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WO2020135581A1 - Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés - Google Patents

Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés Download PDF

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WO2020135581A1
WO2020135581A1 PCT/CN2019/128686 CN2019128686W WO2020135581A1 WO 2020135581 A1 WO2020135581 A1 WO 2020135581A1 CN 2019128686 W CN2019128686 W CN 2019128686W WO 2020135581 A1 WO2020135581 A1 WO 2020135581A1
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seq
nucleotide
sirna
chain
nucleotide sequence
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Chinese (zh)
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张鸿雁
高山
康代武
孔丽娜
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Suzhou Ribo Life Science Co Ltd
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Suzhou Ribo Life Science Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to a nucleic acid capable of inhibiting the expression of angiopoietin-like protein 3 (ANGPTL3) gene and compositions and conjugates containing the nucleic acid.
  • ANGPTL3 angiopoietin-like protein 3
  • the present disclosure also relates to the preparation methods and uses of these nucleic acids, compositions and conjugates.
  • Dyslipidemia also known as hyperlipidemia, is a systemic disease in which fat metabolism or functioning abnormally causes plasma lipids to be higher than normal and is seriously threatening the health of patients worldwide.
  • Existing drugs for treating dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probucol, fibrates, and niacin and their derivatives.
  • Angiopoietin-like protein 3 is a secreted protein expressed mainly in the liver, and is named for its genetic structure similar to that of angiopoietin.
  • ANGPTL3 regulates lipid metabolism by binding to adipose tissue and inhibiting the activity of lipoprotein lipase.
  • Low expression of ANGPTL3 can slow down atherosclerosis caused by dyslipidemia. Therefore, if it is possible to silence gene expression at the gene level and block the generation of ANGPTL3, it will undoubtedly be the most ideal treatment.
  • Small interfering RNA siRNA
  • siRNAi can be based on the mechanism of RNA interference (RNAi) to inhibit or block the expression of any gene of interest in a sequence-specific manner to achieve the purpose of treating diseases.
  • siRNA stabilization modification and its delivery system are two key technologies in the development of small RNA drugs.
  • the present disclosure provides an siRNA capable of inhibiting the expression of the ANGPTL3 gene
  • the siRNA contains a sense strand and an anti-sense strand
  • each nucleotide in the siRNA is independently a modified or unmodified core Glycosides
  • the sense strand contains a nucleotide sequence I
  • the antisense strand contains a nucleotide sequence II
  • the nucleotide sequence I and the nucleotide sequence II are at least partially reversed Complementary to form a double-stranded region
  • the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and no more than 3 nucleotide differences
  • the nucleotide sequence II The length of the nucleotide sequence shown in SEQ ID NO: 2 is equal, and no more than 3 nucleotide differences:
  • Za1 is A
  • Za2 is U
  • nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length, and are not more than 3 nucleotides different, and the nucleotide sequence II and SEQ The nucleotide sequences shown in ID NO:62 are equal in length and no more than 3 nucleotide differences:
  • Z b1 is A
  • Z b2 is U
  • the nucleotide sequence I includes a nucleotide Z b3 corresponding to a position Z b1
  • the nucleotide sequence II includes a nucleotide Z b4 corresponding to a position Z b2
  • the Z b4 is the reaction The first nucleotide at the 5'end of the sense strand.
  • the present disclosure provides a pharmaceutical composition containing the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure provides an siRNA conjugate containing the siRNA provided by the present disclosure and a conjugate group conjugated to the siRNA.
  • the present disclosure provides the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing dyslipidemia caused by abnormal expression of the ANGPTL3 gene the use of.
  • the present disclosure provides a method of treating and/or preventing dyslipidemia, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to dyslipidemia Subject.
  • the present disclosure provides a method of inhibiting ANGPTL3 gene expression in hepatocytes, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver Cell contact.
  • the present disclosure provides a kit containing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
  • the siRNA provided by the present disclosure the composition containing the siRNA and the siRNA conjugate have good stability, higher gene inhibitory activity, and/or can significantly reduce blood lipid levels.
  • the siRNA provided by the present disclosure may have higher stability and/or higher activity in vivo.
  • the siRNA conjugate provided by the present disclosure has good stability, and maintains consistent stability both in vitro lysosomal lysate and human plasma.
  • the siRNA conjugates provided by the present disclosure exhibit significant downregulation of blood lipid levels.
  • conjugate 1 and conjugate 5 can continuously and stably reduce blood lipid levels within 49 days of a single administration. After 49 days of administration, siRNA conjugate 1 and conjugate 5 provided by the present disclosure to ANGPTL3 mRNA The inhibition rates reached 84.7% and 78.1% respectively.
  • a single subcutaneous administration of 3 mg/kg conjugate 2 the maximum inhibition rate of triglyceride (TG) is 90.5%, the maximum inhibition rate of total cholesterol (CHO) is 85.1%, 56 days after administration, the The inhibition rate can be maintained above 70%, and the inhibition rate against CHO can be maintained above 54%.
  • the siRNA conjugates provided by the present disclosure show a more excellent gene suppression rate and a stronger ability to lower blood lipids.
  • the maximum TG inhibition rates were 91.7% and 86.4%, respectively, and the CHO maximum inhibition rates were 74.1% and 71.9%, respectively.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure can inhibit the expression of ANGPTL3 gene, effectively treat and/or prevent dyslipidemia caused by overexpression of ANGPTL3 gene, and have good application prospects.
  • Figures 1-2 show the stability of siRNA of the present disclosure in lysosomes in vitro.
  • Figure 3 shows the detection of the stability of the conjugates 1-8 of the present disclosure in human plasma.
  • Figures 4A-4B show the inhibitory effect of conjugates 1 and 5 of the present disclosure on normal mouse BALB/c blood lipid levels.
  • 4C-4D show the inhibitory effect of conjugates 1 and 5 of the present disclosure on the mRNA expression of ANGPTL3 in normal mouse BALB/c liver.
  • 5A-5D show the inhibitory effect of conjugates 1 and 5 of the present disclosure on changes in serum triglycerides and total cholesterol over time within 49 days after a single administration of high-fat model mice.
  • 5E-5F show the inhibitory effect of conjugate 2 of the present disclosure on the change of serum triglyceride and total cholesterol over time within 98 days after a single administration of high-fat model mice.
  • 5G-5J show the inhibitory effects of conjugates 9 and 10 of the present disclosure on changes in serum triglycerides and total cholesterol over time within 98 days after a single administration of high-fat model mice.
  • Figure 6A shows the inhibitory activity of siRNA of the present disclosure in the psiCHECK system in vitro.
  • Figure 6B shows the inhibitory activity of conjugates F1, F2, F5 and F6 of the present disclosure in Huh7 cells in vitro.
  • the sequence of ANGPTL3 mRNA is the sequence shown in Genbank accession number NM_014495.3.
  • target gene used in this disclosure refers to a gene expressing the above-mentioned ANGPTL3 mRNA
  • target mRNA refers to the above-mentioned ANGPTL3 mRNA.
  • capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left side of the letter m is methoxy Modified nucleotides; lowercase letter f means that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; lowercase letter s means between two nucleotides adjacent to the letter s It is a phosphorothioate group connection; P1 means that the adjacent one nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, and the letter combination VP means the letter combination VP A nucleotide adjacent to the right is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), and the letter combination Ps represents a nucleoside adjacent to
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is replaced by fluorine
  • non-fluoro-modified nucleotide refers to A nucleotide or nucleotide analog formed by substitution of a hydroxyl group at the 2'position of a ribose group of a nucleotide with a non-fluorine group.
  • Nucleotide analog refers to a nucleic acid that can replace nucleotides but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus A group of pyrimidine deoxyribonucleotides. Such as isonucleotide, bridged nucleotide (bridged nucleic acid, BNA for short) or acyclic nucleotide.
  • the "methoxy-modified nucleotide” refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group.
  • the expression "complementary” or “reverse complementation” can be used interchangeably and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each different from the other The bases on the pair are paired in a complementary manner.
  • the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) is always matched with the pyrimidine base Cytosine (G) is paired.
  • Each base pair includes a purine and a pyrimidine.
  • adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine
  • the two chains are considered to be complementary to each other and from their complementary chains
  • the sequence of the chain can be inferred from the sequence.
  • mis means in the art that in double-stranded nucleic acids, the bases at corresponding positions are not paired in a complementary manner.
  • substantially reverse complementarity means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementarity” ⁇ Means that there is no more than one base mismatch between the two nucleotide sequences; “fully reverse complementary” means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence, which means that the base type of the nucleotide at the same position has changed in the former compared with the latter, For example, when one nucleotide base in the latter is A, and the corresponding nucleotide base at the same position of the former is U, C, G, or T, it is regarded as one of the two nucleotide sequences There is a nucleotide difference at this position. In some embodiments, when the nucleotide at the original position is replaced with an abasic nucleotide or its equivalent, it may also be considered that a nucleotide difference has occurred at that position.
  • nucleoside monomer refers to The types and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase synthesis of phosphoramidite (unmodified or modified RNA, phosphoramidites, sometimes RNA is also known as Nucleoside phosphoramidites). Phosphoramidite solid-phase synthesis is a method used in RNA synthesis known to those skilled in the art.
  • the nucleoside monomers used in this disclosure are all commercially available.
  • siRNA conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, “conjugate” is Refers to the compound formed by the covalent connection between the various chemical moieties.
  • siRNA conjugate means a compound formed by one or more chemical moieties with specific functions covalently attached to siRNA.
  • the siRNA conjugate of the present disclosure is sometimes simply referred to as "conjugate”.
  • siRNA conjugate should be understood as the general term of siRNA conjugate, the general term of siRNA conjugate shown in formula (305) and formula (307), or formula (305), formula (307), formula (308) SiRNA conjugate shown.
  • a "conjugated molecule” should be understood as a specific compound that can be conjugated to an siRNA through a reaction, ultimately forming an siRNA conjugate of the present disclosure.
  • a dash (“-”) that is not between two letters or between two symbols is used to indicate the point of attachment of a substituent.
  • -C 1 -C 10 alkyl-NH 2 is connected through C 1 -C 10 alkyl.
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, the number is usually 1 to 20 carbon atoms, for example, 1 to 10 carbon atoms, such as 1 to 8 Or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl groups contain straight-chain and branched-chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residues with a certain number of carbons it is intended to cover all branched and straight chain forms with that number of carbons; therefore, for example, "butyl” means including n-butyl, sec-butyl , Isobutyl and tert-butyl; "propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl, and refers to residues that are the same as alkyl but have two points of attachment.
  • alkenyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, which is derived from the adjacent carbon atom of the parent alkyl group Obtained by removing one molecule of hydrogen.
  • the group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1- Group, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and the like.
  • the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8 or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to residues that are the same as alkenyl but have two points of attachment.
  • alkynyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, which is derived from the adjacent carbon atom of the parent alkyl group Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like.
  • an alkynyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to residues that are the same as alkynyl but have two points of attachment.
  • alkoxy refers to an alkyl group of a specified number of carbon atoms connected through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, Sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentoxy etc.
  • the alkoxy group usually has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, ie, contains a ring according to Hückel theory 3. Delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include but are not limited to phenyl, fluorenyl and naphthyl groups.
  • Arylene is a subset of aryl, and refers to residues that are the same as aryl but have two points of attachment.
  • cycloalkyl refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring may be saturated, or have one or more carbon-carbon double bonds.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and cage-like cyclic groups such as norbornane.
  • halogen substituent or “halo” refers to fluoro, chloro, bromo, and iodo, and the term “halogen” includes fluorine, chlorine, bromine, and iodine.
  • haloalkyl refers to an alkyl group as defined above that has a specified number of carbon atoms replaced by one or more, up to the maximum allowable number of halogen atoms.
  • haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise stated in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic system, which may include a fused ring or a bridged ring system.
  • the heteroatom in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized.
  • the heterocyclic group is partially saturated or fully saturated.
  • the heterocyclic group may be connected to the rest of the molecule through any ring atom.
  • heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] disulfonyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidine Group, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithionyl (trithianyl ), tetrahydropyranyl, thiomorph
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, where at least one ring in the ring system is completely unsaturated, ie, contains a cyclic delocalization (4n according to Hückel theory +2) ⁇ -electronic system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepanyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl, benzod
  • hydroxyl protecting groups can be used in this disclosure.
  • the protecting group makes the chemical functional group insensitive to specific reaction conditions, and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule.
  • Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greeneand Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, cited by The above documents are incorporated into this article in their entirety.
  • the protecting group is stable under basic conditions, but can be removed under acidic conditions.
  • non-exclusive examples of hydroxy protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl) xanthene-9-yl (Mox).
  • non-exclusive examples of hydroxy protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include but are not limited to humans, non-human primates (eg, rhesus monkeys or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry .
  • treatment means eradicating or improving the underlying obstacles to be treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder so that an improvement is observed in the subject, although the subject may still suffer from the underlying disorder.
  • prevention and “prevention” are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits.
  • the conjugate or composition may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of the disease, even if a diagnosis of the disease is possible Not yet made.
  • the present disclosure provides an siRNA capable of inhibiting the expression of ANGPTL3 gene.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and it is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the siRNA of the present disclosure contains a sense strand and an anti-sense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, so
  • the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and SEQ ID
  • the length of the nucleotide sequence shown in NO: 1 is equal, and there is no more than 3 nucleotide differences, and the length of the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 are equal, and More than 3 nucleotide differences:
  • Za1 is A
  • Za2 is U
  • position correspondence refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence.
  • the first nucleotide at the 3'end of nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO:1.
  • the sense strand contains only nucleotide sequence I and the antisense strand contains only nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:2.
  • the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at position Za4 , and Za4 is selected from A, C, or G.
  • the nucleotide difference as a difference at a position Z a4, Z a4 is selected from A, C or G.
  • Z a3 and Z a4 is complementary to nucleotides.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two cores There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complement refers to there are no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement It means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:
  • Z a4 is an antisense strand 5 'end of the first nucleotide
  • Z a3 is selected from A, U, G or C
  • Z a4 and Z a3 is complementary to nucleotides; in some embodiments, In, Za3 is U, Za4 is A;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 20-26 nucleotides.
  • the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the length of nucleotide sequence III and nucleotide sequence IV are each independently 1-4 Nucleotides; the nucleotide sequence III is connected to the 5'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, the nucleotide sequence III
  • the length of the nucleotide sequence IV is equal.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleosides
  • the base composition of the acid sequence III is AA, and the base composition of the nucleotide sequence IV is UU; in this case, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV All are 3 nucleotides.
  • the base composition of nucleotide sequence III is CAA, and the base composition of nucleotide sequence IV is UUG;
  • the length ratio of the sense strand is 22/22; alternatively, the lengths of the nucleotide sequences III and IV are each 4 nucleotides.
  • the base composition of the nucleotide sequence III is: CCAA, the base composition of the nucleotide sequence IV is UUGG; at this time, the length ratio of the sense strand and the antisense strand is 23/23.
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides
  • the base composition of the nucleotide sequence III is AA according to the direction from the 5′ end to the 3′ end ,
  • the base composition of the nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and nucleotide sequence IV are the same, and are completely reverse complementary, therefore, the bases of nucleotide sequence III are given, and the bases of nucleotide sequence IV are also It’s ok.
  • the sense strand and the anti-sense strand are different in length, and the siRNA further contains a nucleotide sequence V, and the nucleotide sequence V is 1 to 3 nucleotides in length, connected to the antisense The 3'end of the strand constitutes the 3'overhang of the antisense strand.
  • the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides, and thus, the length ratio of the sense strand and the anti-sense strand of the siRNA provided by the present disclosure may be 19/21, 21/23, or 23 /25.
  • Each nucleotide in the nucleotide sequence V may be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, and at this time, the siRNA of the present disclosure has better mRNA silencing activity.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5
  • the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 6:
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7
  • the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:
  • Z a4 is an antisense strand 5 'end of the first nucleotide
  • Z a3 is selected from A, U, G or C
  • Z a4 and Z a3 is complementary to nucleotides.
  • the siRNA of the present disclosure is siANa1 or siANa2:
  • Antisense chain 5'-AACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 10);
  • Antisense chain 5'-AACAUAGCAAAUCUUGAUUUUGG-3' (SEQ ID NO: 12).
  • the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified nucleotides.
  • the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, core
  • the siRNA of the present disclosure contains at least 1 modified nucleotide.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analog formed by the substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with another group, or having Modified base nucleotides.
  • the modified nucleotide does not cause the function of siRNA to inhibit gene expression to be significantly impaired or lost.
  • the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug DiscoToday, 2008, 13 (19-20): 842-55 can be selected.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester having a modification group
  • at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain of the sense strand and the antisense strand is a phosphate group having a modifying group and/or Or a ribose group with a modifying group.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
  • the inventor of the present disclosure has surprisingly found that the siRNA described in the present disclosure achieves a high balance of plasma stability and gene silencing efficiency in animal experiments.
  • the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, according to the direction from the 5′ end to the 3′ end, the nucleotide sequence I
  • the nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides; according to the direction from the 5'end to the 3'end, the nuclei at positions 2, 6, 14, and 16 of the nucleotide sequence II Glycosides are fluoro-modified nucleotides.
  • the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I, In addition, according to the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides; the fluoride in the nucleotide sequence II There are no more than 7 generations of modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides.
  • the nucleus at position 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I Glycosides are fluoro-modified nucleotides, and the nucleotides in the rest of the sense strand are non-fluoro-modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand ,
  • the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense strand
  • the nucleotides in the remaining positions are non-fluorinated nucleotides.
  • fluoro-modified nucleotide refers to a nucleotide formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with fluorine, which has a structure represented by the following formula (7).
  • Non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analog formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • each non-fluoro-modified nucleotide is independently selected from the group consisting of nucleotides or nucleotide analogs in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with a non-fluoro group One kind.
  • nucleotides formed by the substitution of the hydroxyl group at the 2′ position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides may be selected from 2′-alkoxy-modified nucleotides, 2′- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (8).
  • the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, such as formula (9 ) As shown.
  • the 2'-amino (2'-NH 2 ) modified nucleotide is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is represented by formula (11):
  • Nucleotide analog refers to the ability to replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxygenation A group of ribonucleotides.
  • the nucleotide analog may be an isonucleotide, bridged nucleotide, or acyclic nucleotide.
  • Bridged nucleotides refer to restricted or inaccessible nucleotides.
  • the BNA may contain a five-membered ring, a six-membered ring or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure.
  • the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide.
  • the BNA may be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by the opening of the sugar ring of nucleotides.
  • the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), where UNA is represented by formula (15) and GNA is represented by formula (16):
  • R is selected from H, OH, or alkoxy (O-alkyl).
  • a heteronucleotide refers to a compound formed by changing the position of a base in a nucleotide on a ribose ring.
  • the isonucleotide may be a compound formed by the base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18):
  • Base represents a nucleic acid base, such as A, U, G, C, or T; R is selected from H, OH, F, or a non-fluoro group as described above.
  • the nucleotide analog is selected from one of isonucleotide, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide.
  • the methoxy-modified nucleotide refers to the 2'of the ribosyl group -Nucleotides formed by substitution of hydroxyl groups with methoxy groups.
  • the siRNAs of the present disclosure are siRNAs with the following modifications: in the direction from the 5′ end to the 3′ end, in the sense strand, positions 7, 8, and 9 of the nucleotide sequence I Or the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand In the nucleotide sequence II, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 are fluoro-modified nucleotides, the antisense The nucleotides in the rest of the chain are methoxy-modified nucleotides.
  • the siRNAs of the present disclosure are siRNAs with the following modifications: according to the direction from the 5′ end to the 3′ end, positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA
  • the nucleotides are fluoro-modified nucleotides
  • the nucleotides at the remaining positions of the sense strand of siRNA are methoxy-modified nucleotides
  • the siRNA’s The nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand are fluoro-modified nucleotides
  • the nucleotides in the remaining positions of the antisense strand of siRNA are methoxy Modified nucleotides;
  • the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense of siRNA The nucleotides in the remaining positions of the strand are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the rest of the antisense strand of siRNA are methoxy-modified nucleotides;
  • the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides
  • the sense strand of the siRNA The nucleotides at the rest of the positions are methoxy-modified nucleotides
  • the second, sixth, fourth and fourth The nucleotide at position 16 is a fluoro-modified nucleotide
  • the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.
  • the siRNA provided by the present disclosure is any one of siANa1-M1, siANa2-M1, siANa1-M2, siANa2-M2, siANa1-M3, siANa2-M3:
  • Antisense strand 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmUm-3' (SEQ ID NO: 14);
  • Antisense chain 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 16);
  • Antisense chain 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 18);
  • Antisense chain 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 20);
  • Antisense chain 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 22);
  • Antisense strand 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 24).
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for the ribonuclease in the blood to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided by the present disclosure are phosphate groups having a modifying group.
  • the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom in the phosphate diester bond of the phosphate group with a sulfur atom; in some embodiments, the The phosphate group having a modification group is a phosphorothioate group having the structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.
  • the phosphorothioate group is linked to at least one of the group consisting of the first and second cores at either end of the sense strand or anti-sense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or antisense strand; or any combination of the above.
  • the phosphorothioate group linkage is present at all of the above positions except the 5'end of the sense strand.
  • the phosphorothioate group linkage is present at all of the above positions except for the 3'end of the sense strand.
  • the phosphorothioate group linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is any one of siANa1-M1S, siANa2-M1S, siANa1-M2S, siANa2-M2S, siANa1-M3S, siANa2-M3S:
  • Antisense chain 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 26);
  • Antisense chain 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 28);
  • Antisense chain 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 30);
  • Antisense chain 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 32);
  • Antisense chain 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 34);
  • Antisense strand 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 36).
  • the 5'terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, and fluorine
  • Base represents a nucleic acid base, selected from A, U, C, G, or T.
  • the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification represented by formula (2), and the nucleotide modified with a 5'-phosphate analog is a vinyl phosphate-containing modification
  • the nucleotides shown in formula (3) or phosphorothioate modified nucleotides are shown in formula (5).
  • the siRNA provided by the present disclosure is siANa1-M1P1, siANa2-M1P1, siANa1-M2P1, siANa2-M2P1, siANa1-M3P1, siANa2-M3P1, siANa1-M1SP1, siANa2-M1SP1, siANa1-M2SP1, siANa2- M2SP1, siANa1-M3SP1, siANa2-M3SP1, siANa1U-M1P1, siANa2U-M1P1, siANa1U-M2P1, siANa2U-M2P1, siANa1U-M3P1, siANa2U-M3P1, siANa1U-M1SP1, siANa2UM1P1, siANa2UM1P1 Any one of siANa1U-M3SP1, siANa2U-M3SP1:
  • Antisense strand 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmUm-3' (SEQ ID NO: 38);
  • Antisense strand 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 40);
  • Antisense chain 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 42);
  • Antisense chain 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 44);
  • Antisense chain 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 46);
  • Antisense chain 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:348);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 50);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 52);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 54);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 56);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUm-3' (SEQ ID NO: 58);
  • Antisense chain 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 60);
  • Antisense strand 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 179);
  • Antisense chain 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 181);
  • Antisense chain 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 183);
  • Antisense chain 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:185);
  • Antisense chain 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 187);
  • Antisense chain 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:189);
  • Antisense chain 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 191);
  • Antisense chain 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO:193);
  • Antisense chain 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO:195);
  • Antisense chain 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 197);
  • Antisense chain 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUm-3' (SEQ ID NO: 199);
  • Antisense strand 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmUmsGmsGm-3' (SEQ ID NO: 201).
  • capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that a nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide ; Lowercase letter f means that the nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right of the letter are connected by a phosphorothioate group; P1 means One nucleotide adjacent to the right side of the letter is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • the inventors of the present disclosure have unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high gene suppression activity.
  • the siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (for example, solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customized services.
  • a modified nucleotide group can be introduced into the siRNA described in this disclosure by using a nucleoside monomer with a corresponding modification, a method of preparing a nucleoside monomer with a corresponding modification, and introducing a modified nucleotide group The method of siRNA is also well known to those skilled in the art.
  • the present disclosure provides an siRNA capable of inhibiting the expression of ANGPTL3 gene.
  • the siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and it is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.
  • the siRNA of the present disclosure contains a sense strand and an anti-sense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, so
  • the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and SEQ ID
  • the length of the nucleotide sequence shown in NO: 61 is equal, and there is no more than 3 nucleotide differences, and the length of the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are equal, and More than 3 nucleotide differences:
  • Z b1 is A
  • Z b2 is U
  • nucleotide sequence I includes a nucleotide Z b3 corresponding to a position Z b1
  • nucleotide sequence II includes a nucleotide Z b4 corresponding to a position Z b2
  • the Z b4 is The first nucleotide at the 5'end of the antisense strand.
  • position correspondence refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence.
  • the first nucleotide at the 3'end of nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO: 61.
  • the sense strand contains only nucleotide sequence I and the antisense strand contains only nucleotide sequence II.
  • nucleotide sequence I there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:62.
  • nucleotide sequence II and SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 comprises a difference Z b4 position, and Z b4 is selected from A, C or G.
  • nucleotide difference is the difference at the Z b4 position, and Z 8 is selected from A, C, or G.
  • Z b3 is a nucleotide complementary to Z b4 .
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two cores There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complement refers to there are no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement It means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63
  • nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64:
  • Z b4 is the first nucleotide at the 5′ end of the antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 ; in some embodiments In, Z b3 is U, Z b4 is A;
  • the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 20-26 nucleotides.
  • the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26.
  • the length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the length of nucleotide sequence III and nucleotide sequence IV are each independently 1-4 Nucleotides; the nucleotide sequence III is connected to the 5'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, the nucleotide sequence III
  • the length of the nucleotide sequence IV is equal.
  • the length of the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, according to the direction from the 5′ end to the 3′ end, the nucleosides
  • the base composition of the acid sequence III is UG, and the base composition of the nucleotide sequence IV is CA; in this case, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV All are 3 nucleotides.
  • the base composition of the nucleotide sequence III is GUG, and the base composition of the nucleotide sequence IV is CAC;
  • the length ratio of the sense strand is 22/22; alternatively, the lengths of the nucleotide sequences III and IV are each 4 nucleotides.
  • the base composition of the nucleotide sequence III is: UGUG, the base composition of the nucleotide sequence IV is CACA; at this time, the length ratio of the sense strand and the antisense strand is 23/23.
  • the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UG according to the direction from the 5′ end to the 3′ end
  • the base composition of the nucleotide sequence IV is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
  • nucleotide sequence III and nucleotide sequence IV are the same, and are completely reverse complementary, therefore, the bases of nucleotide sequence III are given, and the bases of nucleotide sequence IV are also It’s ok.
  • the sense strand and the anti-sense strand are different in length, and the siRNA further contains a nucleotide sequence V, and the nucleotide sequence V is 1 to 3 nucleotides in length, connected to the antisense The 3'end of the strand constitutes the 3'overhang of the antisense strand.
  • the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26.
  • the length of the nucleotide sequence V is 2 nucleotides, and thus, the length ratio of the sense strand and the anti-sense strand of the siRNA provided by the present disclosure may be 19/21, 21/23, or 23 /25.
  • Each nucleotide in the nucleotide sequence V may be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, and at this time, the siRNA of the present disclosure has better mRNA silencing activity.
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65
  • the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66:
  • the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67
  • the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:
  • Z b4 is the first nucleotide at the 5′ end of the antisense strand
  • Z b3 is selected from A, U, G, or C
  • Z b4 is a nucleotide complementary to Z b3 .
  • the siRNA of the present disclosure is siANb1 or siANb2:
  • Antisense chain 5'-CCAUUUAGGUUGUUUUCUCCA-3' (SEQ ID NO: 70);
  • Antisense strand 5'-CCAUUUAGGUUGUUUUCUCCACA-3' (SEQ ID NO: 72).
  • the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified nucleotides.
  • the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, core
  • the siRNA of the present disclosure contains at least 1 modified nucleotide.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analog formed by the substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with another group, or having Modified base nucleotides.
  • the modified nucleotide does not cause the function of siRNA to inhibit gene expression to be significantly impaired or lost.
  • the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug DiscoToday, 2008, 13 (19-20): 842-55 can be selected.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester having a modification group
  • at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain of the sense strand and the antisense strand is a phosphate group having a modifying group and/or Or a ribose group with a modifying group.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
  • the inventor of the present disclosure has surprisingly found that the siRNA described in the present disclosure achieves a high balance of plasma stability and gene silencing efficiency in animal experiments.
  • the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, according to the direction from the 5′ end to the 3′ end, the nucleotide sequence I
  • the nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides; according to the direction from the 5'end to the 3'end, the nuclei at positions 2, 6, 14, and 16 of the nucleotide sequence II Glycosides are fluoro-modified nucleotides.
  • the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I, In addition, according to the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides; the fluoride in the nucleotide sequence II There are no more than 7 generations of modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides.
  • the nucleus at position 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I Glycosides are fluoro-modified nucleotides, and the nucleotides in the rest of the sense strand are non-fluoro-modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand ,
  • the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense strand
  • the nucleotides in the remaining positions are non-fluorinated nucleotides.
  • fluoro-modified nucleotide refers to a nucleotide formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with fluorine, which has a structure represented by the following formula (7).
  • Non-fluorine-modified nucleotide refers to a nucleotide or nucleotide analog formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group.
  • each non-fluoro-modified nucleotide is independently selected from the group consisting of nucleotides or nucleotide analogs in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with a non-fluoro group One kind.
  • nucleotides formed by the substitution of the hydroxyl group at the 2′ position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides may be selected from 2′-alkoxy-modified nucleotides, 2′- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy-modified nucleotide is a 2'-methoxy-modified nucleotide, as shown in formula (8).
  • the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl-modified nucleotide, as shown in formula (9).
  • the 2'-amino modified nucleotide is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is represented by formula (11):
  • Nucleotide analog refers to the ability to replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxygenation A group of ribonucleotides.
  • the nucleotide analog may be an isonucleotide, bridged nucleotide, or acyclic nucleotide.
  • Bridged nucleotides refer to restricted or inaccessible nucleotides.
  • the BNA may contain a five-membered ring, a six-membered ring or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure.
  • the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide.
  • the BNA may be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by the opening of the sugar ring of nucleotides.
  • the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), where UNA is represented by formula (15) and GNA is represented by formula (16):
  • R is selected from H, OH, or alkoxy (O-alkyl).
  • a heteronucleotide refers to a compound formed by changing the position of a base in a nucleotide on a ribose ring.
  • the isonucleotide may be a compound formed by the base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18):
  • Base represents a nucleic acid base, such as A, U, G, C, or T; R is selected from H, OH, F, or a non-fluoro group as described above.
  • the nucleotide analog is selected from one of isonucleotide, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide.
  • the methoxy-modified nucleotide refers to the 2'of the ribosyl group -Nucleotides formed by substitution of hydroxyl groups with methoxy groups.
  • the siRNAs of the present disclosure are siRNAs with the following modifications: in the direction from the 5′ end to the 3′ end, in the sense strand, positions 7, 8, and 9 of the nucleotide sequence I Or the nucleotides at positions 5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand In the nucleotide sequence II, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 are fluoro-modified nucleotides, the antisense The nucleotides in the rest of the chain are methoxy-modified nucleotides.
  • the siRNAs of the present disclosure are siRNAs with the following modifications: according to the direction from the 5′ end to the 3′ end, positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA
  • the nucleotides are fluoro-modified nucleotides
  • the nucleotides at the remaining positions of the sense strand of siRNA are methoxy-modified nucleotides
  • the siRNA’s The nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand are fluoro-modified nucleotides
  • the nucleotides in the remaining positions of the antisense strand of siRNA are methoxy Modified nucleotides;
  • the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense of siRNA The nucleotides in the remaining positions of the strand are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, and 14th nucleotide sequences of the nucleotide sequence II of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the rest of the antisense strand of siRNA are methoxy-modified nucleotides;
  • the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides
  • the sense strand of the siRNA The nucleotides at the rest of the positions are methoxy-modified nucleotides
  • the second, sixth, fourth and fourth The nucleotide at position 16 is a fluoro-modified nucleotide
  • the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.
  • the siRNA provided by the present disclosure is any one of siANb1-M1, siANb2-M1, siANb1-M2, siANb2-M2, siANb1-M3, siANb2-M3:
  • Antisense chain 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 74);
  • Antisense chain 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 76);
  • Antisense chain 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 78);
  • Antisense chain 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO:80);
  • Antisense chain 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 82);
  • Antisense strand 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 84).
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for the ribonuclease in the blood to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided by the present disclosure are phosphate groups having a modifying group.
  • the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom in the phosphate diester bond of the phosphate group with a sulfur atom; in some embodiments, the The phosphate group having a modification group is a phosphorothioate group having the structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.
  • the phosphorothioate group is linked to at least one of the group consisting of the first and second cores at either end of the sense strand or anti-sense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or antisense strand; or any combination of the above.
  • the phosphorothioate group linkage is present at all of the above positions except the 5'end of the sense strand.
  • the phosphorothioate group linkage is present at all of the above positions except for the 3'end of the sense strand.
  • the phosphorothioate group linkage is present in at least one of the following positions:
  • the siRNA provided by the present disclosure is any one of siANb1-M1S, siANb2-M1S, siANb1-M2S, siANb2-M2S, siANb1-M3S, siANb2-M3S:
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 86);
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 88);
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 90);
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 92);
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 94);
  • Antisense chain 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 96).
  • the 5'terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides may have the following structure:
  • R is selected from H, OH, methoxy, and fluorine
  • Base represents a nucleic acid base, selected from A, U, C, G, or T.
  • the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification represented by formula (2), and the nucleotide modified with a 5'-phosphate analog is a vinyl phosphate-containing modification
  • the nucleotides shown in formula (3) or phosphorothioate modified nucleotides are shown in formula (5).
  • the siRNA provided by the present disclosure is siANb1-M1P1, siANb2-M1P1, siANb1-M2P1, siANb2-M2P1, siANb1-M3P1, siANab2-M3P1, siANb1-M1SP1, siANb2-M1SP1, siANb1-M2SP1, siANb2- M2SP1, siANb1-M3SP1, siANb2-M3SP1, siANb1U-M1P1, siANb2U-M1P1, siANb1U-M2P1, siANb2U-M2P1, siANb1U-M3P1, siANab2U-M3P1, siANb1U-M1SP1, siANb2Ub1, M1SP1, siANb2Ub1 Any one of siANb1U-M3SP1, siANb2U-M3SP1:
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 98);
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 100);
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 102);
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 104);
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 106);
  • Antisense chain 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 108);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 110);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 112);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 114);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 116);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 118);
  • Antisense chain 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 120);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 203);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 205);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO: 207);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 209);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAm-3' (SEQ ID NO:211);
  • Antisense chain 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmCmAm-3' (SEQ ID NO: 213);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 215);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO:217);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 219);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 221);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsAm-3' (SEQ ID NO: 223);
  • Antisense chain 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmAmsCmsAm-3' (SEQ ID NO: 225).
  • the capital letter C, G, U, A represents the base composition of nucleotides;
  • the lowercase letter m represents that the adjacent one nucleotide on the left side of the letter m is a methoxy-modified nucleotide;
  • the lowercase letter f represents The nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides on the left and right of the letter are phosphorothioate groups;
  • P1 indicates the phase on the right side of the letter
  • the adjacent one nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • the inventors of the present disclosure have unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high gene suppression activity.
  • the siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (for example, solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customized services.
  • a modified nucleotide group can be introduced into the siRNA described in this disclosure by using a nucleoside monomer with a corresponding modification, a method of preparing a nucleoside monomer with a corresponding modification, and introducing a modified nucleotide group The method of siRNA is also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon nanotubes ( carbon nanotubes), mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, polylysine Acid (poly(L-lysine), PLL), chitosan, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly D Type or L type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer (PLGA), poly(aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and poly( One or more of poly(2-dimethyla
  • the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1:( 1-500), in some embodiments, the above weight ratio is 1: (1-50).
  • the pharmaceutical composition may further include other pharmaceutically acceptable auxiliary materials, and the auxiliary materials may be one or more of various preparations or compounds conventionally used in the art.
  • the other pharmaceutically acceptable auxiliary materials may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.
  • the pH buffer may be a trimethylolaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate with a pH of 5.5-8.5 Buffer.
  • the protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure adjusting agent makes the osmotic pressure of the pharmaceutical composition 200-700 milli-osmoles/kg (mOsm/kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the pharmaceutical composition may be a liquid preparation, such as an injection solution; it may also be a lyophilized powder injection, which is mixed with a liquid adjuvant when administered to prepare a liquid preparation.
  • the liquid preparation may be, but not limited to, for subcutaneous, intramuscular, or intravenous administration, but may also be, but not limited to, administration to the lungs by spraying or administration to other organs (eg, liver) by spraying through the lungs.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposome preparation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter may also be referred to as an organic amine), auxiliary lipids, and/or pegylation Lipid.
  • the organic amine, auxiliary lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in CN103380113A (the entirety of which is incorporated herein by reference) or their pharmaceutically acceptable One or more of the accepted salts or derivatives, auxiliary lipids, and pegylated lipids.
  • the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof:
  • Each X 101 and X 102 is independently O, S, NA, or CA, where A is hydrogen or a C 1 -C 20 hydrocarbon chain;
  • Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain lipid Groups, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic groups, substituted or unsubstituted, branched or linear acyl groups, substituted Or unsubstituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer from 1-10;
  • R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
  • g, e and f are each independently an integer of 1-6, "HCC” represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkane The group or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 may be any of the following formula (204)-formula (213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows R 103 and in formula (201) A possible connection point for the nitrogen atom in, where each H at any * position can be replaced to achieve a connection with the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in CN103380113A.
  • the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, an analogue of cholesterol and/or a derivative of cholesterol;
  • the pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
  • the molar ratio between the organic amine, the auxiliary lipid, and the pegylated lipid is (19.7-80): (19.7-80 ): (0.3-50), for example, (50-70): (20-40): (3-20).
  • the particles of the pharmaceutical composition formed from the siRNA of the present disclosure and the above-described amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, and more generally, the liposome
  • the average diameter of the particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70 , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.
  • the weight of the siRNA and all lipids is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18.
  • each component of the pharmaceutical composition may be present independently when sold, and may be present in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure can replace the existing siRNA; In an embodiment, it can be prepared as follows:
  • Organic amine, auxiliary lipid and pegylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the resulting lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, ethanol.
  • the siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an siRNA aqueous solution.
  • the concentration of the buffered salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M, adjust the pH of the buffered salt solution to 4.0-5.5, for example, it can be 5.0-5.2, the amount of the buffered salt solution is such that the concentration of siRNA does not exceed 0.6mg /mL, for example, 0.2-0.4 mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.
  • the lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the liposome preparation after incubation.
  • the volume ratio of lipid solution and siRNA aqueous solution is 1: (2-5).
  • encapsulation rate is not less than 80%
  • particle size is 40-200nm
  • polydispersity index is not higher than 0.30
  • osmotic pressure is 250-400mOsm/kg
  • physical and chemical parameters can be pH value 7.2-7.6
  • encapsulation rate is not less than 90%
  • particle size is 60-100nm
  • more The dispersion index is not higher than 0.20
  • the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution may be performed before, after, or simultaneously with the removal of impurities.
  • Various methods can be used to remove impurities.
  • a phase-cut flow system a hollow fiber column can be used, and ultrafiltration is performed at 100 KDa.
  • the ultrafiltration exchange solution is phosphate buffered saline (PBS) at pH 7.4.
  • PBS phosphate buffered saline
  • sterilization can be performed by filtering on a 0.22 ⁇ m filter.
  • the present disclosure provides an siRNA conjugate containing the above siRNA and a conjugate group conjugated to the siRNA.
  • the conjugation group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker, and the targeting group are sequentially connected.
  • the targeting group is 1-6.
  • the targeting groups are 2-4.
  • the siRNA molecule may be non-covalently or covalently conjugated to the conjugation group, for example, may be covalently conjugated to the conjugation group.
  • the conjugation site of the siRNA and the conjugation group may be at the 3'end or 5'end of the sense strand of the siRNA, or at the 5'end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and conjugation group is at the 3'end of the sense strand of the siRNA.
  • the conjugation group can be attached to a phosphate group, a 2'-position hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugation group can also be attached to the 3'-position hydroxyl group, in which case a 2'-5' phosphodiester bond is used to connect the nucleotides.
  • the conjugation group is usually connected to the phosphate group of the nucleotide; when the conjugation group is connected to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose ring or base.
  • connection methods can be referred to: Muthiah, Manoharan, et.al.
  • the siRNA and the conjugation group can be connected by acid-labile or reducible chemical bonds, which can be degraded under the acidic environment of cell endosomes, thereby making the siRNA into a free state.
  • the conjugation group can be attached to the sense strand of siRNA, thereby minimizing the impact of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group may be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.
  • the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, polylactose, mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by liver parenchymal cells, such as asialoglycoproteins, asialoglycosan residues, lipoproteins (such as high density Lipoprotein, low density lipoprotein, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (such as
  • each ligand described is independently selected from a ligand capable of binding to a cell surface receptor.
  • at least one ligand is a ligand capable of binding to a hepatocyte surface receptor.
  • at least one ligand is a ligand capable of binding to a mammalian cell surface receptor.
  • at least one ligand is a ligand capable of binding to a receptor on the surface of human hepatocytes.
  • at least one ligand is a ligand capable of binding to asialoglycoprotein receptor (ASGPR) on the liver surface.
  • ASGPR asialoglycoprotein receptor
  • the pharmaceutically acceptable targeting group may be any ligand that binds to the asialoglycoprotein receptor on the surface of mammalian hepatocytes.
  • each ligand is independently a asialoglycoprotein, such as asialorosomucoid (ASOR) or asialofetuin (ASF).
  • the ligand is a sugar or a derivative of sugar.
  • At least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar.
  • each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives.
  • each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives Substances, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
  • each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylofuranose, L-xylulose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-furan mannose, ⁇ -D-furan mannose, ⁇ -D-furan mannose, ⁇ -D-mannose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose Galactose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranofuran
  • the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent , Second price, third price, fourth price.
  • the monovalent, bivalent, trivalent, and tetravalent described herein refer to siRNA molecules and conjugation groups containing galactose or N-acetylgalactosamine molecules as targeting groups to form siRNA conjugates.
  • the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the siRNA described in this disclosure when the siRNA described in this disclosure is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in this disclosure is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • a suitable linker for the types of these linkers, targeting groups, and the connection method with siRNA, please refer to the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • a suitable linker may be a structure as shown in formula (301):
  • k is an integer of 1-3;
  • L A is a chain-like portion containing an amide bond having the structure shown in formula (302), and each of the L A is connected to one of the targeting group and the L C portion through ether bonds at both ends thereof. connection:
  • L B having the formula (303) comprises a pyrrolidine N- acyl chain portion shown structure, the linear portion having a carbonyl group at one end thereof and connected with the L C moiety through an amide bond, at the other end It has an oxygen group and is connected to the siRNA through a phosphate bond:
  • L C is a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trishydroxymethylaminomethane.
  • the L C is connected to each of the L A moieties via an ether bond via an oxygen atom. connection, and connected by an amide bond via a nitrogen atom and L B of the portion.
  • L C is a tetravalent methylaminomethane-based tetravalent linking group, connected by -(L A ) 3 trimethylolaminomethane-L B -as a linker
  • the siRNA conjugate formed by N-acetylgalactosamine molecules and siRNA molecules has the structure shown in the following formula (304):
  • the double helix structure represents siRNA.
  • the conjugation site of the siRNA and the conjugation group can be at the 3'end or 5'end of the sense strand of the siRNA, or at the 5'end of the antisense strand, or in the internal sequence of the siRNA.
  • an siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3 is obtained, which may also be referred to as (GalNAc) 3 -siRNA in the following, and its structure is shown in the following formula (305):
  • the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.
  • a suitable linker may be a structure represented by formula (306):
  • l is an integer of 0-3;
  • # Indicates the site on the linker connected to the siRNA through a phosphate bond.
  • the siRNA conjugate has the structure shown in formula (307):
  • the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.
  • WO2015006740A2 describes in detail the preparation methods of various conjugates.
  • the siRNA conjugate of the present disclosure is obtained in a manner well known to those skilled in the art.
  • WO2014025805A1 the preparation method of the structure represented by formula (305) is described, and Rajeev et al. describe the preparation method of the structure represented by formula (307) in ChemBioChem 2015, 16,903-908.
  • the siRNA conjugate has the structure shown in formula (308):
  • n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
  • Each of m1, m2 and m3 is independently an integer selected from 2-10;
  • Each R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently H, or selected from the group consisting of C 1 -C 10 alkyl, C 1 -C 10 haloalkyl and C 1 -C 10 alkoxy;
  • R 3 is a group represented by the formula A59:
  • E 1 is OH, SH or BH 2
  • Nu is siRNA of the present disclosure
  • L 1 may be selected from the group consisting of A1-A26 groups or any combination of connections thereof, wherein the structure and definition of A1-A26 are as follows:
  • Each R' is independently C 1 -C 10 alkyl
  • Each Ra is independently selected from the group consisting of groups of formula A27-A45:
  • Each Rb is independently C 1 -C 10 alkyl; Represents the site where the group is covalently attached.
  • L 1 is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and/or substitutions.
  • the length of L 1 is the number of atoms in the chain connecting two connection points.
  • a ring obtained by replacing the carbon atom of the linear alkylene group (such as a heterocyclylene group or a heteroarylene group) is counted as one atom.
  • each M 1 represents a targeting group, and its definition and selectable range are the same as the above targeting group.
  • each M 1 is independently selected from one of the ligands that has an affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.
  • n1 may be an integer of 1-3 and n3 may be an integer of 0-4 , To ensure that the number of M 1 targeting groups in the conjugate is at least 2; in some embodiments, n1+n3 ⁇ 2, so that the number of M 1 targeting groups is at least 3, thereby This makes it easier for the M 1 targeting group to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the entry of the conjugate into the cell through endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • each of m1, m2, and m3 is independently selected from an integer of 2-10, the spatial position between multiple M 1 targeting groups can be adapted to the M 1 targeting group and the liver surface
  • each of R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, And one of the C 1 -C 10 alkoxy groups does not change the properties of the conjugate of the present disclosure, and can all achieve the purpose of the present disclosure.
  • each of R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently selected from H, methyl and ethyl.
  • each R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is H.
  • R 3 is a group of the structure represented by Formula A59, wherein E 1 is OH, SH, or BH 2. Based on the availability of raw materials for preparation, in some embodiments, E 1 is OH or SH.
  • R 2 The choice of R 2 is to achieve the connection between the N atom on the nitrogen-containing skeleton and A59.
  • nitrogen-containing skeleton refers to a chain-like structure in which carbon atoms to which R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected, and N are interconnected. Therefore, R 2 may be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner.
  • the R 2 group in the case of preparing the siRNA conjugate represented by formula (308) by the process of solid phase synthesis, the R 2 group needs to also contain a linking site to the N atom on the nitrogen-containing backbone And the connection site to the P atom in R 3 .
  • the site connected to the N atom on the nitrogen-containing skeleton in R 2 forms an amide bond with N
  • the site connected to the P atom on R 3 forms a phosphate bond with the P atom
  • R 2 can be B5, B6, B5', or B6':
  • the value range of q 2 may be an integer of 1-10. In some embodiments, q 2 is an integer of 1-5.
  • L 1 is to connect the M 1 targeting group to the N atom on the nitrogen-containing backbone to provide liver targeting for the siRNA conjugate of formula (308).
  • L 1 is selected from one or more linking combinations of groups of Formulae A1-A26.
  • L 1 is selected from one or more connection combinations of A1, A4, A5, A6, A8, A10, A11, and A13.
  • L 1 is selected from a combination of at least 2 of A1, A4, A8, A10, and A11.
  • L 1 is selected from a combination of at least 2 of A1, A8, and A10.
  • L 1 may be 3-25 atoms in length, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, the length of L 1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atom.
  • j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, in some embodiments, j2 is an integer of 3-5.
  • R ' is C 1 -C 4 alkyl, in some embodiments, R' is a methyl, ethyl and isopropyl group of one.
  • Ra is one of A27, A28, A29, A30, and A31. In some embodiments, Ra is A27 or A28.
  • Rb is C 1 -C 5 alkyl, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl.
  • each of j1, j2, R′, Ra, Rb is selected in formulas A1-A26 to achieve the connection of the M 1 targeting group to the N atom on the nitrogen-containing backbone and the M 1 target
  • the spatial position between the directional groups is more suitable for the M 1 targeting group to bind to the asialoglycoprotein receptor on the liver surface.
  • the conjugate has formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412) ), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422)
  • the P atom in Formula A59 can be linked to any possible position in the siRNA sequence, for example, the P atom in Formula A59 can be linked to any nucleotide of the sense or antisense strand of siRNA; In some embodiments, the P atom in Formula A59 is attached to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is attached to the end of the sense or antisense strand of siRNA; in some embodiments, the P atom in formula A59 is attached to the end of the sense strand of siRNA. The terminus refers to the first 4 nucleotides from the one end of the sense strand or the antisense strand.
  • the P atom in formula A59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is attached to the 3'end of the sense strand of siRNA.
  • the siRNA conjugate shown in formula (308) enters the cell, upon unwinding, a separate siRNA antisense strand can be released to block ANGPTL3 mRNA translation
  • the protein process inhibits the expression of angiopoietin-like protein 3 gene.
  • the P atom in Formula A59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5′ position of the nucleotide, the 2′ position of the nucleotide, the 3 of the nucleotide 'Position or nucleotide base.
  • the P atom in Formula A59 may be linked to the 2′ position, 3′ position, or 5′ position of the nucleotide in the siRNA by forming a phosphodiester bond.
  • the P atom in formula A59 is attached to an oxygen atom formed after the 3'hydroxyl of the 3'terminal nucleotide of the siRNA sense strand is dehydrogenated (in this case, the P atom in A59 can also be regarded as siRNA
  • the P atom in the phosphate group contained in ) or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the positive strand of siRNA, or the P in formula A59
  • the atom is connected to the nucleotide by replacing the hydrogen in the 5'hydroxyl group of the 5'terminal nucleotide of the sense strand of the siRNA.
  • the inventors of the present disclosure have unexpectedly discovered that the siRNA conjugate of the present disclosure has significantly improved plasma stability and low off-target effect, while also exhibiting ANGPTL3 mRNA silencing activity that is not significantly reduced, and also has a higher Lipid inhibition. Therefore, in some embodiments, the siRNA in the siRNA conjugate of the present disclosure is shown in Table 1 or Table 2.
  • Table 1 The first siRNA sequence in the conjugate of the present disclosure
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging of the phosphodiester bond or the phosphorothioate diester bond
  • the oxygen atom or sulfur atom has a negative charge, and it may exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ion in the hydroxyl group or the mercapto group may be partially or completely replaced by a cation.
  • the cation may be any cation, such as one of a metal cation, an ammonium ion NH 4 + , and an organic ammonium cation.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations.
  • the alkali metal ion may be K + and/or Na +
  • the cation formed by the tertiary amine may be ammonium ion formed by triethylamine and/or ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form.
  • the non-bridged oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond is at least partially bound to the sodium ion, and the siRNA or siRNA conjugate of the present disclosure uses a sodium salt or a partial sodium salt Form exists.
  • modified nucleotide groups can be introduced into the siRNAs described in this disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
  • the siRNA conjugate represented by formula (308) can be prepared by any reasonable synthetic route.
  • the siRNA conjugate represented by formula (308) can be prepared by a method including the nucleotides of the sense strand and anti-sense strand of the siRNA under the conditions of solid-phase synthesis of phosphoramiditekinds and order, connect the nucleoside monomers in sequence according to the 3'to 5'direction.
  • the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfuration; the sense strand of siRNA is isolated And the antisense strand, annealed, wherein the siRNA is the siRNA of the present disclosure described above;
  • the method further includes contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid phase carrier in the presence of a coupling reaction condition and a coupling reagent to make formula (321)
  • the compound shown is linked to the nucleotide sequence via a coupling reaction.
  • the compound represented by formula (321) is also referred to as a conjugated molecule.
  • R 4 is a group capable of binding to siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R 4 is a group capable of covalently binding to the siRNA represented by Nu. In some embodiments, R 4 is a group capable of being conjugated to any functional group of siRNA represented by Nu through phosphodiester bond through reaction;
  • Each S 1 is independently a group formed by replacing all active hydroxyl groups in M 1 with YCOO- groups, wherein each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, and monofluoromethyl
  • Y is independently selected from methyl, trifluoromethyl, difluoromethyl, and monofluoromethyl
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 and M 1 are as described above.
  • R 4 is to achieve the connection with the N atom on the nitrogen-containing backbone and provide a suitable reaction site for the siRNA conjugate shown in the synthetic formula (308).
  • R 4 includes an R 2 linking group or a protected R 2 linking group, and a functional group that can react with siRNA to form the structure shown in A59.
  • R 4 includes a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu, and a second functional group that can react with a hydroxyl group or an amino group to form a covalent bond or contains The solid carrier supported by the covalent bond.
  • the first functional group is phosphoramidite, hydroxyl, or protected hydroxyl.
  • the second functional group is phosphoramidite, carboxyl, or carboxylate.
  • the second functional group is a solid-phase carrier connected to other parts of the molecule via a covalent bond, the covalent bond being formed by a hydroxyl group or an amino group.
  • the solid phase carrier is connected via a phosphate bond, a carboxylate bond, or an amide bond.
  • the solid support is a resin.
  • the first functional group contains a hydroxyl group, -OR k, or a group represented by formula (C3);
  • the second functional group contains formulas (C1), (C2), (C3), (C1' ) Or (C3'):
  • q 1 is an integer of 1-4
  • X is O or NH
  • M + is a cation
  • R k is a hydroxy protecting group
  • SPS represents a solid phase support
  • the first functional group contains a phosphoramidite group, as shown in formula (C3)
  • the phosphoramidite group can be linked to a hydroxyl group at any position on the nucleotide, such as a hydroxyl group at the 2′ position or
  • the 3'hydroxyl group undergoes a coupling reaction to form a phosphite, and is oxidized or vulcanized to form a phosphodiester bond or a phosphorothioate bond represented by Formula A59, and the conjugate molecule is conjugated to the siRNA.
  • the compound of formula (321) can be conjugated to the nucleotide, without affecting the acquisition of the siRNA conjugate represented by formula (308).
  • the compound of formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and the subsequent During the oxidation or sulfidation process, phosphodiester bond linkages or phosphorothioate linkages are formed, conjugating the compound of formula (321) to siRNA.
  • the first functional group contains a protected hydroxyl group.
  • the second functional group includes a group that can react with a solid support, and the reaction provides a conjugated molecule that includes the solid support.
  • the second functional group contains a carboxyl group, carboxylate or phosphoramidite, as shown in formula (C1), (C2) or (C3), when the second functional group contains a carboxyl group or carboxylate.
  • the compound of formula (321) undergoes an esterification reaction or an amidation reaction with a solid phase carrier, such as a hydroxyl group or an amino group on a resin, to form a conjugated molecule containing the solid phase carrier connected by a carboxylate bond.
  • the compound of formula (321) undergoes a coupling reaction with a universal solid-phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a solid-phase carrier-containing compound connected by a phosphodiester bond Conjugated molecules.
  • a universal solid-phase carrier such as a hydroxyl group on a resin
  • the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or anti-sense strand of the siRNA to which the conjugation group is connected.
  • the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under the coupling reaction conditions.
  • the first functional group contains a hydroxyl group or a protected hydroxyl group
  • the second functional group contains a solid-phase carrier connected by a carboxylate bond, a solid-phase carrier connected by an amide bond, or connected by a phosphate bond
  • the solid phase carrier is shown in formula (C1') or (C3').
  • the carboxylate salt may be represented as -COO - M + , where M + is a cation, for example one selected from a metal cation, an ammonium cation NH 4 + , and an organic ammonium cation.
  • M + is a cation, for example one selected from a metal cation, an ammonium cation NH 4 + , and an organic ammonium cation.
  • the metal ion is selected from one of alkali metal ions, such as K + or Na + .
  • the organic ammonium ion is an ammonium cation or a quaternary ammonium cation formed from a tertiary amine, for example, an ammonium ion formed from triethylamine or N,N-di Ammonium ions formed by isopropylethylamine.
  • the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.
  • R 4 contains a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11'), or (B12'):
  • q 1 is an integer of 1-4
  • q 2 is an integer of 1-10
  • X is O or NH
  • M + is a cation
  • R k is a hydroxyl protecting group
  • SPS represents a solid phase support
  • q 1 is 1 or 2.
  • q 2 is an integer of 1-5.
  • R 4 contains the structure represented by formula (B9) or (B10).
  • R 4 contains the structure represented by formula (B11) or (B12).
  • R k is Tr(trityl), MMTr(4-methoxytrityl), DMTr(4,4′-bismethoxytrityl), TMTr(4 , 4', 4'-trimethoxytrityl) one or more.
  • R k may be DMTr, that is, 4,4′-bismethoxytrityl (4,4′-dimethoxytrityl).
  • L 1 The definition of L 1 is as described above.
  • L 1 is used to connect the M 1 targeting group to the N atom on the nitrogen-containing backbone, thereby providing liver targeting for the siRNA conjugate of formula (308).
  • L 1 comprises any one of A1-A26 or a combination thereof.
  • the first functional group and the optional second functional group can be used to connect the conjugated molecule.
  • the siRNA conjugate represented by formula (308) to any possible position of the nucleotide sequence for example, the conjugate molecule is attached to the end of the nucleotide sequence, and the conjugate molecule is attached to the end of the nucleotide sequence.
  • each S 1 is independently M 1 . In some embodiments, each S 1 is independently a group formed by at least one active hydroxyl group in M 1 protected by a hydroxyl protecting group. In some embodiments, each S 1 is independently a group formed by protecting all of the active hydroxyl groups present in M 1 with a hydroxy protecting group. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group in M 1 .
  • the protected hydroxyl group may be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C 1 -C 10 alkyl and C 6 -C 10 aryl, the C The 1 -C 10 alkyl group and the C 6 -C 10 aryl group are optionally substituted with one or more substituents selected from the group consisting of halogen and C 1 -C6 alkyl group.
  • each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl , Monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C 1 -C 6 alkylphenyl.
  • each S 1 is independently selected from the group consisting of formulas A46-A54:
  • S 1 is formula A49 or A50.
  • each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n- One of propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.
  • the preparation method of the siRNA conjugate represented by formula (308) further includes the following steps: synthesizing another strand of siRNA (for example, when the above-mentioned step synthesizes the siRNA sense strand to which the conjugated molecule is attached, Including the synthesis of antisense strand of siRNA according to solid phase synthesis method, and vice versa), separation of sense strand and antisense strand, and annealing.
  • the solid phase carrier attached to the nucleotide sequence and/or conjugated molecule is cleaved, and the necessary protecting groups are removed (at this time, each S in the compound of formula (321) 1 group is converted to the corresponding M 1 targeting group) to obtain the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand) connected to the conjugated molecule, the sense strand and the antisense strand are annealed
  • a double-stranded RNA structure is formed to obtain the siRNA conjugate represented by formula (308).
  • the method for preparing the siRNA conjugate represented by formula (308) includes the following steps: In the presence of the coupling reaction conditions and the coupling reagent, the compound represented by formula (321) is combined with the sense strand or antisense The first nucleoside monomer at the 3'end of the chain is contacted to connect the compound represented by formula (321) to the first nucleotide in the sequence, under the conditions of phosphoramidite solid-phase synthesis, according to the desired sense strand or The types and sequence of antisense strand nucleotides, the nucleoside monomers are connected sequentially in the direction of 3'to 5'to synthesize the sense strand or antisense strand of siRNA; wherein, the compound represented by formula (321) is contained in R 4
  • the first functional group and the second functional group the first functional group contains a protected hydroxyl group, the second functional group has a structure as shown in formula (C1') or (C3'), before connecting to the first nucleoside monomer, the formula
  • the preparation method of the siRNA conjugate represented by formula (308) includes the following steps: according to the nucleotide types and order of the sense strand or anti-sense strand in the double-stranded siRNA, according to 3'to 5' The direction of the nucleoside monomer is connected in sequence to synthesize the sense strand and the antisense strand.
  • each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfidation to obtain the The sense strand and the antisense strand attached to the solid support; in the presence of the coupling reaction conditions and the coupling reagent, the compound represented by formula (321) and the sense strand attached to the solid support or the solid support
  • the compound of formula (321) is connected to the sense chain or antisense chain, wherein the compound of formula (321) is a formula containing the first functional group in R 4 and the first functional group is a phosphoramidite group ( 321) Compound; deprotection group and cleavage with solid phase carrier, separate purification, to obtain the sense strand or anti-sense strand of siRNA, annealing, wherein the sense strand or anti-sense strand of the siRNA is connected with a conjugation group .
  • the P atom in Formula A59 is attached to the 3'end of the sense strand in the siRNA, and the preparation method of the siRNA conjugate represented by Formula (308) includes:
  • the compound of formula (321) is removed (wherein the compound of formula (321) contains the first functional group and the second functional group in R 4 , the first functional group contains the protected hydroxyl group OR k , and the second functional group has the formula (C1 ') or the compound of the structure shown in (C3')) hydroxyl protecting group R k ; in the presence of coupling reaction conditions and coupling reagents, the deprotected product is contacted with the nucleoside monomer to obtain A nucleoside monomer linked to a solid phase carrier;
  • the sense strand of siRNA is synthesized by the phosphoramidite solid-phase synthesis method in the 3'-5' direction;
  • siRNA conjugate represented by formula (308).
  • the method for removing the protecting group R k in the compound of formula (321) includes contacting the compound of formula (321) with a deprotection reagent under deprotection conditions.
  • Deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent may be selected from trifluoroacetic acid , One or more of trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the compound of formula (321) is 10:1 to 1000:1, and in some embodiments 50:1 to 500:1.
  • any conditions and reagents suitable for the above coupling reaction can be used.
  • the same conditions and reagents as the coupling reaction in the solid phase synthesis method employed can be used.
  • the conditions of the coupling reaction include a reaction temperature of 0-50°C, and in some embodiments 15-35°C.
  • the molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1-1:50, in some embodiments, 1:2-1:5; the molar ratio of the compound of formula (321) and the coupling reagent may be 1:1-1:50, in some embodiments 1:3-1:10, reaction time is 200-3000 seconds, in some embodiments 500-1500 seconds.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, in some embodiments 5-ethylsulfide Radical 1H-tetrazolium.
  • the coupling reaction may be performed in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile.
  • the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • step (2) by the method of phosphoramidite nucleic acid solid-phase synthesis, using the nucleoside monomer prepared by the above steps and connected to the solid-phase carrier through a conjugated molecule, the first synthesis is carried out in the 3'-5' direction The sense strand S of the two siRNA conjugates. At this point, the conjugation group is attached to the 3'end of the resulting sense strand.
  • conditions for the solid-phase synthesis described in steps (2) and (3) include deprotection conditions for nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reactions
  • deprotection conditions for nucleoside monomers include deprotection conditions for nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reactions
  • Conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, sulfidation reaction conditions, types and amounts of vulcanizing reagents use various reagents, amounts and conditions conventionally used in the art.
  • the solid phase synthesis described in steps (2) and (3) may use the following conditions:
  • Nucleoside monomer deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, deprotection reagents can be selected From one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support may be 2:1-100:1, and in some embodiments 3:1-50:1 .
  • the coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, and the molar ratio of the nucleic acid sequence linked to the solid phase carrier to the nucleoside monomer may be 1:1-1:50, in In some embodiments, it is 1:5-1:15; the molar ratio of the nucleic acid sequence linked to the solid phase carrier and the coupling reagent is 1:1-1:100, in some embodiments 1:50-1:80
  • the choice of reaction time and coupling reagent is the same as above.
  • the capping reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 5-500 seconds, and in some embodiments 10-100 seconds, the choice of capping reagents is the same as previously described.
  • the molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is from 1:100 to 100:1, and in some embodiments from 1:10 to 10:1.
  • the capping reagent uses an equimolar amount of acetic anhydride and N-methylimidazole
  • the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence linked to the solid phase carrier can be 1:1:10-10: 10:1, in some embodiments 1:1:2-2:2:1.
  • the oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidation reagent in some embodiments is iodine (In some embodiments, provided in the form of iodized water).
  • the molar ratio of the oxidizing reagent to the nucleic acid sequence attached to the solid phase support in the coupling step may be 1:1-100:1, and in some embodiments 5:1-50:1.
  • the vulcanization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a sulfidation reagent in some embodiments as hydrogenation Xanthogen.
  • the molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid phase carrier in the coupling step is 10:1 to 1000:1, and in some embodiments 10:1 to 500:1.
  • the method After linking all nucleoside monomers and before annealing, the method also includes separating the sense and antisense strands of the siRNA.
  • the method of separation is well known to those skilled in the art, and generally includes cleaving the synthesized nucleotide sequence from the solid phase carrier, removing the protecting group on the base, phosphate group and ligand, purifying and desalting .
  • the synthesized nucleotide sequence can be cleaved from the solid phase carrier, and the protecting groups on the base, phosphate group and ligand can be removed according to the conventional cleaving and deprotecting methods in siRNA synthesis.
  • the obtained nucleotide sequence connected to a solid phase carrier is contacted with concentrated ammonia; during the deprotection process, the protective group YCOO- of the A46-A54 group is converted into a hydroxyl group, and the S 1 group is converted into the corresponding
  • the M 1 group forms the conjugate represented by formula (308).
  • the concentrated ammonia water may be 25-30% by weight ammonia water, and the amount of the concentrated ammonia water may be 0.2ml/ ⁇ mol-0.8ml/ ⁇ mol compared with the target siRNA sequence.
  • the method further includes contacting the nucleotide sequence from which the solid phase carrier has been removed with triethylamine trihydrofluoride to remove the 2'-TBDMS protection.
  • the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxyl group.
  • the amount of triethylamine trihydrofluoride pure product can be 0.4ml/ ⁇ mol-1.0ml/ ⁇ mol. In this way, the siRNA conjugate represented by formula (308) can be obtained.
  • a preparative ion chromatography purification column can be used to complete the nucleic acid purification by gradient elution with NaBr or NaCl; after the product is collected and combined, a reverse phase chromatography purification column can be used for desalting.
  • the non-bridged oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between the nucleotides is basically bound to the sodium ion, formula ( 308)
  • the siRNA conjugate shown basically exists as a sodium salt.
  • a well-known ion exchange method can be used to replace the sodium ion with hydrogen ions and/or other cations to obtain other forms of siRNA conjugates represented by formula (308). The cation is as described above.
  • the purity and molecular weight of the nucleic acid sequence can be detected at any time to better control the synthesis quality.
  • detection methods are well known to those skilled in the art.
  • the purity of nucleic acids can be detected by ion exchange chromatography and the molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).
  • the method of annealing is also well known to those skilled in the art.
  • the synthesized sense chain (S chain) and antisense chain (AS chain) can be simply mixed in equimolar ratio and heated to 70-95°C in water for injection, followed by cooling at room temperature to form a double bond through hydrogen bonding Chain structure.
  • the siRNA conjugate represented by formula (308) can be obtained.
  • the synthesized siRNA conjugate represented by formula (308) can also be characterized by molecular weight detection using methods such as liquid chromatography/mass spectrometry, It is determined that the synthesized siRNA conjugate is the siRNA conjugate represented by the target design formula (308), and the synthesized siRNA sequence is the desired siRNA sequence, for example, the sequence listed in Table 1 or Table 2. one.
  • the compound represented by the formula (321) can be obtained by the following preparation method: the method includes, in an organic solvent, under the conditions of the esterification reaction, and in the presence of a base and an esterification catalyst, the compound represented by the formula (313) and the cyclic The acid anhydride is contacted, ion exchanged, and the compound represented by formula (321) is isolated:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1, S 1 are as described above;
  • R 6 is a group that provides R 4 in formula (321); in some embodiments, R 6 has the structure shown in formula (A61):
  • R i is any group capable of connecting to the N atom on the nitrogen-containing skeleton, connecting to R k O and having a free hydroxyl group, and R k is a hydroxyl protecting group.
  • R 4 contains a first functional group and a second functional group as a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C1) or (C2).
  • the esterification reaction conditions include a reaction temperature of 0-100°C and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions are a reaction temperature of 10-40°C and a reaction time of 20-30 hour.
  • the organic solvent comprises an epoxy-based solvent, an ether-based solvent, a halogenated alkyl-based solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine One or more of them.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the haloalkane solvent is dichloromethane, One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is dichloromethane. Relative to the compound represented by formula (313), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments, succinic anhydride.
  • the molar ratio of the cyclic acid anhydride to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
  • the esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine.
  • the molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
  • the base may be any inorganic base, organic base, or a combination thereof. Considering solubility and product stability, the base may be, for example, a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1-20:1, and in some embodiments, 3:1-10:1.
  • the ion exchange function is to convert the compound of formula (321) into the desired form of carboxylic acid or carboxylate.
  • the method of ion exchange is well known to those skilled in the art, and suitable ion exchange solution and exchange conditions can be used to obtain The conjugated molecule of M + cation will not be detailed here.
  • the ion exchange reaction is performed using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2-0.8M.
  • the triethylamine phosphate solution The concentration of 0.4-0.6M, relative to the compound of formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, in a further embodiment is 4-5L/mol.
  • the compound of formula (321) can be isolated from the reaction mixture using any suitable separation method.
  • the solvent can be removed by evaporation, and then the compound of formula (321) can be separated by chromatographic methods.
  • the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in the subsequent reaction.
  • the method for preparing the compound of formula (321) further includes, under condensation reaction conditions, in an organic solvent, in the presence of a condensing agent and a tertiary amine, the product obtained by the above ion exchange reaction is further combined with The solid-phase carrier of amino group or hydroxyl group is contacted.
  • R 4 contains a first functional group and a second functional group
  • the first functional group contains a hydroxy protecting group
  • the second functional group contains a compound of formula (321) having a structure represented by formula (C1′).
  • the solid phase carrier is one of the carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art.
  • the solid phase carrier may be selected from solid phase carriers containing active hydroxyl or amino functional groups.
  • the solid phase carrier is an amino resin or a hydroxyl resin.
  • the amino or hydroxy resin has the following parameters: particle size 100-400 mesh (mesh), surface amino or hydroxy loading 0.2-0.5mmol/g.
  • the ratio of the compound represented by the formula (321) to the solid phase carrier is 10-400 ⁇ mol of compound per gram of solid phase carrier ( ⁇ mol/g). In some embodiments, the ratio of the compound represented by formula (321) to the solid phase carrier is 50-200 ⁇ mol/g.
  • the organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art.
  • the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the epoxy solvent is dioxane and/or tetrahydrofuran
  • the ether solvent is diethyl ether and/or methyl tert-butyl ether
  • the haloalkane solvent is dichloromethane, One or more of methyl chloride and 1,2-dichloroethane.
  • the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 20-200 L/mol, and in some embodiments, 50-100 L/mol.
  • the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3- Benzazole 4(3H)-one and/or O-benzotriazole-tetramethylurea hexafluorophosphate, in some embodiments, the condensing agent is O-benzotriazole-tetramethyl Urea hexafluorophosphate.
  • the molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in a further embodiment is 1:1-5:1.
  • the tertiary amine is triethylamine and/or N,N-diisopropylethylamine, in some embodiments, N,N-diisopropylethylamine; the tertiary The molar ratio of amine to compound represented by formula (321) is 1:1-20:1, and in some embodiments is 1:1-5:1.
  • the method for preparing the compound of formula (321) may further include, under capping reaction conditions, in an organic solvent, contacting the obtained condensation product with a capping reagent and an acylation catalyst to isolate to obtain formula (321) Compound.
  • the function of the capping reaction is to remove any reactive functional groups that have not yet been completely reacted, so as to avoid unnecessary by-products in subsequent reactions.
  • the conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-10h, and in some embodiments 3-6h.
  • the capping reagent can be a capping reagent used in siRNA solid phase synthesis, and the capping reagent used in siRNA solid phase synthesis is well known to those skilled in the art.
  • the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylmethylimidazole, and in some embodiments, N-methylimidazole Is provided in the form of a mixed solution of pyridine/acetonitrile, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments, 1:3-1:1, and the total volume of pyridine and acetonitrile is The volume ratio of imidazole is 1:1-10:1, in some embodiments 3:1-7:1.
  • the capping reagent 2 is acetic anhydride.
  • the capping reagent 2 is provided in the form of an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is 1:1-1:10, in a further embodiment 1:2-1: 6.
  • the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound of formula (321) is 5 ml/g-50 ml/g, and in some embodiments 15 ml/g- 30ml/g.
  • the ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is 0.5 ml/g-10 ml/g, and in some embodiments 1 ml/g-5 ml/g.
  • the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole.
  • the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine.
  • the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.
  • the acylation catalyst may be selected from any catalyst that can be used for esterification condensation or amidation condensation, such as basic heterocyclic compounds.
  • the acylation catalyst is 4-dimethylaminopyridine.
  • the mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, and in some embodiments is 0.01:1 to 0.1:1.
  • the compound of formula (321) can be isolated from the reaction mixture using any suitable separation method.
  • the compound of formula (321) can be obtained by washing thoroughly with an organic solvent and filtering to remove unreacted reactants, excess capping reagents, and other impurities.
  • the organic solvent is selected from acetonitrile and dichloromethane , Methanol, and in some embodiments acetonitrile.
  • the preparation method of the conjugated molecule represented by formula (321) includes combining the compound represented by formula (313) with phosphorous in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent The acyldiamine is contacted to isolate the compound represented by formula (321). At this time, what is obtained is that R 4 contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C3).
  • the coupling reaction conditions include that the temperature may be 0-50°C, for example 15-35°C, and the molar ratio of the compound of formula (313) to phosphoramidite may be 1:1-1:50, For example, 1:5-1:15; the molar ratio of the compound of formula (313) and the coupling reagent can be 1:1-1:100, for example 1:50-1:80; the reaction time can be 200-3000 seconds , For example, 500-1500 seconds.
  • the phosphorous diamine for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine can be used, which is commercially available or synthesized according to a method known in the art.
  • the coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, for example, 5-ethylthio 1H-tetrazole Azole.
  • the coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile.
  • the amount of the organic solvent is 3-50 L/mol, for example, 5-20 L/mol.
  • the hydroxyl group in the compound of formula (313) reacts with the phosphoramidite to form the phosphoramidite group.
  • the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in the subsequent reaction.
  • the method for preparing the compound of formula (321) further includes the steps of: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the isolated product is further The solid support is contacted. Subsequently, the compound of formula (321) is isolated by cap reaction and oxidation reaction. At this time, what is obtained is a compound of formula (321) in which R 4 contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group has a structure represented by formula (C3′).
  • the solid phase carrier is a solid phase carrier known in the art that can be used for nucleic acid solid phase synthesis.
  • it can be a commercially available universal solid phase carrier after deprotection reaction ( UnyLinker TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, structure shown in formula B80):
  • the deprotection conditions include a temperature of 0-50°C, for example 15-35°C; a reaction time of 30-300 seconds, for example 50-150 seconds.
  • the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid.
  • the deprotection reagent is dichloroacetic acid.
  • the molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1.
  • the coupling reaction conditions and the selection of coupling reagents can be as described above.
  • the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
  • the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping reagent.
  • the selection and amount of capping reagent can be as described above.
  • the oxidation reaction conditions include a temperature of 0-50°C, for example, 15-35°C, a reaction time of 1-100 seconds, for example, 5-50 seconds, and an oxidation reagent, for example, iodine (in some embodiments, iodine Provided in the form of water).
  • an oxidation reagent for example, iodine (in some embodiments, iodine Provided in the form of water).
  • the molar ratio of the oxidizing reagent to the nucleic acid sequence attached to the solid support is 1:1-100:1, for example, it can be 5:1-50:1.
  • R 6 is one of the groups of formula B7 or B8,
  • the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under the amidation reaction conditions, and in the presence of the amidation reaction condensing agent and tertiary amine, the formula (314) The compound is contacted with the compound represented by formula (A-1) or the compound of formula (A-2), and then separated:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 , q 2 and R k each have their own definitions and selectable ranges such as As mentioned earlier.
  • the amidation reaction conditions may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. In some embodiments, the amidation reaction condition is a reaction temperature of 10-40°C and a reaction time of 2- 16 hours.
  • the organic solvent is an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diiso One or more of propylethylamine.
  • the alcoholic solvent is one or more of methanol, ethanol, and propanol in some embodiments, and ethanol in some embodiments.
  • the epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments.
  • the ether solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether.
  • the halogenated alkyl solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane.
  • the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent is 3-50 L/mol, in a further embodiment 3-20 L/mol.
  • the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxy Carboxyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethylurea hexafluorophosphate, in a further embodiment 3-diethoxyphosphoryl-1 , 2,3-Benzazole 4(3H)-one.
  • the molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) may be 1:1-10:1, and in some embodiments 2.5:1-5:1.
  • the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in a further embodiment is N,N-diisopropylethylamine.
  • the molar ratio of the tertiary amine to the compound represented by formula (314) is 3:1-20:1, and in some embodiments is 5:1-10:1.
  • compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means.
  • R k is a DMTr group
  • the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride, and then Then, the compound of formula (A-2) is prepared by reacting with DMTrCl, and the cyclic acid anhydride may be a cyclic acid anhydride having 4-13 carbon atoms, and in some embodiments, 4-8.
  • the compound of formula (313) can also be prepared by sequentially reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl. It is easily understood by those skilled in the art that these modifications do not affect the structure and function of the compound of formula (313), and these modifications are easily realized by those skilled in the art based on the above method.
  • any suitable separation method can be used to isolate the compound of formula (313) from the reaction mixture.
  • the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in the subsequent reaction.
  • the compound represented by formula (314) can be obtained by the following preparation method: the method includes in an organic solvent, in the presence of an amidation reaction condensing agent and a tertiary amine, under the condensation reaction conditions, the formula ( 320) The compound represented by formula (316) is contacted with the compound represented by formula (316), and then separated:
  • n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are as described above.
  • the compound of formula (316) may use, for example, the compound disclosed in J. Am. Chem. Soc. 2014, 136, 16959-16961, or the compound of formula (316) may be prepared by a person skilled in the art by various methods, for example, Certain compounds of formula (316) were prepared with reference to the method disclosed in Example 1 of US Patent No. 8,106,022 B2, and the entire contents of the above documents were incorporated herein by reference in their entirety.
  • the condensation reaction conditions include a reaction temperature of 0-100°C, a reaction time of 0.1-24 hours, and in some embodiments, a reaction temperature of 10-40°C and a reaction time of 0.5-16 hours.
  • the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl
  • the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments
  • the ether solvent is diethyl ether and/or methyl tert-butyl in some embodiments Ether
  • the halogenated alkyl solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in some embodiments
  • the organic solvent is two Methyl chloride.
  • the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
  • the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate, 4-(4,6-dimethoxytriazin-2-yl)
  • DEPBT 3-Benzazole 4(3H)-one
  • O-benzotriazole-tetramethylurea hexafluorophosphate 4-(4,6-dimethoxytriazin-2-yl)
  • -4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole in a further embodiment benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexa
  • benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexa A mixture of fluorophosphates and 1-
  • the tertiary amine may be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and in some embodiments, N-methylmorpholine; the tertiary amine and the formula ( 316)
  • the molar ratio of the compound shown may be 2:1-10:1, in some embodiments 2:1-5:1.
  • any suitable separation method can be used to isolate the compound of formula (314) from the reaction mixture.
  • the solvent can be removed by evaporation, and then the compound of formula (314) can be separated by chromatographic methods.
  • the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in the subsequent reaction.
  • siRNA conjugate of the present disclosure can also be used in combination with other pharmaceutically acceptable excipients, which can be one or more of various formulations or compounds conventionally used in the art.
  • pharmaceutically acceptable excipients which can be one or more of various formulations or compounds conventionally used in the art.
  • pharmaceutically acceptable excipients which can be one or more of various formulations or compounds conventionally used in the art.
  • SiRNA of the present disclosure pharmaceutical composition containing the siRNA and application of conjugate
  • the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for the treatment and/or prevention of dyslipidemia.
  • the present disclosure provides a method for preventing and/or treating dyslipidemia, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in need Subject.
  • the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used for preventing and/or treating dyslipidemia, or for preparing a medicament for preventing and/or treating dyslipidemia.
  • the dyslipidemia refers to dyslipidemia caused by the overexpression of the ANGPTL3 gene in liver cells, usually manifested by increased levels of any or all of the lipids and/or lipoproteins such as triglycerides and cholesterol in the blood, and high levels of blood lipids Highly related to hypertension, cardiovascular disease, diabetes and other pathological conditions.
  • Hypertriglyceridemia is associated with atherosclerosis and can also cause pancreatitis.
  • the dyslipidemias described in this disclosure include but are not limited to hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.
  • administering/administering refers to a method or route by which at least partly localizes the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to a desired site to produce a desired effect
  • the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure are placed into a subject.
  • Suitable administration routes for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in delivery of more siRNA conjugates to specific sites compared to the subject's systemic circulation; whereas systemic administration results in delivery of siRNAs, pharmaceutical compositions, and/or siRNA conjugates of the present disclosure To the subject's basic systemic circulation.
  • an administration method capable of delivering a drug to the liver is adopted.
  • the subject may be administered to the subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including buccal administration and sublingual administration).
  • oral or parenteral routes such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including buccal administration and sublingual administration).
  • the frequency of administration may be daily, weekly, bi-weekly, tri-weekly, monthly, bi-monthly, quarterly, semi-annual, or 1 or more times per year.
  • the dosage of the siRNA, the pharmaceutical composition or the siRNA conjugate described in the present disclosure may be a conventional dosage in the art, and the dosage may be determined according to various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be measured by standard pharmaceutical procedures in cell culture or experimental animals, such as the determination of LD 50 (lethal dose that kills 50% of the population) and ED 50 (in dose response refers to the dose that causes 50% of the maximum response intensity, (In qualitative response, it refers to the dose that can cause 50% of the test subjects to have a positive reaction).
  • the range of human dosage can be derived based on data obtained from cell culture analysis and animal studies.
  • siRNA conjugate the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight, in other embodiments 0.1-15 mg/kg body weight, in other embodiments 0.1-10 mg/kg body weight;
  • pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier The amount of siRNA can be 0.001-50 mg/kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, in some embodiments 0.1-3 mg/kg body weight body weight.
  • the present disclosure provides a method of inhibiting ANGPTL3 gene expression in hepatocytes, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver After cell contact, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are introduced into the hepatocytes, and the purpose of inhibiting the expression of the ANGPTL3 gene in the hepatocytes is achieved through the mechanism of RNA interference.
  • the hepatocytes may be selected from liver cancer cell lines such as Hep3B, HepG2, Huh7 or isolated primary liver cells. In some embodiments, the cells are Huh7 liver cancer cells.
  • the method provided by the present disclosure is used to inhibit the expression of ANGPTL3 gene in cells.
  • the amount of siRNA in the modified siRNA, pharmaceutical composition and/or siRNA conjugate provided is generally such an amount that it is sufficient to reduce the expression of the target gene, and This results in an extracellular concentration of 1 pM to 1 ⁇ M or 0.01 nM to 100 nM or 0.05 nM to 50 nM or 0.05 nM to about 5 nM at the target cell surface.
  • the amount required to achieve this local concentration will vary with various factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or systemic), etc. .
  • the concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.
  • the present disclosure provides a kit comprising an effective amount of at least one of the modified siRNA of the present disclosure, a pharmaceutical composition, and an siRNA conjugate.
  • kits described herein can provide modified siRNA in one container.
  • the kits described herein can include a container that provides a pharmaceutically acceptable excipient.
  • the kit may also contain other ingredients, such as stabilizers or preservatives.
  • the kits described herein may contain at least one other therapeutic agent in a container other than the container that provides the modified siRNA described herein.
  • the kit may include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or excipients or other ingredients, if any.
  • the modified siRNA and a pharmaceutically acceptable carrier and/or adjuvant as well as the modified siRNA, pharmaceutical composition and/or siRNA conjugate and/or conjugate, and/ Or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dried form or lyophilized form.
  • the modified siRNA and pharmaceutically acceptable carriers and/or excipients as well as the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable excipients are substantially pure and/or Sterile.
  • sterile water may be provided in the kit of the present disclosure.
  • reagents and media used in the following examples are all commercially available products, and the operations of nucleic acid electrophoresis and real-time PCR used are all described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) Method.
  • Huh7 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences and cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 1% non-essential amino acids (NEAA, Corning) at 37°C Incubate in an incubator with 5% CO 2 /95% air.
  • DMEM complete medium Hyclone
  • FBS fetal bovine serum
  • NEAA non-essential amino acids
  • siRNA conjugate against ANGPTL3 gene or siRNA siRNA conjugate as a negative control
  • Lipofectamine TM 2000 (Invitrogen) as the transfection reagent, please refer to the instructions provided by the manufacturer for specific operations .
  • the animal models used are as follows:
  • BALB/c mice 6-8 weeks old, purchased from Beijing Viton Lihua Laboratory Animal Technology Co., Ltd.;
  • APOC3 transgenic mice B6; CBA-Tg (APOC3) 3707 Bres/J, purchased from Jackson Laboratory, USA;
  • conjugates 1, 9, and 3 (hereinafter, also referred to as L10-siANa1M3SVP, L10-siANa1M3Sp, and L10-siANa1M3S, respectively) were synthesized.
  • the aforementioned conjugate is a conjugate formed by conjugating L-9 conjugate molecules with siRNAs numbered siANa1M3SVP, siANa1M3Sp and siANa1M3S. See Table 4 for the sequence of siRNA conjugated in this conjugate.
  • L-10 compound was synthesized:
  • GAL-1 N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol
  • 100.0g GAL-1 N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol
  • 100.0g GAL-1 N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol
  • step (1-1-1b) GAL-3 (26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1,2-dichloroethane, and dried 30g molecular sieve powder, then add 9.0g 5-hexene-1-ol (CAS No. 821-41-0, purchased from Adamas-beta, 89.9mmol), stir at room temperature for 30 minutes, add under ice bath and nitrogen protection 9.08g TMSOTf (40.9mmol), the reaction was stirred at room temperature overnight.
  • 9.0g 5-hexene-1-ol CAS No. 821-41-0, purchased from Adamas-beta, 89.9mmol
  • GAL-4 (14.9g, 34.7mmol,) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml of dichloromethane and 77ml of acetonitrile, and 103ml of deionized water and 29.7g were added respectively Sodium periodate (CAS No. 7790-28-5, purchased from Aladdin Company, 138.8 mmol), stirred for 10 minutes in an ice water bath, and added ruthenium trichloride (CAS No. 14898-67-0, purchased from Anai Ji Company, 238 mg, 1.145 mmol), reacted at room temperature overnight.
  • the reaction solution was diluted with 300 ml of water and stirred, and saturated sodium bicarbonate was added to adjust the pH to about 7.5.
  • the organic phase was separated and discarded.
  • the aqueous phase was extracted three times with dichloromethane, 200 ml each time, and the organic phase was discarded.
  • the pH of the aqueous phase was adjusted to about 3 with citric acid solid, and extracted three times with dichloromethane, 200 ml each time.
  • the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a white foamy solid product GAL-5 6.85g .
  • step (1-1-2) Combine L-8 (40g, 27.09mmol, obtained from multiple batches of products) obtained in step (1-1-2) and A-1 (41.418g, 81.27) obtained in step (1-1-3a) mmol), dissolve in 271ml of dichloromethane, add 3-diethoxyphosphoryl-1,2,3-benzazole 4(3H)-one (DEPBT) (24.318g, 81.37mmol), then add diisocyanate Propylethylamine (21.007g, 162.54mmol), stirred at 25°C for 1.5h, washed the organic phase with 800ml of saturated sodium bicarbonate, the aqueous phase was extracted three times with dichloromethane, 50ml each time, and washed with 150ml of saturated saline The organic phase and the aqueous phase were extracted once with 50 ml of dichloromethane.
  • DEPBT 3-diethoxyphosphoryl-1,2,3-benzazole 4(3H)-one
  • the L-10 compound was prepared by attaching the L-9 conjugated molecule to a solid support.
  • CapA and CapB are capping reagent solutions
  • CapA is a 20 volume% N-methylimidazole pyridine/acetonitrile mixed solution, and the volume ratio of pyridine to acetonitrile is 3:5
  • CapB is a 20 volume% acetic anhydride acetonitrile solution.
  • the difference between the sense strand of conjugate 1 and the sense strand of conjugate 9 or 3 is only that the last nucleotide at the 3'-end is different, and the last nucleotide at the 3'-end of the conjugate 1 is a base
  • the base A, the last nucleotide at the 3'-end of the conjugate 9 or 3 sense strand is the base U.
  • the preparation methods of conjugates 1, 9 and 3 are the same except that the nucleoside monomers initially synthesized are different.
  • the L-10 compound prepared by the above steps is used to start the cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides.
  • Each connected nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfidation.
  • phosphate ester connection between two nucleotides when connecting the latter nucleoside monomer, it includes deprotection, coupling, capping, and oxidation four-step reaction.
  • phosphorothioate connection between two nucleotides when connecting the latter nucleoside monomer, it includes four steps of protection, coupling, capping and sulfidation.
  • the synthesis conditions are given as follows:
  • the nucleoside monomer is provided in a 0.1M acetonitrile solution.
  • the deprotection reaction conditions are the same at each step, that is, the temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is dichloroacetic acid in dichloromethane (3% v/v), the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.
  • the coupling reaction conditions are the same in each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence linked to the solid phase carrier to the nucleoside monomer of 1:10, and a molar ratio of the nucleic acid sequence linked to the solid phase carrier and the coupling reagent
  • the ratio is 1:65
  • the reaction time is 600 seconds
  • the coupling reagent is a 0.5M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).
  • the capping conditions are the same at each step, including a temperature of 25°C and a reaction time of 15 seconds.
  • the oxidation reaction conditions are the same in each step, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidation reagent of iodine water with a concentration of 0.05M.
  • the molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling step is 30:1.
  • the conditions of the vulcanization reaction in each step are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfidation reagent of hydrogenated xanthan.
  • the molar ratio of the sulfurizing reagent to the nucleic acid sequence connected to the solid support in the coupling step is 120:1.
  • the cleavage and deprotection conditions are as follows: the synthetic carrier-linked nucleotide sequence is added to ammonia water with a concentration of 25wt%, the amount of ammonia water is 0.5ml/ ⁇ mol, the reaction is performed at 55°C for 16h, the liquid is removed, and the residue is concentrated in vacuo to dry.
  • IEX-HPLC Ion-exchange chromatography
  • LC-MS liquid-mass spectrometry
  • Detection Purity is detected by ion exchange chromatography (IEX-HPLC); molecular weight is analyzed by liquid-mass spectrometry (LC-MS). The measured value is consistent with the theoretical value, indicating that the synthesized is the antisense strand AS with the target sequence.
  • VP-Um vinyl phosphate modified 2'-methoxy modified uracil nucleoside monomer
  • VP-U-2 molecule was synthesized:
  • the antisense strand of conjugate 9 differs from the antisense strand of conjugate 1 only in that the first nucleotide modification at the 5'-end is different.
  • the last connected nucleoside monomer is 2'-methoxy modified adenine nucleoside monomer (Am), which is then deprotected, coupled, capped, and oxidized.
  • the step reaction connects the CPR-I monomer (Suzhou Gema, Cat# 13-2601-XX) to the 5'end of the antisense strand to form a 5'-phosphate modification.
  • IEX-HPLC Ion-exchange chromatography
  • LC-MS Liquid-mass spectrometry
  • the antisense strand of conjugate 3 differs from the antisense strand of conjugate 1 only in the first nucleotide modification at the 5'-end.
  • the last nucleoside monomer is 2'-methoxy modified adenine nucleoside monomer (Am).
  • IEX-HPLC Ion-exchange chromatography
  • LC-MS Liquid-mass spectrometry
  • conjugate 1 the S chain and AS chain were dissolved in water for injection to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, and cooled at room temperature to obtain the annealed product, lyophilized, Get lyophilized powder.
  • Use ultrapure water (Milli-Q ultrapure water meter self-made, resistivity 18.2M ⁇ *cm (25 °C)) to dilute the conjugate to a concentration of 0.2mg/mL, using liquid-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier), for molecular weight detection.
  • LC-MS liquid-mass spectrometry
  • the measured value is consistent with the theoretical value, indicating that the synthesized conjugate 1 is the double-stranded nucleic acid sequence with the L-9 conjugated molecule designed by the target.
  • conjugates 9 and 3 The same method was used to prepare conjugates 9 and 3, except that the sense strands of conjugates 9 and 3 prepared above were used instead of the sense strands of conjugate 1, and the conjugates 9 and 3 prepared above were used.
  • the antisense strand replaces the antisense strand of conjugate 1, and the molecular weights of the obtained conjugates 9 and 3 are detected respectively.
  • the measured value is consistent with the theoretical value, indicating that the synthesized conjugate is the target design with L-9 conjugate Synthetic double-stranded nucleic acid sequence.
  • the structures of conjugates 1, 9 and 3 are shown in formula (403).
  • conjugates 2, 4-8, and 10 were synthesized, except that the siRNA shown in Table 4 corresponded to conjugates 2, 4-8, and 10, respectively. sequence.
  • comparative conjugate 1 was synthesized, and the sequence of the siRNA conjugated in the conjugate is shown as the sequence numbered (GalNAc) 3 -ANG65695 in Table 4.
  • the conjugate has the same structure as the compound AD-65695 in WO2016168286A1.
  • the compound 30 was synthesized according to the method described in Example 17 of WO2014025805A1, that is, containing the linker-(L A ) 3 trishydroxymethylaminomethane-L B -as described above and N-acetylgalactose as a targeting group
  • Conjugation molecules of amine molecules (wherein each L A can be connected to one N-acetylgalactosamine molecule, and thus one linker can be connected to three N-acetylgalactosamine molecules) are referred to as (GalNAc) 3 conjugated molecules
  • the synthetic chemical reaction formula and the structure of (GalNAc) 3 conjugated molecule are as follows:
  • the conjugated molecule connected to the solid phase carrier was prepared, except that the (GalNAc) 3 conjugated molecule was used instead of the L-9 conjugated molecule to obtain the connection (GalNAc) 3 conjugated molecule on solid support.
  • Comparative Conjugate 1 was prepared by the same method as steps (1-2), (1-3C) and (1-4) in Preparation Example 1, except that: 1) obtained in step (3-2) The compound serves as a starting point to start the synthesis of the sense strand; 2) The conjugated siRNA has the sequence shown in Table 4 as (GalNAc) 3 -ANG65695.
  • LC-MS Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier
  • the measured value is consistent with the theoretical value, and it is determined that the synthesized conjugate is the compound of the target design, and its structure is shown in formula (305).
  • siRNA sense strand and anti-sense strand listed in Table 5 were obtained by solid phase synthesis method, DEPC water was used to dissolve an equimolar mixture of sense strand and anti-sense strand, and then annealed to form an siRNA double strand.
  • conjugates F1-F8 were synthesized, and the sequence of siRNA conjugated in the conjugate is shown in Table 4.
  • the FIN-2 conjugated molecule was synthesized according to the following process route.
  • PRO-6 L-hydroxyproline, CAS No. 51-35-4, purchased from Angie, 22.4mmol
  • 22.5ml 1,4-dioxane (1,4-dioxane Ring, CAS No.: 123-91-1)
  • 34ml of 10% (w/w) aqueous solution of Na 2 CO 3 in the state of suspension
  • 6.95g of Fmoc-Cl (chloroformic acid-9-fluorenylmethyl) Ester CAS No.: 28920-43-6, purchased from Angie, 26.8 mmol
  • 34 ml of 1,4-dioxane was dissolved in 34 ml of 1,4-dioxane, added to the above suspension under an ice bath, and naturally raised to room temperature to react overnight.
  • PRO-7 (22.2mmol) was dissolved in 80ml THF (CAS number: 109-99-9), the oil bath was heated to 65 °C, 36.6ml 2mol/L BH 3 -Me 2 S was added under reflux
  • the THF solution (CAS No. 13292-87-0, purchased from Bellingwell, 73.2 mmol) was continued to reflux for 3 hours. Pour out the reaction solution, dissolve the remaining solid with methanol, add methanol with stirring until the reaction solution is gas-free and continue to stir for 30 minutes. After distilling off the solvent under reduced pressure, purify it three times with petroleum ether to obtain white solid product PRO-87.1g.
  • GAL-5 (4.5g, 10mmol) obtained according to the method described in (1-1-1) was dissolved in 40ml DMF, and 3.9g DIEA (N,N-diisopropylethylamine, CAS number: 7087-68-5, purchased from Aladdin, 30mmol) and 3.8g HBTU (benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, CAS number: 94790-37- 2.
  • the silica gel column was pre-alkalized with pyridine and loaded. It was eluted with a solution of 1 volume% triethylamine and 1 volume% methanol in dichloromethane (DCM). The product eluate was collected and decompressed. The solvent was evaporated to dryness to obtain 6.5 g of light yellow foamy solid product FIN-1.
  • the silica gel column was pre-alkalized with pyridine and the crude product was dissolved in DCM and loaded with ethyl acetate. The product eluent was collected and the solvent was distilled off under reduced pressure to obtain a colorless syrup-like crude product. 4.5g. The crude product was dissolved with 50% by volume of acetonitrile aqueous solution until completely dissolved, with C-18, 330g, A medium-pressure purification column was used to purify the sample. The column was first basified with a 1% by volume pyridine in acetonitrile. The product peak was collected by gradient elution. The solvent was distilled off under reduced pressure to obtain 2.2 g of white powder product FIN-2 conjugated molecule. 31 P NMR (162 MHz, CDCl 3 ) ⁇ 148.04, 147.94, 147.62, 147.19, phosphorus spectrum purity 92%; C18RP-HPLC purity 90.54%.
  • the FIN-2 conjugated molecule obtained in step (11-1-3) was connected to a universal solid phase carrier (UnyLinker TM loaded through three cycles ) Solid Supports), so that the conjugation group (FIN_FIN_FIN) is connected to the 3'end of the RNA sense strand.
  • a universal solid phase carrier UnyLinker TM loaded through three cycles ) Solid Supports
  • the FIN conjugated molecules connected to the solid phase carrier are obtained; the FIN conjugated to the solid phase carrier is removed
  • the hydroxyl protecting group DMTr on the coupling molecule is coupled with the FIN-2 conjugated molecule to perform capping and oxidation reactions, and repeat the above deprotection-coupling-cap-oxidation steps to connect the third FIN -2 Conjugate the molecule to obtain the conjugation group (FIN_FIN_FIN) attached to the solid support.
  • reaction conditions of deprotection, coupling, capping, oxidation, the amount of solvent and reagents are the same as the nucleic acid solid phase synthesis method described in the previous step (1-2).
  • the title conjugate was prepared by the same method as steps (1-2), (1-3A) or (1-3C) and (1-4) in Preparation Example 1, except that: 1) 11-2) The obtained compound is used as a starting point to start the sense strand synthesis; 2)
  • the conjugated siRNA has the sequence shown in Table 4 corresponding to the conjugates F1-F8.
  • the siRNA solution or siRNA conjugate solution refers to a solution of a desired concentration obtained by dissolving siRNA or siRNA conjugate with DEPC water.
  • the siRNA solution or siRNA conjugate solution refers to a solution of the desired concentration obtained by dissolving the siRNA or siRNA conjugate in 1 ⁇ PBS (pH 7.4) buffer.
  • Experimental Example 1-1 Stability testing of siRNA in lysosomes.
  • Reference sample preparation without lysosomal lysate treatment Take 1.5 ⁇ l of siRNA 3, 4, 7 or 9 solution (20 ⁇ M) each and mix with 7.5 ⁇ L sodium citrate aqueous solution (pH 5.0) and 1 ⁇ L deionized water, Add 30 ⁇ L of 9M urea solution to denature, then add 8 ⁇ L of 6 ⁇ loading buffer to mix, immediately freeze in -80°C refrigerator to stop the reaction, and obtain each reference sample.
  • Each siRNA reference sample is labeled Con in the electropherogram.
  • a 16% by weight non-denatured polyacrylamide gel was prepared, and 20 ⁇ l of each of the above test sample and the reference sample was applied to the above gel. After electrophoresis at 20 mA constant current for 10 min, electrophoresis was continued at 40 mA constant current for 30 min. After the electrophoresis was completed, the gel was placed on a shaker and stained with Gelred dye (BioTium, Catalog No. 13G1203) for 10 min. Observe and take pictures with gel imaging. The results are shown in Figure 1.
  • the modified siRNA provided by the present disclosure can stably exist in a mouse-derived lysosome for at least 48 hours without degradation.
  • the modified siRNA provided by the present disclosure can stably exist in a mouse-derived lysosome for at least 6 hours without degradation.
  • Experimental Example 1-2 Detection of the stability of siRNA conjugate in human plasma.
  • siRNA or siRNA conjugate concentrations are 20 ⁇ M, 12 ⁇ l, the conjugate is based on the amount of siRNA
  • human plasma Human plasma, PBS dilution
  • 10 ⁇ L samples were taken at 0, 2, 4, 6, 24, 48, and 72 hours respectively, immediately subjected to quick freezing in liquid nitrogen, and frozen in a refrigerator at -80°C. After sampling at each time point, the frozen samples were diluted 5 times with 1 ⁇ PBS (pH 7.4), and 10 ⁇ L was taken from each dilution to prepare test samples.
  • siRNA conjugate provided by the present disclosure has not been degraded in human plasma until 72h, showing excellent stability in human plasma.
  • This experimental example investigates the inhibitory rate of conjugates 1 and 5 on ANGPTL3 mRNA in liver tissues and the effect on blood lipids in normal mouse BALB/c.
  • mice BALB/c normal mice of 6-8 weeks old were randomly divided into groups of 6 and each group was given conjugates 1, 5 and PBS. All animals calculated the dose according to their body weight and used a single subcutaneous injection.
  • the dose of siRNA conjugate (based on the amount of siRNA) was 3mg/kg (also labeled as 3mpk) and 0.3mg/kg (also labeled as 0.3mpk) Two dose groups, the administration volume is 10mL/kg.
  • Each siRNA conjugate is provided in an aqueous PBS solution, and the drug concentration to which the conjugate should be configured is calculated according to the dose and volume administered.
  • the animals were bled orbitally and blood serum was collected to detect the serum lipid level. All mice were sacrificed on the 7th day after the administration, and the liver was collected to detect the expression of ANGPTL3 mRNA in the liver.
  • mice in each group on the 7th day after administration is shown in FIGS. 4A-4B relative to the blood lipid content before administration.
  • the tested siRNA conjugate can significantly reduce the blood lipid level of normal mice.
  • mice were sacrificed 7 days after administration, and the liver was collected and stored with RNA (Sigma Aldrich); then the liver tissue was homogenized with a tissue homogenizer, and then extracted with Trizol (Thermo Fisher) according to the standard operating procedures for total RNA extraction Total liver RNA was obtained.
  • Real-time fluorescence quantitative PCR was used to detect the expression level of ANGPTL3 mRNA in liver tissue, specifically: using reverse transcription kit (Promega Company, Catalog No. A3500) to reverse transcribe cDNA according to the operation method of its instructions. Using 2 ⁇ Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0956) kit, using cDNA as a template, the ANGPTL3 mRNA expression was detected according to the steps of the instructions.
  • the PCR primers used to amplify ANGPTL3 and GAPDH as internal reference genes are shown in Table 6.
  • the inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate is (1-ANGPTL3 mRNA expression) ⁇ 100%.
  • the control group is a control group of mice administered with PBS in this experiment, and each test group is a group of mice administered with different siRNA conjugates.
  • the siRNA conjugate provided by the present disclosure at a dose of 3 mg/kg inhibited ANGPTL3 mRNA by as much as 94.0% or more.
  • the tested siRNA conjugates also showed a strong inhibitory effect on the ANGPTL3 mRNA of normal mouse liver tissue, the inhibition rates were 68.9% and 57.9%, respectively.
  • APOC3 transgenic mice Tg (APOC3) 3707Bre were randomly grouped according to serum TG content> 2mmol/L, 6 mice in each group, and conjugate 1, 5 and PBS blank control were given to each group of mice. All animals calculated the dose according to their body weight and used a single subcutaneous injection.
  • the dose of siRNA conjugate (based on the amount of siRNA) was 3 mg/kg and 1 mg/kg, and the volume was 5 ml/kg.
  • Each siRNA conjugate is provided as a PBS aqueous solution, and the concentration of the conjugate should be calculated according to the dose and volume administered.
  • Blood was taken from the orbital venous plexus of the mice before administration (reported as day 0) and on days 7, 14, 21, 28, 35, 42, and 49 after administration.
  • Standardized blood lipid level (post-administration test group blood lipid content/pre-administration test group blood lipid content) x 100%.
  • Inhibition rate of blood lipid level (1-blood lipid content in test group after administration / blood lipid content in test group before administration) ⁇ 100%.
  • Blood lipid refers to total cholesterol (CHO) or triglyceride (TG).
  • Figures 5A and 5B are serum CHO levels at doses of 3 mg/kg and 1 mg/kg, respectively, and Figures 5C and 5D are serum TG levels at doses of 3 mg/kg and 1 mg/kg, respectively.
  • conjugates 1 and 5 can significantly reduce TG and CHO, indicating that conjugates 1 and 5 can continue to steadily and efficiently reduce within 49 days of a single administration Blood lipid levels.
  • ANGPTL3 mRNA in the liver was detected by the same method as Experimental Example 2, and the inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate was calculated.
  • the PCR primers used to amplify ANGPTL3 and GAPDH as internal reference genes are shown in Table 7.
  • Conjugate Numbering Dosage Inhibition rate Conjugate 1 L10-siANa1M3SVP 3mg/kg 84.7% Conjugate 5 L10-siANb1M3SVP 3mg/kg 78.1% Conjugate 1 L10-siANa1M3SVP 1mg/kg 42.2% Conjugate 5 L10-siANb1M3SVP 1mg/kg 53.6%
  • Figure 5E shows the inhibitory effect of conjugate 2 on TG at two doses at different time points after administration.
  • the maximum inhibition rate of TG reached 90.5% for 21 days after a single administration; the inhibition rate of TG remained above 70% for up to 56 days after administration.
  • the maximum TG inhibition rate appeared 73.6% 21 days after administration.
  • Figure 5F shows the inhibitory effect of conjugate 2 on CHO at two doses at different time points after administration.
  • the maximum inhibition rate of CHO reached 85.1% after a single administration for 28 days; the inhibition rate of CHO always remained above 54% for 56 days after administration.
  • the maximum CHO inhibition rate appeared at 28 days after administration, at 68.9%.
  • Figures 5G and 5H show the inhibitory effect of conjugate 9 and conjugate 10 on TG at two doses at different time points after administration.
  • the maximum TG inhibition rate of conjugate 9 and conjugate 10 reached 91.7% and 86.4%, respectively, for 14 days after a single administration; the inhibition rate of TG was maintained for up to 56 days after administration Above 50%.
  • the maximum inhibition rates of conjugate 9 and conjugate 10 appeared at 14 and 21 days after administration, respectively, at 75.5 and 70.9%, respectively.
  • Figure 5I and Figure 5J show the inhibitory effect of conjugate 9 and conjugate 10 on CHO at two doses at different time points after administration.
  • the maximum CHO inhibition rates of conjugate 9 and conjugate 10 reached 74.1% and 71.9%, respectively, for 21 days after a single administration; the CHO inhibition rate was maintained for up to 42 days after administration Above 50%.
  • the maximum inhibition rates of conjugate 9 and conjugate 10 appeared at 14 and 21 days of administration, 65.7% and 49.4%, respectively.
  • Quantitative Real-Time PCR was used to detect the expression level of ANGPTL3 mRNA in Huh7 cells transfected with siRNA conjugates of various concentrations. The specific steps are: after culturing the transfected cells for 24 hours, use Trizol (Thermo Fisher) to extract the total RNA in the cells according to the standard operating procedures for total RNA extraction; take 1 ⁇ g of total RNA and use a reverse transcription kit (Promega Corporation) , Article number A3500) reverse transcription according to the instructions in its instructions to obtain cDNA. Using 2 ⁇ Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No.
  • the inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate is (1-ANGPTL3 mRNA expression) ⁇ 100%.
  • each test group was Huh7 cells treated with siRNA conjugates of various concentrations, and the control group was Huh7 cells that were not treated with siRNA conjugates.
  • IC 50 value of the targeted mRNA is calculated as follows:
  • Y is the expression level of residual mRNA
  • X is the logarithm of the concentration of transfected siRNA conjugate
  • Bot is the Y value at the bottom of the steady state period
  • Top is the Y value at the top of the steady state period
  • LogIC 50 is the X value when Y is halfway between the bottom and the top, and HillSlope is the slope of the curve.
  • the IC 50 values of Conjugate 9 and Conjugate 10 in Huh7 cells in vitro were 0.1791 nM and 0.1928 nM, respectively. It can be seen that the conjugate 9 and conjugate 10 provided by the present disclosure also have high inhibitory activity in in vitro cell lines.
  • This experimental example examined the inhibitory activity of siRNA 6, 11 and comparative siRNA 1 in the psiCHECK system in vitro.
  • the target sequence (5'-TGGAGAAAACAACCTAAATGG-3', SEQ ID NO. 171) was cloned into the Xho I/Not I site of the psiCHECK TM -2 (Promega TM ) plasmid.
  • the target sequence contains a nucleotide sequence fragment that is completely complementary to the antisense strand of the siRNA to be tested.
  • siRNA and the above-mentioned detection plasmid were co-transfected, wherein 10 ng of plasmid was transfected into each well, and 0.2 ⁇ L of Lipofectamine TM 2000 was used.
  • the final concentration of siRNA is 0.1 nM, 0.03 nM and 0.01 nM.
  • Each group has 3 complex holes. For each specific concentration of siRNA test group, the group without siRNA treatment was used as a control.
  • NC is the universal negative control B01001 which has no homology with the target gene sequence.
  • the dual luciferase reporter gene detection kit (Dual Luciferase reporter kit, Promega Corporation, cat.E2940) was used to lyse HEK293A cells according to the instruction manual to detect the dual luciferase reporter gene.
  • the Renilla luciferase protein level (Ren) was normalized to the firefly luciferase protein level (Fir). This represents the remaining expression level of the target gene after being inhibited by siRNA, thus reflecting the inhibitory activity of siRNA.
  • the results are shown in Figure 6A.
  • the inhibition rate of siRNA 11 (77%) is the inhibition of the comparative siRNA 1 Twice the rate (38%); at 0.03nM concentration, the inhibition rate of siRNA 11 (51%) is 4 times the inhibition rate of comparative siRNA 1 (13%); at a concentration of 0.01 nM, compared to siRNA 1, there is no inhibitory activity, The inhibition rate of siRNA 11 was 62%.
  • the siRNA 6 provided by the present disclosure has an inhibition rate of up to 87% for target sequences at various concentrations, wherein at a concentration of 0.1 nM, the inhibition rate of siRNA 6 for target sequences is 97%.
  • This experimental example was measured F1-F2 F5-F8 50 values IC siRNA conjugates in vitro and psiCHECK system.
  • the experimental example 5-1 was used to construct the detection plasmid and the method of transfection and detection. The difference was that different concentrations of siRNA conjugate were applied. Based on the amount of siRNA, starting from 5nM, 3 times diluted to 0.00008nM, a total of 11 concentrations, 3 replicates per group. The IC 50 value of each siRNA conjugate was calculated using the method of Experimental Example 4, and the results are shown in Table 10.
  • This experimental example investigated the inhibitory activity of siRNA conjugates F1, F2, F5 and F6 in Huh7 cells in vitro.

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Abstract

La présente invention concerne un ARNsi permettant d'inhiber l'expression du gène de la protéine 3 de type angiopoïétine ainsi qu'une composition pharmaceutique et un conjugué contenant l'ARNsi. Chaque nucléotide de l'ARNsi est un nucléotide indépendamment modifié ou non modifié. L'ARNsi contient un brin de sens et un brin antisens. Le brin de sens contient une séquence nucléotidique I. La longueur de la séquence nucléotidique I est égale à celle d'une séquence nucléotidique représentée par SEQ ID NO : 1, et le nombre de différences de nucléotides est de trois maximum. Le brin antisens contient une séquence nucléotidique II. La longueur de la séquence nucléotidique II est égale à celle d'une séquence nucléotidique représentée par SEQ ID NO : 2, et le nombre de différences de nucléotides est de trois maximum. L'ARNsi et la composition pharmaceutique et le conjugué associé proposés dans la présente invention permettent de traiter et/ou de prévenir efficacement la dyslipidémie.
PCT/CN2019/128686 2018-12-28 2019-12-26 Acide nucléique, composition et conjugué contenant un acide nucléique, procédé de préparation et utilisation associés Ceased WO2020135581A1 (fr)

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US11918600B2 (en) 2018-08-21 2024-03-05 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof
US12083142B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US12084661B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
WO2024188173A1 (fr) * 2023-03-10 2024-09-19 苏州瑞博生物技术股份有限公司 Acide nucléique, composition et conjugué le comprenant, et utilisation associée
US12428642B2 (en) 2017-12-01 2025-09-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, preparation method and use thereof
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WO2024234831A1 (fr) * 2023-05-15 2024-11-21 苏州时安生物技术有限公司 Arn double brin pour réguler l'expression du gène de l'angiotensinogène, et conjugué, composition pharmaceutique et utilisation de celui-ci
CN117568350B (zh) * 2024-01-15 2024-04-30 苏州时安生物技术有限公司 一种用于调节血管紧张素原基因表达的双链rna、其缀合物、药物组合物及用途
WO2025140565A1 (fr) * 2023-12-27 2025-07-03 苏州瑞博生物技术股份有限公司 Peptide porteur, conjugué comprenant un peptide porteur, composition, procédé de préparation et utilisation

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US11492620B2 (en) 2017-12-01 2022-11-08 Suzhou Ribo Life Science Co., Ltd. Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof
US11660347B2 (en) 2017-12-01 2023-05-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
US12428642B2 (en) 2017-12-01 2025-09-30 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, preparation method and use thereof
US12274752B2 (en) 2017-12-01 2025-04-15 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing same, preparation method, and use thereof
US12084661B2 (en) 2017-12-01 2024-09-10 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
US11918600B2 (en) 2018-08-21 2024-03-05 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof
US11896674B2 (en) 2018-09-30 2024-02-13 Suzhou Ribo Life Science Co., Ltd. SiRNA conjugate, preparation method therefor and use thereof
US12496347B2 (en) 2018-12-28 2025-12-16 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof
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WO2023098908A1 (fr) * 2021-12-03 2023-06-08 Microbio (Shanghai) Co. Ltd. Motifs de modification pour petites molécules d'arn interférent à stabilité et activité en matière d'inactivation génique élevées
WO2024188173A1 (fr) * 2023-03-10 2024-09-19 苏州瑞博生物技术股份有限公司 Acide nucléique, composition et conjugué le comprenant, et utilisation associée

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