目的是获得它们对生物环境抗降解的稳定性以及改进药理学性质，例如药效性质。RNAi剂可以包含非天然存在的碱基，或者非天然存在的糖，如非糖类化合物环形载体分子，用于RNAi试剂的非天然存在的糖的典型特性。RNAi试剂可以包括核苷酸之间的键(例如手性硫代磷酸酯键)，用于增加核酸酶抗性。RNAi试剂此外还可包含，或者可选择地包含，核糖类似物以增加核酸酶抗性。
经修饰的核苷酸包括但不限于脱氧核苷酸、核苷酸模拟物、非碱基核苷酸(在本申请中表示为X、Ab)、2’-修饰的核苷酸、3’至3键联(反向)核苷酸(在本申请中表示为invdN、invN、invn、invX、invAb、包含非天然碱基的核苷酸、桥接核苷酸、肽核酸(PNA)、2’，3’-断核苷酸模拟物(解锁核碱基类似物，在本申请中表示为NUNA或NUNA)、锁定核苷酸(在本申请中表示为NLNA或NLNA)、3’-O-甲氧基(2’核苷间连接的)核苷酸(在本申请中表示为3’-OMen)、2’-F-阿拉伯糖核苷酸(在本申请中表示为NfANA或NfANA)、5’-Me，2’-氟核苷酸(在本申请中表示为5Me-Nf)、吗啉代核苷酸、膦酸乙烯酯脱氧核糖核苷酸(在本申请中表示为vpdN)、含膦酸乙烯酯的核苷酸、含膦酸环丙酯的核苷酸(cPrpN)、磷酰胺酯、双环核酸、异源核苷酸、EVP、LNA、GNA和UNA。
其中2’-修饰的核苷酸(即在五元糖环的2′位置处具有除了羟基基团之外的基团的核苷酸)包括但不限于2’-O-甲基修饰核苷酸(在本申请中表示为核苷酸序列中的小写字母n)、2’-脱氧-2’-氟核苷酸(在本申请中表示为Nf，在本申请中还表示为2′-氟核苷酸)、2’-脱氧核苷酸(在本申请中表示为dN)、2’-甲氧基乙基(2’-O-2-甲氧基乙基)修饰核苷酸(在本申请中表示为NM或2’-MOE)、2'-O-烯丙基修饰核苷酸、2'-烷氧基修饰的核苷酸、2’-氨基核苷酸和2’-烷基核苷酸。具体地，可以在单个ANGPTL3 RNAi剂中或甚至在其单核苷酸中掺入多于一种的修饰。可通过本领域已知的方法合成和/或修饰ANGPTL3 RNAi剂正义链和反义链。一个核苷酸处的修饰独立于另一个核苷酸处的修饰。
在某些实施方案中，所述修饰的核苷酸为2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸。
在某些实施方案中，按照5'末端到3'末端的方向，正义链的第7、9、10和11位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸或2'-脱氧-修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、4、5、6、8、9、10、12、14和16位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、6、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、5、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、5、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、4、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、6、12、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、6、10、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
在某些实施方案中，按照5'末端到3'末端的方向，反义链的第2、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
在某些实施方案中，所述正义链和/或反义链独立地包含一个或多个硫代磷酸酯键；优选地，所述正义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键，或者，所述反义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键。
配体
在某些实施方案中，其中所述ANGPTL3 RNAi剂还包含配体。
在某些实施方案中，配体改变了与其结合的RNAi剂的分布、靶向或寿命。在某些实施例中，与缺乏配体的种类相比较，配体增强了所选择的靶的亲和力。所选择的靶，例如为分子、细胞或细胞类别和诸如细胞或器官腔隙的腔隙、组织、器官或身体的某个区域。
在某些实施方案中，配体能够促进运输、杂交和特异性的特性，并能够促进所获天然的或修饰的寡核糖核苷酸的核酸酶抗性，或者促进含有任一在本申请所描述的单体和/或天然或修饰的核糖核苷酸的结合体的多聚体分子的核酸酶抗性。
例如，配体还可以包括靶向基团，例如，细胞或组织靶向试剂，如凝集素、糖蛋白、脂或蛋白质，例如结合到某一个特定的细胞如肝细胞或空肠细胞的抗体。靶向基团可以为促甲 状腺素、促黑细胞激素、凝集素、糖蛋白、表面活性剂蛋白A、粘液素碳水化合物、多价乳糖、多价半乳糖、N-乙酰半乳糖胺、N-乙酰葡萄糖胺、多价甘露糖、多价海藻糖、糖基化聚氨基酸、多价半乳糖、铁传递蛋白、二膦酸酯、聚谷氨酸酯、聚天冬氨酸酯、脂、胆固醇、类固醇、胆酸、叶酸、维生素B12或生物素。
在某些实施方案中，所述配体包括靶向基团。
在某些实施方案中，该靶向基团可以是单价、二价、三价、四价的或具有更高的化合价。代表性靶向基团包括对细胞表面分子具有亲和力的化合物、细胞受体配体、半抗原、抗体、单克隆抗体、抗体片段和对体细胞表面分子具有亲和力的抗体模拟物。
在某些实施方案中，所述配体包含N-乙酰基-半乳糖胺。
在某些实施方案中，其中所述靶向基团能够靶向肝细胞或肝脏。
在某些实施方案中，其中所述靶向基团包括脱唾液酸糖蛋白受体配体。
在某些实施方案中，脱唾液酸糖蛋白受体配体包括一种或多种半乳糖衍生物或半乳糖簇或由一种或多种半乳糖衍生物或半乳糖簇组成。本申请中，术语半乳糖衍生物包括半乳糖，和具有对脱唾液酸糖蛋白受体的亲和力(等于或大于半乳糖的亲和力)的半乳糖衍生物两者。半乳糖衍生物包括：半乳糖、半乳糖胺、N-甲酰基半乳糖胺、N-乙酰基-半乳糖胺、N-丙酰基-半乳糖胺、N-正-丁酰基-半乳糖胺和N-异-丁酰基半乳糖-胺。半乳糖衍生物已经用于在体内通过结合至在肝细胞表面上的脱唾液酸糖蛋白受体(ASGPr)使分子靶向至肝细胞。ASGPr配体与ASGPr的结合促进细胞-特异性靶向至肝细胞并将分子内吞至肝细胞中。可以使用本领域已知的方法将半乳糖簇附接至RNAi多核苷酸的3’或5’末端。
在某些实施方案中，其中使所述配体缀合至所述正义链和/或反义链。例如，所述配体可以缀合至正义链和/或反义链的5’端和/或3’端。
递送媒介物
在某些实施方案中，递送媒介物可用于将ANGPTL3 RNAi剂递送至细胞或组织。递送媒介物是一种改善ANGPTL3 RNAi剂向细胞或组织递送的化合物。递送媒介物可包括以下或由以下组成：聚合物，诸如两亲性聚合物，可逆修饰的聚合物或肽，或可逆修饰的膜活性聚胺。
在某些实施方案中，ANGPTL3 RNAi剂可以与本领域可获得的脂质、纳米颗粒、聚合物、脂质体、胶束或其它递送系统组合。还可使ANGPTL3 RNAi剂化学缀合至靶向基团、脂质(包括胆固醇和胆固醇基衍生物)、纳米颗粒、聚合物或本领域可获得的其它递送系统。
本申请描述了用于体内递送ANGPTL3 RNAi剂至哺乳动物的肝细胞的方法。在某些实施方案中，可以使用递送媒介物。递送媒介物可以是但不限于：聚合物(诸如两亲性聚合物，膜活性聚合物)，配体等。在某些实施方案中，使ANGPTL3 RNAi剂连接至包括脱唾液酸糖 蛋白配体的靶向配体。例如，可以使ANGPTL3 RNAi剂连接至包含半乳糖簇或由半乳糖簇组成的靶向配体。
如上所述，本申请提供一种siRNA缀合物，其具有如下通式的结构，
Z-X   (I)，或者
通式(I)中：
X为核苷酸序列，可以为siRNA的正义链或反义链，Z为核苷酸序列的第一接头部分，其中Z与X可以直接连接，也可以通过化学基团连接，且Z的通式如(Z-1)所示，其中O基一端与核苷酸序列连接：
其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、或者取代或未取代的杂环或取代或未取代的环烷基；
其中R2为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-还可以任选被选自卤素、烷基、烷氧基和烷氨基中的一种取代基取代；所述a1和a2相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数，进一步优选1到5的整数。
优选地，X代表的核苷酸序列为上述第一核苷酸序列。
优选地，所述第一接头部分Z与第二接头部分L1连接，所述第二接头部分L1与分支点基团E连接。
优选地，所述分支点基团E与a个靶向组合体连接，所述a为选自0到10的整数，优选1到5的整数；其中所述靶向组合体包括1：1比例的栓系部分L2和靶向部分T。
一个实施方案中，所述所述ANGPTL3 RNA干扰剂结构式可以表示为：

优选地，通式(II)中：
L3包括第三接头部分，L4为靶向部分，具体地，通式(I)中的T和通式(II)中的L3和L4可以相同或者不同，优选地，L3和L4分别为以下结构：
X为核苷酸序列，可以为正义链或反义链，
Y是O或S，
其中b,c,d和e分别选自0到10的整数，且b和e不同时为0。在一个实施例中，b为0，d＝2,e＝1。
另一方面，所述第二接头部分L1具有以下结构：
或者
其中f,g，h和i分别是从1至20的整数，优选为1至10的整数。
本发明另一方面，所述栓系部分L2具有如下结构式：
其中j,k,l,m,n和o分别是从1至20的整数，优选为1至10的整数。
本发明另一方面还提供一种ANGPTL3 RNA干扰剂，包括：正义链和反义链，以及上述siRNA缀合物。
本发明实施例的另一目的在于提供一种包括上述RNA干扰剂的组合物。
本发明实施例提供的siRNA缀合物可以提高靶向部分结合细胞或细胞受体的效率，增 加RNA干扰的作用效果。
一个优选实施方案中，所述靶向配体中化合物Z的通式如下(Z-2)所示：
优选地R2为-NH-，此时化合物Z为：
优选地R2为-C(O)-，此时化合物Z为
优选地，L1为以下结构：
或者
一个实施方案中，在通式(I)中，所述化合物E选自以下五种结构中的一种：
一个实施例中，通式(I)中的化合物L2为
其中j,k,l,m,n和o分别是从1至20的整数；
优选地，L2具有以下结构式：
或者
优选实施方案中，通式(I)所述靶向配体(不含X部分)具有以下结构：


一个实施方案中，所述靶向配体中T选自N-乙酰基-半乳糖胺、半乳糖、半乳糖胺、N-甲酰基-半乳糖胺、N-丙酰基-半乳糖胺、N-正丁酰基半乳糖胺和N-异丁酰基-半乳糖胺中的一种，优选为N-乙酰基-半乳糖胺，结构式如下：
优选实施例中，通式(I)中的靶向配体通过磷酸酯基团或硫代磷酸酯基团或膦酸基团与siRNA末端连接。
优选实施例中,所述靶向配体具有以下结构:





在关于通式(II)的一个实施方案中，所述靶向配体中H—O—L3为:

其中R2各自独立的是-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，-NH(CO)NH-，所述-CH2-还可以任选被卤素或烷基取代，其中所述烷基任选进一步被选自羟基、氨基、卤素、烷氧基和烷氨基中的一种取代基取代)；
所述p,q,r,s,t和u分别独立地选自0到20的整数，优选为1到10的整数。
在优选的实施例中，H—O—L3具有以下结构：

在通式(II)的另一优选实施方案中，所述靶向配体中H—O—L4为：
在一个优选实施例中，H—O—L4具有以下结构：
一个优选实施方案中，通式(II)具有以下结构：




其中Y为O或者S。
另一优选实施方案中，所述siRNA缀合物具有以下结构：





其中Y为O或者S。
另一方面，上述结合至3’端的结构均可以结合至X的5’端。
一个优选实施方案中，所述配体包含N-乙酰基-半乳糖胺，且所述配体连接至siRNA的正义链3'末端或5'末端。
在某些实施方案中，所述配体包含一种或多种选自以下的结构：



药物组合物实施例
另一方面，本申请提供了一种药物组合物，其包含前述的ANGPTL3 RNAi剂以及任选地药学上可接受的赋形剂。
在某些实施方案中，所述ANGPTL3 RNAi剂可用于抑制如受试者中的细胞、细胞群或组织中ANGPTL3的表达。在某些实施方案中，ANGPTL3 RNAi剂用于配制用于施用给受试者的组合物，即药物组合物或药物。例如，药物组合物或药物可以包含药理学有效量的至少一种所述ANGPTL3 RNAi剂和一种或多种药学上可接受的赋形剂。药学上可接受的赋形剂(赋形剂)是经过适当安全评价且有意包含在药物递送系统中的除了活性药物成分(API，治疗性产物，如ANGPTL3 RNAi剂)以外的物质。赋形剂在预定剂量下不发挥或不意在发挥治疗效果。赋形剂可用于a)在制造期间有助于药物递送系统的处理，b)保护、支持或增强API的稳定性、生物利用度或患者可接受性，c)有助于产物鉴别和/或d)增强在储存或使用期间递送API的整体安全性、有效性的任何其它属性。药学上可接受的赋形剂可以或可以不是惰性物质。
赋形剂包括：吸收促进剂、抗粘剂、消泡剂、抗氧化剂、粘合剂、粘合剂、缓冲剂、载剂、包衣剂、颜料、递送促进剂、递送聚合物、葡聚糖、右旋糖、稀释剂、崩解剂、乳化剂、增量剂、填充剂、香味剂、助流剂、保湿剂、润滑剂、油、聚合物、防腐剂、盐水、盐、溶剂、糖、助悬剂、持续释放基质、甜味剂、增稠剂、张力剂、媒介物、憎水剂和润湿剂。
药物组合物可以含有药物组合物中常见的其它另外组分。此类另外的组分包括但不限于：止痒剂、收敛剂、局部麻醉剂或抗炎剂(如抗组胺剂、苯海拉明等)。还设想表达或包含本申请定义的RNAi剂的细胞、组织或分离的器官可以用作“药物组合物”。
在某些实施方案中，将所述ANGPTL3 RNAi剂与一种或多种另外的治疗剂或治疗组合，所述治疗剂或治疗包括但不限于：他汀类药物、PCSK9 RNAi抑制剂、PCSK9抗体抑制剂和 PCSK9小分子抑制剂。可将所述RNAi剂及包含本申请公开的ANGPTL3 RNAi剂的药物组合物包装或包含在试剂盒、容器、包装或分配器中。可将ANGPTL3 RNAi剂和包含所述ANGPTL3 RNAi剂的药物组合物包装在预充式注射器或小瓶中。
本申请药物组合物可用于抑制细胞、组织或生物体中ANGPTL3基因的表达。在某些实施方案中，所述药物组合物用于治疗患有将从ANGPTL3表达减少或抑制受益的疾病、疾患或病症的受试者。在某些实施方案中，所述药物组合物用于治疗处于发展将受益于ANGPTL3表达减少或抑制的疾病、疾患或病症的风险的受试者。将受益于ANGPTL3表达减少或抑制的疾病、疾患或病症包括但不限于：高甘油三酯血症、肥胖、高脂血症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症或他汀类药物抗性高胆固醇血症，肝源性疾病，炎症、心脑血管、心肌梗塞、或代谢疾病。在某些实施方案中，受试者是哺乳动物，包括但不限于人。
考虑包含本申请所述的至少一种ANGPTL3 RNAi剂的细胞、组织和非人类生物体。通过以本领域可获得的任何方式将ANGPTL3 RNAi剂递送至细胞、组织或非人类生物体来制备该细胞、组织或非人类生物体。在某些实施方案中，细胞是哺乳动物细胞，包括但不限于人类细胞。细胞、组织或非人类生物体可用于研究或作为研究工具(如药物测试或诊断)。
用途
另一方面，本申请提供前述的ANGPTL3 RNAi剂或前述的药物组合物在制备药物中的用途，其中所述药物用于减少哺乳动物中ANGPTL3 mRNA或蛋白质表达，或者用于预防和/或治疗代谢失调或者与ANGPTL3过量表达相关疾病或病症、或者降低疾病或病症的风险。
在某些实施方案中，所述代谢失调为脂代谢作用失调，例如高血脂或高甘油三酯血症或高低密度脂蛋白(LDL)。
在某些实施方案中，所述疾病是心脏代谢疾病。
在某些实施方案中，所述心脏代谢疾病是高甘油三酯血症、肥胖、高脂血症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症或他汀类药物抗性高胆固醇血症，肝源性疾病，炎症、心脑血管、心肌梗塞、或代谢疾病；
优选地，所述心血管代谢疾病包括高脂血症、中风、动脉粥样硬化、血栓形成、冠心病，心卒中，脑卒中或主动脉瓣狭窄。
另一方面，本申请提供一种预防和/或治疗疾病或病症的方法，使细胞或组织与有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物的接触，并维持接触状态一段时间使得该 接触时间足够ANGPTL3 mRNA降解，从而实现RNAi抑制细胞中ANGPTL3基因表达。
在某些实施方案中，本申请所述的ANGPTL3 RNAi剂可用于治疗通过降低ANGPTL3表达来受益的疾病或病症。该疾病或病症的实例包括但不限于：高甘油三酯血症、肥胖、高脂血症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症或他汀类药物抗性高胆固醇血症，肝源性疾病，炎症、心脑血管、心肌梗塞、或代谢疾病。在某些实施方案中，该方法包括施用组合物，诸如包含本申请所述的ANGPTL3 RNAi剂的药物组合物给目标哺乳动物。
在某些实施方案中，将治疗有效量的一种或多种所述ANGPTL3 RNAi剂施用给受试者，从而抑制受试者中ANGPTL3的表达(如，有效抑制受试者中ANGPTL3表达的量)。
在某些实施方案中，该方法还包括施用第二治疗剂或治疗的步骤。在某些实施方案中，第二治疗剂是另一种ANGPTL3 RNAi剂(如，靶向ANGPTL3靶标内不同序列的ANGPTL3 RNAi剂)。在其它实施方案中，第二治疗剂可选自：他汀类药物、PCSK9 RNAi抑制剂、PCSK9抗体抑制剂和PCSK9小分子抑制剂。
施用途径是使RNAi剂与身体接触的途径。通常，用于治疗受试者的药物和核酸的施用方法在本领域中是众所周知的，并且可以应用于本申请所述的组合物的施用。本申请所述的化合物可以经由任何适合的途径在针对特定途径适当定制的制剂中施用。因此，本申请所述的化合物可以通过注射，例如静脉内、肌内、皮内、皮下或腹膜内施用。
在某些实施方案中，可以使用本领域已知的第一核苷酸序列递送技术将本申请所述的ANGPTL3 RNAi剂或组合物递送至细胞、细胞群、组织或受试者。通常，本领域公认的用于递送核酸分子(体外或体内)的任何适合的方法可以适用于本申请所述的ANGPTL3 RNAi剂。例如，递送可通过局部施用(local administration)(如，直接注射、植入或局部施用(topical administering)，全身施用，或皮下、静脉内、经口、腹膜内或肠胃外途径，包括颅内(如心室内、实质内和鞘内)、肌内、透皮、气道(气雾剂)、经鼻、经直肠或局部(包括经颊和舌下)施用。在某些实施方案中，通过皮下或静脉内输注或注射施用组合物。
在某些实施方案中，ANGPTL3 RNAi剂可以与本领域可获得的脂质、纳米颗粒、聚合物、脂质体、胶束或其它递送系统组合。还可将RNAi剂化学缀合至靶向基团、脂质(包括但不限于胆固醇和胆固醇基衍生物)、纳米颗粒、聚合物、脂质体、胶束或本领域可获得的其它递送系统。可使ANGPTL3 RNAi剂缀合至递送聚合物。在某些实施方案中，递送聚合物是可逆掩蔽/修饰的两亲性膜活性聚胺。
表达的抑制
另一方面，本申请提供一种用于减少细胞或组织中ANGPTL3 mRNA或蛋白质表达的方法，其包括：使细胞或组织与有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物的接触。
在某些实施方案中，其中所述细胞是肝细胞。在某些实施方案中，其中所述组织是肝脏组织。
在某些实施方案中，其中所述细胞和组织是离体的。在另一些实施方案中，其中所述细胞和组织是在体内的。
另一方面，本申请提供一种用于减少受试者中ANGPTL3 mRNA或蛋白质表达的方法，其包括：向目标受试者施用有效量的前述的ANGPTL3 RNAi剂或前述的药物组合物。
如本申请所用，术语“沉默”、“减少”、“抑制”、“下调”或“敲低基因表达”当提及ANGPTL3基因时，意指当将细胞、细胞群或组织用所述ANGPTL3 RNAi剂处理时，相较于在施用ANGPTL3 RNAi剂之前相同的细胞、细胞群或组织，基因的表达(如通过其中转录ANGPTL3基因的细胞、细胞群或组织中从基因转录的RNA的水平或者从mRNA翻译的多肽、蛋白质或蛋白质亚基的水平测量的)降低。
在某些实施方案中，施用所述ANGPTL3 RNAi剂的受试者中ANGPTL3的基因表达水平和/或mRNA水平，相对于施用ANGPTL3 RNAi剂之前的受试者或未接受ANGPTL3 RNAi剂的受试者减少至少约5％以上，例如5％至98％。受试者中的基因表达水平和/或mRNA水平可在受试者的细胞、细胞群和/或组织中减少。在某些实施方案中，施用所述ANGPTL3 RNAi剂的受试者中ANGPTL3的蛋白质水平，相对于施用ANGPTL3 RNAi剂之前的受试者或未接受ANGPTL3 RNAi剂的受试者减少5％以上，例如5％至98％。受试者中的蛋白质水平可以在受试者的细胞、细胞群、组织、血液和/或其它流体中减少。可以通过本领域已知的任何方法评估基因表达、mRNA或蛋白质水平的减少。ANGPTL3 mRNA水平和/或蛋白质水平的减少或降低在本申请中被统称为ANGPTL3的减少或降低，或抑制或减少ANGPTL3的表达。
引入细胞当提及ANGPTL3 RNAi剂时意指将ANGPTL3 RNAi剂功能性地递送至细胞中。功能性递送意指RNAi剂被递送至细胞并具有预期的生物活性(如，基因表达的序列-特异性抑制)。
以下通过具体实施例对本申请进行进一步描述。
实施例1化合物GENO-Gal-6合成
(1)合成路线
(2)具体合成步骤
1)化合物Int-11-2的制备
将Int-11-1(10g)溶于(140mL)DCM(Dichloromethane)并降温至0℃,滴加TMSCN(Trimethylsilyl cyanide，三甲基腈硅烷(TMSCN))(4.01g)和BF3.Et2O(2.83mL),反应10min。TLC板监测反应(使用Hexane:EtOAc＝5:1,KMnO4显色，原料Rf＝0.3,α构型Rf＝0.28，β构型Rf＝0.27)原料反应完全。反应完全后，将100mL饱和NaHCO3水溶液加入到反应液中，另加入100ml DCM分出有机相，将有机相用饱和食盐水洗一次，使有机相浓缩，然后溶于EtOAc(200mL)，用NaHCO3水溶液(100mL)洗一次，饱和食盐水洗一次，有机相使用无水硫酸钠干燥，浓缩。然后用正向硅胶柱纯化，缓慢增加极性，其中Hexane/EtOAc＝20％时出α构型，25％时出β构型，α构型得到(5.3g)白色固体,β构型得到(3.7g)无色油状。1H NMR(400MHz,DMSO)δ6.03～6.06(dt,1H),5.91～5.94(dt,1H),5.35(dq,1H),5.12(m,1H),4.30(dd,1H),4.30(dd,1H),3.82(ddd,1H),2.10～2.12(2s,6H).
2)化合物Int-11-3的制备
将HCl水溶液(1M,17.2mL)加入到Int-11-2(3.7g，β构型，无色油状)和10％Pd/C(377mg)的悬浮液中,加入乙酸乙酯/2-丙醇/乙醇(2:1:1,共90mL)的混合物中，在氢气(40Psi)下搅拌反应48小时。利用TLC监测反应，通过硅藻土过滤去除催化剂，减压浓缩溶剂。将得到的粗品用甲苯带洗两遍，然后溶于(10mL甲醇中，加入28％NH3.H2O(30mL),室温搅拌16小时，反应液浓缩后，用甲苯乙腈1:1混合溶液(50mL)带洗3次。将上述粗品取1g溶于(40mL)H2O降温至0℃,加入NaHCO3(1.4g),Na2CO3(0.88g),将FmocOSu(2.13g)溶于二氧六环(dioxane)(40mL)溶剂，然后滴加到上述水溶液中，室温反应1小时。TLC监测反应(DCM:MeOH＝10:1,254nm,Rf＝0.5).正向柱纯化，MeOH/DCM＝5％出产物，得Int-11-3(340mg)无色固体。1H NMR(CDCl3):δ7.76-7.78(d,2H),7.58-7.60(d,2H),7.39-7.42(t,2H),7.30-7.34(t,2H),5.12(t,1H),4.42-4.52(m,2H),4.22(t,1H),3.84(br.s,2H). 3.47-3.57(m,3H),3.11-3.24(m,2H),1.30-1.56,1.67-1.72,2.05-2.22(m,4H)。
3)化合物Int-11-4的制备
将Int-11-3(340mg)溶于2ml吡啶，降温至0℃,将DMTrCl(450mg)溶于2ml吡啶后逐滴滴加到上述溶液中，TLC监测，原料反应完全。加5ml水溶液淬灭反应，反向柱C18纯化MeCN/H2O＝80％出产物int-11-4(290mg)。
4)化合物GENO-Int-11的制备
将Int-11-4(700mg,1.02mmol)溶于2mL二氯甲烷，然后将DBU(310mg,2.04mmol)加到上述溶液中，TLC监测，原料反应完全。加5ml饱和Na2CO3溶液淬灭反应，用二氯甲烷20mL萃取，有机相浓缩用DCM:MeOH＝10:1,(254nm,Rf＝0.5)。正向柱纯化，MeOH/DCM＝5％出产物，得GENO-Int-11(550mg)黄色固体。
5)化合物Int-6-1的制备
将Gal-3-5B(1.45g，3.24mmol)溶于无水DMF(10mL)溶剂中，然后加入HATU(1.64g，4.32mmol),3A分子筛(1g)和DIEA(1.07mL，6.47mmol),反应液室温下搅拌30min,然后把GENO-int-11(1g，2.16mmol)溶于DMF(10mL)加入到上述反应液中，反应液在氩气保护下室温搅拌过夜。反应液过滤，通过反向柱C18纯化MeCN/H2O＝60％出产物得到Int-6-1(1.8g)。1H NMR:(400MHz,DMSO-d6)δ7.80(d,J＝9.2Hz,1H),7.71-7.73(m,1H),7.39-7.41(m,2H),7.24-7.30(m,6H),7.18-7.21(m,2H),6.87(d,J＝8.8Hz,4H),5.21(d,J＝3.6Hz,1H),4.95-4.98(m,1H),4.59(d,J＝6.4Hz,1H),4.47(d,J＝8.4Hz,1H),4.00-4.05(m,2H),3.83-3.90(m,1H),3.64-3.73(m,7H),3.23-3.38(m,8H),2.97-3.14(m,3H),2.04-2.14(m,5H),1.98(s,3H),1.89(s,3H),1.77(s,3H),1.64-4.66(m,1H)1.42-1.50(m,4H).
5)化合物Geno-Gal-6的制备
将Int-6-1(2.1g，2.35mmol)溶于DCM(30mL)，加入Tetrazole(33mg,0.47mmol),NMI(77.2mg，0.94mmol)和2g分子筛，反应液用氩气置换三次，在室温下搅拌20min，然后把磷试剂(920.81mg，3.06mmol)溶于少量二氯甲烷加入到反应液中，室温下搅拌1小时。反应液用饱和NaHCO3水溶液洗两次，水洗一次，食盐水洗一次，室温浓缩浓缩，通过反向柱C18纯化MeCN/H2O＝65％出产物得到GENO-Gal-6(1.5g)。1H NMR:(400MHz,CD3CN)δ7.48-7.50(m,2H),7.20-7.36(m,7H),6.83-6.87(m,4H),6.45-6.51(m,1H),5.28(d,J＝3.2Hz,1H),4.98-5.02(m,1H),4.49(d,J＝8.4Hz,1H),3.90-4.12(m,4H),3.24-3.76(m,20H),3.02-3.11(m,1H),2.49-2.59(m,1H),2.36-2.39(m,1H),2.08-2.25(m,9H),1.97(s,3H),1.91(s,3H),1.83(s,3H),1.71-1.74(m,1H),1.46-1.63(m,5H),0.85-1.39(m,20H).
6)化合物Gal-5-1的制备
将1,12-十二烷二酸单苄酯(387mg，1.2mmol)溶于无水DMF(N,N-Dimethylformamide)(10mL)溶剂中，然后加入HBTU(O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate)(546mg，1.44mmol),DIEA(N,N-Diisopropylethylamine)(0.65mL，3.6mmol),HOBT(1-Hydroxybenzotriazole)(324mg，2.4mmol)和GENO-Int-11(560mg，1.2mmol，然后在氩气保护下室温搅拌过夜。反应液通过反向柱C18纯化MeCN/H2O＝70％出产物得到Gal-5-1(200mg)。1H NMR(400MHz,DMSO)δ7.68(t,1H),7.49–7.12(m,14H),6.86(d,4H),5.07(s,2H),4.58(d,1H),3.73(s,6H),3.38(dd,1H),3.28–3.16(m,3H),3.22-2.85(m,3H),2.41–2.25(m,2H),2.11–2.00(m,2H),1.92(dd,1H),1.64(d,1H),1.57–1.41(m,4H),1.27–1.06(m,14H)。
7)化合物Gal-5-2的制备
将Gal-5-1(200mg，0.26mmol)溶于5mL乙酸乙酯中，加入50毫克钯炭和三乙胺(0.11mL，0.78mmol)，反应液在氢气(15psi))，室温下搅拌16小时，反应液过滤浓缩得Gal-5-2(150mg)。
8)化合物Gal-5-3的制备
将Gal-5-2(对应式I-1)(150mg，0.22mmol)溶于5mL无水DMF中，向此溶液中加HBTU(101mg，0.27mmol),DIEA(0.16mL),3A分子筛(1g)和Gal-3-4C(441mg.0.22mmol)。然后上述混合物于室温下搅拌16小时，反应液通过反向柱C18纯化MeCN/H2O＝60％出产物得到Gal-5-3(380mg)。1H NMR(400MHz,DMSO)δ7.91–7.67(m,10H),7.42(d,2H),7.34–7.24(m,6H),7.20(d,1H),6.99(s,1H),6.87(d,4H),5.21(d,3H),4.97(dd,3H),4.60(s,1H),4.48(d,3H),4.09–3.95(m,10H),3.93–3.82(m,3H),3.77–3.66(m,9H),3.61–3.48(m,12H),3.45–3.20(m,16H),3.09–2.95(m,15H),2.27(t,6H),2.10(s,9H),2.05(dd,10H),1.99(s,9H),1.89(s,9H),1.76(d,9H),1.63–1.38(m,24H),1.16(s,14H)。
9)化合物Gal-5-4的制备
将Gal-5-3(370mg，0.15mmol)，丁二酸酐(75mg，0.76mmol),3A分子筛(0.5g) 和DMAP(4-Dimethylaminopyridine)(46mg，0.38mmol)溶于5mL THF(Tetrahydrofuran)中，然后反应液40℃搅拌过夜，反应液通过反向柱C18纯化MeCN/H2O＝40％出产物得到Gal-5-4(220mg)。
10)连接到固相载体上的氨基半乳糖化合物GENO-Gal-5合成
将Gal-5-4(220mg，0.086mmol)悬浮于4ml CH3CN和2mL DMF中，滴加DIEA(0.035mL，0.216mmol)和HBTU(49.07mg，0.129mmol)，室温搅拌5分钟，向反应液中加入氨甲基树脂(99.51mg，100-200目，氨基载量250umol/g，25℃下进行摇床反应，转速220转/分钟，反应16h后过滤，滤饼以DCM淋洗3次，每次30ml，乙腈淋洗3次，每次30ml，30ml正己烷淋洗3次，真空油泵干燥2h，随后加入混合试剂(CapB1,4-二甲氨基吡啶,N-甲基咪唑和乙腈，11.2mL/12.4mg/0.50mL/4.32mL)进行盖帽反应。25℃下置于摇床上，转速220转/分钟，反应16h，反应液过滤，滤饼用乙腈淋洗3次，每次30ml，抽滤至干，真空油泵减压下干燥过夜，得目标产物，GNEO-Gal-5化合物160mg。
实施例2 siRNA的合成
siRNA(具体序列见表1，表2和表3，其中表1为未修饰的序列，表2为已修修饰的序列，表3为缀合配体的序列)的合成过程简要描述如下：于Dr.Oligo 48合成器(Biolytic)上，以Universal CPG载体为起始，根据合成程序逐个连接核苷亚磷酰胺单体，其中2’-F RNA、2’-O-甲基RNA等核苷亚磷酰胺单体原料购自芜湖华仁和上海兆维。采用5-乙基硫-1H-四唑(ETT)作为活化剂(0.6M乙腈溶液)，使用0.22M的PADS溶于1:1体积比的乙腈和三甲基吡啶(上海凌江)混合溶剂中，得到的溶液作为硫化试剂，使用碘吡啶/水溶液(上海凌江)作为氧化剂。
在第一核苷酸序列中，核苷酸单体通过5’-3’-磷酸二酯键相互连接，包含硫代磷酸酯连键和磷酸二酯连键。
对序列进行修饰的具体操作：固相合成完成后，寡核糖核苷酸自该固体支撑物裂解，采用3:1的28％氨水和乙醇溶液在50℃条件下浸泡15小时。然后离心，将上清液转移到另一个离心管中，浓缩蒸发干后，使用C18反向色谱纯化，流动相为0.1M TEAA和乙腈，并使用3％三氟乙酸溶液脱除DMTr。目标第一核苷酸序列收集后冻干，并经LC-MS鉴定为目标产物，再经过UV(260nm)定量。
所得到的单链第一核苷酸序列，根据等摩尔比，按照互补配对，退火，最后所得到的双链siRNA溶于1×PBS或无菌水中，并调整至实验所需浓度备用。
表1，表2和表3的序列中，各符号分别用于表示如下修饰的核苷酸(酯)：
A＝腺苷-3’-磷酸酯
C＝胞苷-3’-磷酸酯
G＝鸟苷-3’-磷酸酯
U＝尿苷-3’-磷酸酯
Am＝2’-O-甲基腺苷-3’-磷酸酯
Ams＝2’-O-甲基腺苷-3’-硫代磷酸酯
Cm＝2’-O-甲基胞苷-3’-磷酸酯
Cms＝2’-O-甲基胞苷-3’-硫代磷酸酯
Gm＝2’-O-甲基鸟苷-3’-磷酸酯
Gms＝2’-O-甲基鸟苷-3’-硫代磷酸酯
Um＝2’-O-甲基尿苷-3’-磷酸酯
Ums＝2’-O-甲基尿苷-3’-硫代磷酸酯
Af＝2’-氟腺苷-3’-磷酸酯
Afs＝2’-氟腺苷-3’-硫代磷酸酯
Cf＝2’-氟胞苷-3’-磷酸酯
Cfs＝2’-氟胞苷-3’-硫代磷酸酯
Gf＝2’-氟鸟苷-3’-磷酸酯
Gfs＝2’-氟鸟苷-3’-硫代磷酸酯
Uf＝2’-氟尿苷-3’-磷酸酯
Ufs＝2’-氟尿苷-3’-硫代磷酸酯
其中：小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；
小写字母s在大写字母中间时表示与该字母s左右相邻的两个核苷酸之间的连接为硫代 磷酸酯基连接；
小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸酯基。
表1靶向ANGPTL3基因的未修饰siRNA编号和序列



表2靶向ANGPTL3基因的修饰siRNA编号和序列






表3缀合配体的靶向ANGPTL3基因的修饰siRNA编号和序列



L96结构式：
Gal-6为实施例1制备的目标产物Geno-Gal-6的简写；
[Gal-6]s[Gal-6]s[Gal-6]结构式：
实施例3 ANGPTL3 RNAi剂的体外筛选
将人类ANGPTL3mRNA序列(登记号NM_014495.4)或其片段从可商购获得的哺乳动物表达载体(Origene)亚克隆到可商购获得的基于报告子的筛选质粒psiCHECK2(Promega)，产生海肾萤光素酶/ANGPTL3融合mRNA。将Huh7细胞(人类肝细胞癌系)以约10,000个细胞/孔以96孔格式铺板。将每种ANGPTL3 RNAi剂(已修饰和未修饰，分别来自表1和表2)以两种浓度(1nM和0.1nM或1nM和0.01nM))与每孔25ng psiCHECK2-ANGPTL3质粒DNA和每孔0.2μL LipoFectamine 2000一起共转染。通过使用双萤光素酶报告子测定(购买自Promega，Madison，WI)测量归一化至同样存在于psiCHECK2质粒上的组成型表达的萤火虫萤光素酶的水平的海肾萤光素酶水平来确定基因敲低(表4)。
如表4所示，90条修饰的序列中的绝大部分(86条)序列在1nM时显示出大于60％的敲低效率，23条修饰序列在0.01nM时显示出大于60％的敲低效率；90条序列中的绝大部分(88条)序列在1nM时显示出大于60％的敲低效率，78条序列在0.01nM时显示出大于60％的敲低效率。
表4.通过双萤光素酶报告子测定确定的ANGPTL3 RNAi剂的体外功效筛选结果


实施例4通过抑制人huh7和Hep3B细胞中ANGPTL3 mRNA表达对非缀合ANGPTL3 RNAi剂的体外筛选
在Huh7或Hep3B细胞中反式转染：向96孔板中的每一个孔中，将每孔4.9μl的Opti-MEM加0.1μl的Lipofectamine RNAiMax(购买自英杰公司，卡尔斯巴德CA.cat#13778-150)添加至5μl的ANGPTL3 RNAi剂，得到混合物。然后将混合物在室温孵育20分钟。将100μl含10000 Huh7或Hep3B细胞的完全生长培养基添加至该siRNA混合物中，孵育48hr后提取RNA并进行纯化。单次剂量实验以1nM ANGPTL3 RNAi剂终浓度进行。使用RNeasy 96试剂盒(购买自凯杰公司，74182)进行总的RNA分离。使用诺维赞cDNA反转录试剂盒和qPCR试剂盒，进行cDNA合成和实时PCR检测(结果见表5)。
表5.在Huh7以及Hep3B细胞中，通过RT-qPCR检测ANGPTL3基因mRNA表达抑制率(％)

实施例5 ANGPTL3 RNA干扰剂的体外筛选
使用与实施例3相同的转染条件在Huh7细胞产生6个浓度的剂量反应，其中ANGPTL3RNAi剂(已修饰)浓度范围为8pM-8nM(检测结果见表6)。
表6.Huh7细胞中，靶向ANGPTL3基因的siRNA活性结果
实施例6 ANGPTL3 RNA干扰剂的体内活性评价
为了评价ANGPTL3 RNAi的体内活性，使用雌性C57 BL/6小鼠。适应环境设施后，根据体重，将小鼠平均分组，每组3只。在给药当天(第0天)，给予小鼠皮下注射ANGPTL3 RNAi溶液，对照组小鼠给予PBS溶液，所有小鼠在颈背部皮下注射给药一次，给药剂量为1mg/Kg，给药体积5ml/kg。给药前3天、给药后第5天、第14天、第21天(仅部分小鼠)和第28天，通过眼眶静脉丛(异氟烷麻醉后)采血用于收集血清，采血结束后，将动物安乐死。
通过使用ELISA方法(mANGPTL3,R&D)检测小鼠血清中mANGPTL3蛋白水平来评价mANGPTL3表达敲低情况。为了归一化，将给定时间点的每只动物的mANGPTL3水平除以在此动物中的预处理水平(Day-3)以确定“归一化至预处理”的表达的比率；然后通过将各只动物的“归一化至预处理”比率除以对照组中所有小鼠的平均“归一化至预处理”比率，将特定时间点的表达归一化至对照组。这就使每个时间点的表达归一化至对照组的表达，归一化之后的具体检测结果如图2所示。
将ANGPTL3 RNAi剂如上所述施用给C57 BL/6小鼠。每只小鼠接受单次皮下注射(SC)剂量1mg/Kg ANGPTL3 RNAi剂溶液，监测血清中的mANGPTL3蛋白水平长达28天。敲低水平和响应持续时间示于表7中。从表7可看出，施用第5天时，9个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于40％的敲低，其中有7个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低；施用第14天时，有11个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于40％的敲低，其中有10个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低，其中有7个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于60％的敲低；施用第28天时，有5个施用的ANGPTL3 RNAi剂对mANGPTL3蛋白水平仍保持大于30％的敲低。
表7.皮下施用1mg/Kg ANGPTL3 RNAi剂后小鼠血清中的相对mANGPTL3水平
实施例7 ANGPTL3 RNA干扰剂的体内活性评价(单剂量)
为了评价ANGPTL3 RNAi的体内活性，使用雌性hANGPTL3 C57 BL/6转基因小鼠。适应环境设施后，在给药前三天收取小鼠血清，根据血清中hANGPTL3水平，将小鼠分成18组，每组3只。在给药当天(第0天)，给予hANGPTL3 C57 BL/6转基因小鼠皮下注射ANGPTL3 RNAi溶液，对照组小鼠给予PBS溶液。所有小鼠在颈背部皮下注射给药一次，给药剂量为1mg/Kg，给药体积5ml/kg。给药前3天、给药当天、给药后第5天和第14天所有小鼠通过眼眶静脉丛(异氟烷麻醉后)采血用于收集血清，部分给药组小鼠在第21天、28天和35天继续采血。采血结束后，将动物安乐死。
通过使用ELISA方法(hANGPTL3,Abcam)检测小鼠血清中hANGPTL3蛋白水平来评价hANGPTL3表达敲低情况。为了归一化，将给定时间点的每只动物的hANGPTL3水平除以在此动物中的预处理水平(Day0)以确定“归一化至预处理”的表达的比率；然后通过将各只动物的“归一化至预处理”比率除以对照组中所有小鼠的平均“归一化至预处理”比率，将特定时间点的表达归一化至对照组。这就使每个时间点的表达归一化至对照组的表达，归一化之后的具体检测结果如图3所示。
将L96缀合的ANGPTL3 RNAi剂如上所述施用给hANGPTL3 C57 BL/6转基因小鼠。每只小鼠接受单次皮下注射(SC)剂量1mg/Kg ANGPTL3 RNAi剂溶液，监测血清中的hANGPTL3蛋白水平长达35天。敲低水平和响应持续时间示于表8中。从表8可看出，施用第5天时，有8个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低，其中有2个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于70％的敲低；施用第14天时，有7个施用 的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低，其中有4个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于70％的敲低；施用第21天时，有7个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于50％的敲低，其中有3个施用的ANGPTL3 RNAi剂1mg/Kg显示出大于70％的敲低；施用第28天时，有4个施用的ANGPTL3 RNAi剂对hANGPTL3蛋白水平仍保持大于50％的敲低；施用第35天时，有4个施用的ANGPTL3 RNAi剂对hANGPTL3蛋白水平仍保持大于50％的敲低。
表8.皮下施用1mg/Kg ANGPTL3 RNAi剂后小鼠血清中的相对hANGPTL3水平
实施例8 ANGPTL3 RNA干扰剂的体内活性评价(多剂量)
为了评价ANGPTL3 RNAi的体内活性，使用雌性hANGPTL3 C57 BL/6转基因小鼠。适应环境设施后，在给药前3天收取小鼠血清，根据血清中hANGPTL3水平，将小鼠分成12组，每组3只。在给药当天(第0天)，给予hANGPTL3 C57 BL/6转基因小鼠皮下注射ANGPTL3 RNAi溶液，对照组小鼠给予PBS溶液。所有小鼠在颈背部皮下注射给药一次，给药剂量为1mg/Kg或者3mg/Kg，给药体积5ml/kg。给药前4天、给药当天、给药后第5天、第14天、第21天和第28天，所有小鼠通过眼眶静脉丛(异氟烷麻醉后)采血用于收集血清，采血结束后，将动物安乐死。
通过使用ELISA方法(hANGPTL3，Abcam)检测小鼠血清中hANGPTL3蛋白水平来评价hANGPTL3表达敲低情况。为了归一化，将给定时间点的每只动物的hANGPTL3水平除以在此动物中的预处理水平(Day0)以确定“归一化至预处理”的表达的比率；然后通过将各只动物的“归一化至预处理”比率除以对照组中所有小鼠的平均“归一化至预处理”比率，将特定时间点的表达归一化至对照组。这就使每个时间点的表达归一化至对照组的表达，归一化后的检测结果如图4A和图4B所示，其中图4A显示了1mg/Kg对应的检测结果，图 4B显示了3mg/Kg对应的检测结果。
将Gal6缀合的ANGPTL3 RNAi剂如上所述施用给hANGPTL3 C57 BL/6转基因小鼠。每只小鼠接受单次皮下注射(SC)剂量1mg/Kg或者3mg/Kg ANGPTL3 RNAi剂溶液，监测血清中的hANGPTL3蛋白水平长达28天。敲低水平和响应持续时间示于表9中。从表9可看出，施用第5天时，ANGPTL3 RNAi剂Geno-2-167M，Geno-2-168M，Geno-2-169M和Geno-2-171M在1mg/Kg和3mg/Kg均显示出大于50％的敲低，其中Geno-2-167M 3mg/Kg均显示出大于80％的敲低，Geno-2-168M，Geno-2-170M和Geno-2-171M 3mg/Kg显示出大于70％的敲低；施用第14天时，ANGPTL3 RNAi剂Geno-2-167M，Geno-2-168M，Geno-2-170M和Geno-2-171M 3mg/Kg均显示出大于80％的敲低；施用第21天时，ANGPTL3 RNAi剂Geno-2-168M和Geno-2-171M 3mg/Kg显示出大于90％的敲低；施用第28天时，ANGPTL3 RNAi剂3mg/Kg仍显示出大于60％的敲低，其中Geno-2-168M，Geno-2-170M和Geno-2-171M 3mg/Kg仍显示出大于70％的敲低，Geno-2-168M，Geno-2-170M和Geno-2-171M 1mg/Kg仍显示出大于60％的敲低。
表9皮下施用1mg/Kg和3mg/Kg ANGPTL3 RNAi剂后小鼠血清中的相对hANGPTL3水平
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (34)

  1. 一种抑制ANGPTL3表达的RNA干扰剂，包含第一核苷酸序列，所述第一核苷酸序列包括SEQ ID NO:1至SEQ ID NO:184中的任一个核苷酸序列或者与之互补的核苷酸序列中的至少12个连续核苷酸，或者与所述至少12个连续核苷酸相差不超过3个核苷酸的序列，所述核苷酸为经修饰或者未被修饰的状态。
  2. 根据权利要求1所述的RNA干扰剂，其特征在于，所述第一核苷酸序列为单链寡核苷酸或双链寡核苷酸。
  3. 根据权利要求1或2所述的RNA干扰剂，其特征在于，所述第一核苷酸序列中的一个或多个核苷酸被修饰以形成经修饰的核苷酸。
  4. 根据权利要求3所述的RNA干扰剂，其特征在于，所述第一核苷酸序列包括表2中经修饰的任意一个核苷酸序列或与之相差不超过3个核苷酸的序列。
  5. 根据权利要求1所述的RNA干扰剂，其特征在于，所述第一核苷酸序列为正义链或反义链。
  6. 根据权利要求1所述的RNA干扰剂，其特征在于，所述第一核苷酸序列包括反义链和正义链的双链结构。
  7. 根据权利要求6所述的RNA干扰剂，其特征在于，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸；优选地，所述正义链的长度为19个核苷酸且所述反义链的长度为21个核苷酸，或所述正义链的长度为21个核苷酸且所述反义链的长度为23个核苷酸，或所述正义链的长度为23个核苷酸且所述反义链的长度为25个；更优选地，所述正义链的长度为21个核苷酸且所述反义链的长度为23个核苷酸。
  8. 根据权利要求6或7所述的RNA干扰剂，其特征在于，还包含具有1至4个未配对核苷酸的突出端，优选地，所述突出端为2个或3个未配对核苷酸。
  9. 根据权利要求8所述的RNA干扰剂，其特征在于，所述突出端位于所述反义链的3'端。
  10. 根据权利要求6所述的RNA干扰剂，其特征在于，所述正义链和/或反义链独立地包含一个或多个经修饰的核苷酸。
  11. 根据权利要求4或10所述的RNA干扰剂，其特征在于，所述修饰的核苷酸相互独立地选自：2'-O-甲基修饰核苷酸、2'-O-甲氧基乙基修饰核苷酸、2'-O-烯丙基修饰核苷酸、2'-烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-氟代修饰的核苷酸、2'-脱氧核苷酸、3'-脱氧-胸腺嘧啶核苷酸、双环核酸、异源核苷酸、EVP、LNA、GNA和UNA 中的至少一种；
    优选地，所述修饰的核苷酸为2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸。
  12. 根据权利要求5或10所述的RNA干扰剂，其特征在于，按照5'末端到3'末端的方向，正义链的第7、9、10和11位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸或2'-脱氧-修饰的核苷酸；
    优选地，按照5'末端到3'末端的方向，正义链的第7、10和11位的核苷酸各自独立地为2'-氟代修饰的核苷酸或2'-脱氧-修饰的核苷酸。
  13. 根据权利要求5或10所述的RNA干扰剂，其特征在于，按照5'末端到3'末端的方向，反义链的第2、4、5、6、8、9、10、12、14和16位的核苷酸部分或全部地、各自独立地为2'-氟代修饰的核苷酸；
    优选地，按照5'末端到3'末端的方向，反义链的第2、6、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、5、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、5、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、4、6、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、6、12、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、6、10、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    还更优选地，按照5'末端到3'末端的方向，反义链的第2、8、9、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸；
    更优选地，按照5'末端到3'末端的方向，反义链的第2、14和16位的核苷酸各自独立地为2'-氟代修饰的核苷酸。
  14. 根据权利要求5或6或10所述的RNA干扰剂，其特征在于，所述正义链和/或反义链独立地包含一个或多个硫代磷酸酯键；优选地，所述正义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键，或者，所述反义链在3'和5'末端的末端核苷酸之间包含两个连续的硫代磷酸酯键。
  15. 根据权利要求6所述的RNA干扰剂，其特征在于，其中所述反义链包括表2中反义链编号栏中任意一个或与其相差不超过3个核苷酸的序列。
  16. 根据权利要求1-15中任一项所述的RNA干扰剂，其中所述RNA干扰剂还包含配体。
  17. 根据权利要求16所述的RNA干扰剂，其中所述配体包括靶向基团，优选地，所述靶向基团包括脱唾液酸糖蛋白受体配体；进一步优选地，所述脱唾液酸糖蛋白受体配体包含半乳糖簇。
  18. 根据权利要求16或17所述的RNA干扰剂，其特征在于，所述配体用于缀合至正义链和/或反义链，优选地，缀合至正义链。
  19. 根据权利要求16-18中任一项所述的RNA干扰剂，所述配体包括如下结构：
    Z-X  (I)，
    其中Z为核苷酸序列的第一接头部分，且Z的通式如(Z-1)所示：
    其中R1是O、S、NR3或者CR3R4,其中R3和R4各自独立地是氢、卤素、取代或未取代的脂族基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的杂环或取代或未取代的环烷基；
    其中R2为-O-，-S-，-NH-，-CH2-，-C(O)-，-OC(O)-，-C(O)O-，-NHC(O)-，-C(O)NH-，-CH2NH-，-CH2O-，-NH-C(O)-CH2-，-C(O)-CH2-NH-，或者-NH(CO)NH-，其中，所述-CH2-还可以任选被选自卤素，烷基，烷氧基，烷氨基的取代基取代；所述a和b相同或者不相同，并且分别选自0到20的整数，优选为1到10的整数。
  20. 根据权利要求19所述的RNA干扰剂，其特征在于，所述配体具有如下结构：
    (I-0)，所述第一接头部分Z与第二接头部分L1连接，所述第二接头部分L1与分支点基团E连接。
  21. 根据权利要求20所述的RNA干扰剂，其特征在于，所述分支点基团E还连接于a个与靶向部分连接的栓系部分L2，所述a为选自0到10的整数，优选1到5的整数。
  22. 根据权利要求19-21中任一项所述的RNA干扰剂，其特征在于，所述配体具有如下 结构：
    通式(II)中：
    其中L4为靶向部分，
    X为核苷酸序列，
    Y是O或S
    其中b,c,d和e分别选自0到10的整数，且b和e不同时为0。
  23. 根据权利要求19-22中任一项所述的RNA干扰剂，其特征在于，所述第二接头部分L1具有以下结构：

    或者
    其中f,g，h和i分别是从1至20的整数，优选为1至10的整数。
  24. 根据权利要求21所述的RNA干扰剂，其特征在于，所述栓系部分L2具有如下结构式：
    其中j,k,l,m,n和o分别是从1至20的整数，优选为1至10的整数。
  25. 根据权利要求16-24中任一项所述的RNA干扰剂，所述配体包括化学式为I-1至I-16 中的至少一种：




    或者结构式为II-1至II-28中的至少一种：










    其中Y为O或者S。
  26. 根据权利要求16所述的RNA干扰剂，所述配体包含N-乙酰基-半乳糖胺，且所述配体连接至siRNA的正义链3'末端或5'末端。
  27. 根据权利要求26所述的RNA干扰剂，所述配体包含一种或多种选自以下的结构：



  28. 根据权利要求16-27中任一项所述的RNA干扰剂，包含表3所示序列中的一种。
  29. 权利要求1-28中任一项所述的RNA干扰剂用于制备药物的用途，其中所述药物用于减少哺乳动物中ANGPTL3 mRNA或蛋白质表达，或者用于预防和/或治疗ANGPTL3表达相关的疾病或病症、或者降低疾病或病症的风险。
  30. 根据权利要求29所述的用途，其特征在于，所述疾病是心脏代谢疾病。
  31. 根据权利要求30所述的用途，其特征在于，所述心脏代谢疾病是高甘油三酯血症、肥胖、高脂血症、异常脂质和/或胆固醇代谢、动脉粥样硬化、II型糖尿病、心血管疾病、冠状动脉疾病、非酒精性脂肪性肝炎、非酒精性脂肪肝病、纯合和杂合家族性高胆固醇血症或他汀类药物抗性高胆固醇血症，肝源性疾病，炎症、心脑血管、心肌梗塞，或代谢疾病；
    优选地，所述心血管代谢疾病包括高脂血症、中风、动脉粥样硬化、血栓形成、冠心病，心卒中，脑卒中或主动脉瓣狭窄。
  32. 根据权利要求29所述的用途，其中所述药物包括药学上可接受的赋形剂，优选地，所述药学上可接受的赋形剂为PBS缓冲液或生理盐水。
  33. 根据权利要求29或32所述的用途，其特征在于，所述药物为组合物，还包含一种或多种另外的治疗剂。
  34. 根据权利要求33所述的用途，其特征在于，所述治疗剂选自他汀类药物、PCSK9 RNAi抑制剂、PCSK9抗体抑制剂和PCSK9小分子抑制剂。
PCT/CN2023/071788 2022-01-11 2023-01-11 抑制angptl3表达的rna干扰剂及其用途 WO2023134705A1 (zh)

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