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WO2024188173A1 - 一种核酸、含有该核酸的组合物与缀合物及其用途 - Google Patents

一种核酸、含有该核酸的组合物与缀合物及其用途 Download PDF

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Publication number
WO2024188173A1
WO2024188173A1 PCT/CN2024/080731 CN2024080731W WO2024188173A1 WO 2024188173 A1 WO2024188173 A1 WO 2024188173A1 CN 2024080731 W CN2024080731 W CN 2024080731W WO 2024188173 A1 WO2024188173 A1 WO 2024188173A1
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nucleotide sequence
sirna
seq
nucleotide
rnai agent
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PCT/CN2024/080731
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English (en)
French (fr)
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甘黎明
高山
梁子才
张鸿雁
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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 pharmaceutical composition
  • a pharmaceutical composition comprising a first RNAi agent that can target ANGPTL3 mRNA and a second RNAi agent that can target APOC3 mRNA, and a method for using the pharmaceutical composition to treat and/or prevent diseases or symptoms associated with dyslipidemia.
  • Hyperlipidemia is a type of dyslipidemia, a systemic disease in which fat metabolism or operation is abnormal, causing plasma lipids to be higher than normal, and is a serious threat to the health of patients around the world.
  • Existing drugs for the treatment of dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probucol, fibrates, and niacin and its derivatives.
  • Angiopoietin-like protein 3 (ANGPTL3) is a secretory protein mainly expressed in the liver. It is named because its gene structure is similar to 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.
  • Apolipoprotein C3 (APOC3) plays an important role in lipid metabolism. The expression of APOC3 in the blood circulation of people carrying the APOC3 mutation gene is reduced by 46%, and the plasma triglyceride level is reduced by 39% compared with ordinary people.
  • siRNA Small interfering RNA
  • RNAi RNA interference
  • siRNA can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases. Therefore, if gene expression can be silenced at the gene level and the production of ANGPTL3 and APOC3 can be blocked, it will undoubtedly be the most ideal treatment.
  • siRNA small interfering RNA
  • the inventors of the present invention unexpectedly discovered that by co-administering two siRNAs that can target ANGPTL3 mRNA and APOC3 mRNA, blood lipids in plasma can be reduced, especially the triglyceride content in plasma can be significantly reduced, and a good balance of high maximum inhibition efficiency and long sustained inhibition time can be achieved at the same time.
  • the present disclosure provides a pharmaceutical composition, comprising a first RNAi agent and a second RNAi agent, wherein the first RNAi agent comprises one or more first siRNAs and/or one or more first siRNA conjugates, wherein the first siRNA comprises a first sense strand and a first antisense strand, wherein each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the first sense strand comprises a nucleotide sequence I a , and the first antisense strand comprises a nucleotide sequence II a , wherein the nucleotide sequence I a and the nucleotide sequence II a are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I a has at least 15 consecutive nucleotides consistent with the nucleotide sequence shown in any one of SEQ ID NOs: 93-97; each of the first siRNA conjugates comprises a first siRNA group
  • the second RNAi agent comprises one or more second siRNAs and/or one or more second siRNA conjugates
  • the second siRNA comprises a second sense strand and a second antisense strand
  • each nucleotide in the second siRNA is independently a modified or unmodified nucleotide
  • the second sense strand contains a nucleotide sequence I b
  • the second antisense strand contains a nucleotide sequence II b
  • the nucleotide sequence I b and the nucleotide sequence II b are at least partially reverse complementary to form a double-stranded region
  • the nucleotide sequence I b has at least 15 consecutive nucleotides consistent with the nucleotide sequence shown in SEQ ID NO: 98 or 99
  • the second siRNA conjugate comprises a second siRNA group formed by the second siRNA and a conjugated group conjugated thereto.
  • the present disclosure also provides a use of the pharmaceutical composition of the present disclosure in preparing a medicament for treating and/or preventing diseases or symptoms associated with dyslipidemia.
  • the present disclosure also provides a method for treating and/or preventing a disease or symptom associated with dyslipidemia, the method comprising administering the above-mentioned first RNAi agent and second RNAi agent to a subject in need thereof.
  • the present disclosure also provides a method for inhibiting the expression levels of ANGPTL3 mRNA and APOC3 mRNA in cells, the method comprising contacting an effective amount of the pharmaceutical composition described in the present disclosure with the cells.
  • the drug conjugate provided by the present invention has good lipid-lowering effect and long-term efficacy.
  • the pharmaceutical composition of the present invention has a comparable maximum inhibition rate to conjugate 2, which is significantly higher than conjugate 1, and the pharmaceutical composition of the present invention has a comparable long-term effect to conjugate 1, and a significantly higher sustained inhibition effect than conjugate 2; for the inhibition of TC levels in mouse plasma, the pharmaceutical composition has a higher maximum inhibition rate than conjugate 1 or conjugate 2 alone, and the pharmaceutical composition has a comparable long-term effect to conjugate 1, and a better sustained inhibition effect than conjugate 2.
  • the pharmaceutical composition of the present invention can achieve a good balance of a higher maximum inhibition effect of blood lipids and a longer-lasting blood lipid inhibition level at the same total siRNA dose, and therefore shows a good potential for preventing and/or treating symptoms or diseases associated with dyslipidemia.
  • FIG1 is a line graph showing relative serum TG levels at different time points after administration of PBS, conjugates or pharmaceutical compositions in human APOC3 transgenic mice.
  • FIG2 is a line graph showing the relative levels of serum TC at different time points in human APOC3 transgenic mice after administration of PBS, conjugate or pharmaceutical composition.
  • ANGPTL3 mRNA or "mRNA expressed by ANGPTL3 gene” refers to mRNA having a sequence as shown in Genbank Accession No. NM_014495.4
  • ANGPTL3 target gene i.e. ANGPTL3 gene refers to a gene that transcribes the above ANGPTL3 mRNA
  • APOC3 mRNA or "mRNA expressed by APOC3 gene” refers to mRNA having a sequence as shown in Genbank Accession No. NM_000040.3
  • APOC3 target gene i.e. APOC3 gene refers to a gene that transcribes the above APOC3 mRNA.
  • C, G, U, and A represent the base composition of nucleotides
  • lowercase letter m represents that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide
  • lowercase letter f represents that the nucleotide adjacent to the left of letter f is a fluorinated-modified nucleotide
  • lowercase letter s represents that the two nucleotides adjacent to the left and right of letter s are connected by phosphorothioate subunits
  • P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide
  • P1 is VP, Ps or P representing a specific modification, wherein the letter combination VP indicates that a nucleotide adjacent to the right side of the letter combination VP is a nucleotide modified with vinyl phosphate
  • fluorinated 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-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. Such as isonucleotides, bridged nucleotides (bridged nuclear acid, abbreviated as BNA) or acyclic nucleotides.
  • BNA bridged nuclear acid
  • methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the expressions "complementary” or “reverse complementary” can be used interchangeably and have the meanings known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one chain are each paired with the bases on the other chain 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 paired with the pyrimidine base cytosine (G).
  • Each base pair includes a purine and a pyrimidine.
  • mismatch means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there are no more than 1 base mismatch between the two nucleotide sequences; and “completely reverse complementary” means that there are no base mismatches between the two nucleotide sequences.
  • the nucleoside monomer refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared.
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are all commercially available.
  • a "substituted” or “substituted” group such as a substituted alkyl, a substituted alkoxy, a substituted amino, a substituted aliphatic group, a substituted heteroaliphatic group, a substituted acyl group, a substituted aryl group or a substituted heteroaryl group.
  • a “substituted” or “substituted” group refers to a group formed by replacing the hydrogen atoms in the group with one or more substituents.
  • a "substituted alkoxy” refers to a group formed by replacing one or more hydrogen atoms in an alkoxy group with a substituent.
  • the substituent is selected from the group consisting of C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 -C 10 haloalkyl, -OC 1 -C 10 alkyl, -OC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-OH, -OC 1 -C 10 haloalkyl, -SC 1 -C 10 alkyl, -SC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-SH, -SC 1 -C 10 haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 alkyl-NH 2 , -N(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), -NH(C 1 -C 10 alkyl ), -N(C 1 -C 10 alky
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C1 - C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a specific number of carbons it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl” is meant to include 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 with two points of attachment.
  • alkenyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group.
  • the group can be in the cis or trans configuration of the double bond.
  • Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc.
  • the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, 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 identical to 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 obtained by removing two molecules of hydrogen from adjacent carbon atoms of the parent alkyl group.
  • 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, etc.
  • the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 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 identical to alkynyl but have two points of attachment.
  • alkoxy refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc.
  • Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
  • aryl refers to a radical 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 carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) ⁇ -electron system according to the Hückel theory.
  • Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl.
  • Arylene is a subset of aryl and refers to residues identical to aryl but having two points of attachment.
  • 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 can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., Contains a cyclic delocalized (4n+2) ⁇ -electron system according to the Hückel theory.
  • Heteroaryl includes fused or bridged ring systems.
  • the heteroatom in the heteroaryl is an oxidized heteroatom.
  • the heteroaryl contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl is a quaternized nitrogen atom.
  • the heteroaryl is attached to the rest of the molecule through any ring atom.
  • heteroaryl examples include, but are not limited to, azacycloheptatrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzo dioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazo
  • halogen substituent or “halo” refers to fluoro, chloro, bromo and iodo.
  • halogen includes fluorine, chlorine, bromine and iodine.
  • haloalkyl refers to an alkyl group as defined above in which a specified number of hydrogen atoms are replaced by one or more, up to the maximum permitted number of halogen atoms.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.
  • hydroxyl protecting groups can be used in the present disclosure.
  • protecting groups make chemical functionalities insensitive to specific reaction conditions and can be added and removed from the functionality in a molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety.
  • the protecting group is stable under alkaline conditions but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)oxanthene-9-yl (Mox).
  • non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), 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 (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.
  • treatment refers to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
  • prevention refers to a method of obtaining a beneficial or desired result, including but not limited to a preventive benefit.
  • a double-stranded siRNA a pharmaceutical composition or a siRNA conjugate can be administered to a subject at risk of a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not yet be made.
  • prevention of a disease or symptom associated with hyperlipidemia means that when the subject begins to show a tendency to increase blood lipids, the subject is prevented from increasing blood lipids to abnormal levels by administering the pharmaceutical composition of the present disclosure to the subject, wherein the abnormal level refers to a total cholesterol (TC) level in a human subject exceeding 6.2 mmol/L, a triglyceride (TG) level exceeding 2.3 mmol/L, and/or a low-density lipoprotein cholesterol (LDL-C) level exceeding 4.1 mmol/L.
  • TC total cholesterol
  • TG triglyceride
  • LDL-C low-density lipoprotein cholesterol
  • the siRNA or siRNA conjugate may exist in a salted form or an unsalted form.
  • the siRNA or siRNA conjugate may exist in the form of a partial or complete water-soluble salt, and the water-soluble salt may be an amine salt or an alkali metal salt thereof.
  • the alkali metal salt may be a potassium salt or a sodium salt.
  • the amine salt may be an ammonium salt, a methylamine salt or a triethylamine salt.
  • each adjacent nucleotide is connected by a phosphodiester bond or a thiophosphorothioate diester bond
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the thiophosphorothioate diester bond carries a negative charge, which may exist in the form of a hydroxyl or a sulfhydryl group, and the hydrogen ions in the hydroxyl or sulfhydryl group may also be partially or completely replaced by cations.
  • the cation may be any cation, such as a metal cation, an ammonium ion NH 4 + , or one of an organic ammonium cation.
  • the organic ammonium cation may be a cation formed by methylamine, a cation formed by triethylamine, or a quaternary ammonium cation.
  • the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation.
  • the alkali metal ion may be K + and/or Na +
  • the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine.
  • the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the thiophosphorothioate diester bond is at least partially bound to a sodium ion
  • the siRNA or siRNA conjugate disclosed herein exists in the form of a sodium salt or a partial sodium salt.
  • compositions of the present disclosure are provided.
  • the present disclosure provides a pharmaceutical composition capable of lowering blood lipid levels, especially capable of lowering the triglyceride content in plasma.
  • the pharmaceutical composition of the present disclosure comprises a first RNAi agent and a second RNAi agent, wherein the first RNAi agent comprises one or more first siRNAs and/or one or more first siRNA conjugates, wherein the first siRNA comprises a first sense strand and a first antisense strand, wherein each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the first sense strand comprises a nucleotide sequence I a , and the first antisense strand comprises a nucleotide sequence II a , wherein the nucleotide sequence I a and the nucleotide sequence II a are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I a has at least 15 consecutive nucleotides consistent with the nucleotide sequence shown in any one of SEQ ID NOs: 93-97; each of the first siRNA conjugates comprises a first siRNA group formed by the first siRNA
  • the second RNAi agent comprises one or more second siRNAs and/or one or more second siRNA conjugates, the second siRNA comprises a second sense strand and a second antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the second sense strand comprises a nucleotide sequence I b , the second antisense strand comprises a nucleotide sequence II b , the nucleotide sequence I b and the nucleotide sequence II b
  • the nucleotide sequence II b is at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I b has at least 15 consecutive nucleotides consistent with the nucleotide sequence shown in SEQ ID NO:98 or 99;
  • the second siRNA conjugate comprises a second siRNA group formed by the second siRNA and a conjugated group conjugated thereto.
  • the weight ratio of the first RNAi agent to the second RNAi agent is 1:(0.1-10) based on the total amount of siRNA and siRNA groups contained in each of the first RNAi agent and the second RNAi agent. In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent is 1:(0.5-5). In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent is 1:(0.5-2). In some embodiments, the weight ratio of the first RNAi agent to the second RNAi agent is 1:1, 1:2 or 2:1.
  • the weight of the first RNAi agent in the pharmaceutical composition is 1 mg-1000 mg, for example, 1 mg-800 mg, 1 mg-600 mg, 1 mg-500 mg, 1 mg-300 mg, 1 mg-250 mg, 1 mg-100 mg, based on the total amount of siRNA and siRNA groups contained in each of the first RNAi agent and the second RNAi agent. In some embodiments, the weight of the first RNAi agent is 5 mg-500 mg. In some embodiments, the weight of the second RNAi agent is 1 mg-1000 mg, for example, 1 mg-800 mg, 1 mg-600 mg, 1 mg-500 mg, 1 mg-300 mg, 1 mg-250 mg, 1 mg-100 mg. In some embodiments, the weight of the first RNAi agent in the pharmaceutical composition is 5 mg-500 mg. In some embodiments, the weight of the first RNAi agent is 5 mg-500 mg, and the weight of the second RNAi agent is 5 mg-500 mg.
  • the first RNAi agent and the second RNAi agent are identical to each other.
  • the pharmaceutical composition provided by the present disclosure comprises a first RNAi agent and a second RNAi agent.
  • the first RNAi agent comprises one or more first siRNAs and/or one or more first siRNA conjugates.
  • the first siRNA comprises a first sense strand and a first antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the first sense strand contains a nucleotide sequence I a , the first antisense strand contains a nucleotide sequence II a , and the nucleotide sequence I a and the nucleotide sequence II a are at least partially reverse-complemented to form a double-stranded region, wherein the nucleotide sequence I a is consistent with the nucleotide sequence shown in any one of SEQ ID NOs: 93-97 for at least 15 consecutive nucleotides.
  • nucleotide sequence Ia is equal in length to the nucleotide sequence shown in SEQ ID NO:1, and differs by no more than 3 nucleotides
  • nucleotide sequence IIa is equal in length to the nucleotide sequence shown in SEQ ID NO:2, and differs by no more than 3 nucleotides:
  • Za1 is U
  • Za2 is A
  • the nucleotide sequence Ia contains the nucleotide Za3 corresponding to the position of Za1
  • the nucleotide sequence IIa contains the nucleotide Za4 corresponding to the position of Za2
  • Za4 is the first nucleotide at the 5' end of the antisense strand.
  • position corresponding means being in the same position in the nucleotide sequence starting from the same end of the nucleotide sequence, for example, the first nucleotide at the 3' end of the nucleotide sequence Ia is the nucleotide whose position corresponds to the first nucleotide of SEQ ID NO:1.
  • the second RNAi agent comprises one or more second siRNAs and/or one or more second siRNA conjugates, the second siRNA comprises a second sense strand and a second antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the second sense strand comprises a nucleotide sequence I b , the second antisense strand comprises a nucleotide sequence II b , the nucleotide sequence I b and the nucleotide sequence II b are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I b has at least 15 consecutive nucleotides consistent with the nucleotide sequence shown in SEQ ID NO: 98 or 99:
  • nucleotide sequence I b is equal in length to the nucleotide sequence shown in SEQ ID NO:46, and differs in no more than 3 nucleotides
  • nucleotide sequence II b is equal in length to the nucleotide sequence shown in SEQ ID NO:47, and differs in no more than 3 nucleotides:
  • Z b1 is U
  • Z b2 is A
  • the nucleotide sequence I b contains the nucleotide Z b3 corresponding to Z b1
  • the nucleotide sequence II b contains the nucleotide Z b4 corresponding to Z b2
  • Z b4 is the first nucleotide at the 5' end of the antisense strand.
  • At least one of the 3rd to 6th nucleotides of the nucleotide sequence II a and the nucleotide sequence II b is a stabilizing modified nucleotide.
  • the acid refers to a nucleotide in which the 2'-hydroxyl group of the ribose of the nucleotide is replaced by a stabilizing modification group.
  • the thermal stability of the first siRNA and the second siRNA comprising the stabilizing modified nucleotide is increased, and the steric hindrance of the stabilizing modification group is greater than that of the 2'-O-methyl group.
  • the 3rd and/or 5th nucleotide in the nucleotide sequence II a and the nucleotide sequence II b is a stabilizing modified nucleotide.
  • the 3rd nucleotide in the nucleotide sequence II a and the nucleotide sequence II b is the stabilizing modified nucleotide.
  • the 5th nucleotide in the nucleotide sequence II a and the nucleotide sequence II b is the stabilizing modified nucleotide.
  • nucleotides from the 3rd to the 9th nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are the stabilizing modified nucleotides in the direction from the 5' end to the 3' end. In some embodiments, from the 5' end to the 3' end, the nucleotides other than the 3rd to the 9th nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are not stabilizing modified nucleotides.
  • the ability of the siRNA to regulate the expression level of the target sequence may be significantly affected.
  • the first siRNA and the second siRNA disclosed in the present invention can achieve a better balance between pharmaceutical activity and low toxicity while also having excellent stability.
  • “increased thermal stability” in the context of the present disclosure refers to an increase in the double-stranded thermal dissociation temperature (Tm) of the siRNA. In some embodiments, “increased thermal stability” refers to an increase in the Tm of the siRNA by at least 0.05°C, and in some embodiments, an increase in the Tm of the siRNA by 0.1-6°C. In some embodiments, an increase in the Tm of the siRNA by 0.5-4°C.
  • the ability of the first antisense strand in the first siRNA of the present disclosure to bind to the mRNA expressed by the ANGPTL3 gene or the second antisense strand in the second siRNA to bind to the mRNA expressed by the APOC3 gene is substantially unaffected, while the binding to the off-target target mRNA is significantly reduced, thereby reducing or even eliminating the off-target effect.
  • each of the stabilizing modifying groups independently has a structure represented by -XR, wherein X is O, NR', S or SiR'2 ; R is one of C2 - C6 alkyl, substituted C2 - C6 alkyl, C6 - C8 aryl, and substituted C6 - C8 aryl; each R' is independently H, C1 - C6 alkyl, substituted C1 - C6 alkyl, C6 - C8 aryl, and substituted C6 - C8 aryl; the substituted C2 - C6 alkyl, substituted C 6 -C 8 aryl or substituted C 1 -C 6 alkyl refers to a group formed by replacing one or more hydrogen atoms in C 2 -C 6 alkyl, C 6 -C 8 aryl or C 1 -C 6 alkyl with a substituent, and the substituent is selected from one or more of the following substituents: C 1 -C
  • each of the stabilizing modification groups is independently selected from 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O-2-N-methylamino-2-oxysubylethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O-2,4-dinitrophenyl.
  • each of the stabilizing modifying groups is 2'-O-methoxyethyl.
  • the third or fifth nucleotide in the nucleotide sequence II a and the nucleotide sequence II b is a 2'-O-methoxyethyl modified nucleotide in the direction from the 5' end to the 3' end.
  • no more than two nucleotides in the third to ninth nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are 2'-O-methoxyethyl modified nucleotides in the direction from the 5' end to the 3' end.
  • the second, sixth, fourteenth, and sixteenth nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are 2'-fluoro modified nucleotides if they are not 2'-O-methoxyethyl modified nucleotides in the direction from the 5' end to the 3' end.
  • all nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are modified nucleotides; from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II a and the nucleotide sequence II b , if not 2'-O-methoxyethyl-modified nucleotides, are 2'-fluoro-modified nucleotides, and the other nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are each independently one of the non-fluorinated modified nucleotides.
  • nucleotide difference between the nucleotide sequence Ia and the nucleotide sequence shown in SEQ ID NO: 1 may include the difference at the Za3 position and/or the nucleotide difference at any other nucleotide position in the nucleotide sequence Ia .
  • nucleotide difference between the nucleotide sequence Ia and the nucleotide sequence shown in SEQ ID NO: 1 may include the nucleotide difference at the Za3 position and/or the nucleotide position adjacent to Za3 .
  • nucleotide sequence Ia is the nucleotide sequence shown in SEQ ID NO:1.
  • nucleotide difference there is no more than one nucleotide difference between the nucleotide sequence IIa and the nucleotide sequence shown in SEQ ID NO: 2.
  • the difference between the nucleotide sequence IIa and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at position Za4 , and Za4 is selected from U, G or C.
  • Za3 is a nucleotide complementary to Za4 .
  • the difference between the nucleotide sequence IIa and the nucleotide sequence shown in SEQ ID NO:2 is the difference at the position of Za4 , and Za4 is selected from U, G or C.
  • each U in the nucleotide sequence of the first siRNA and the second siRNA of the present disclosure can be arbitrarily replaced by T.
  • These nucleotide differences do not significantly reduce the target gene inhibition ability of siRNA or increase the off-target effect of siRNA.
  • these siRNAs containing nucleotide differences are also within the scope of protection of the present disclosure.
  • nucleotide sequence Ia and nucleotide sequence IIa are substantially reverse complementary, substantially reverse complementary or completely reverse complementary.
  • Substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; substantially reverse complementary means that there are no more than 1 base mismatches between the two nucleotide sequences; completely reverse complementary means that there are no mismatches between the two nucleotide sequences.
  • the nucleotides at positions 3-19 of the nucleotide sequence II a are completely reverse complementary to the nucleotides at positions 1-17 of the nucleotide sequence I a in the direction from the 5' end to the 3' end. In some embodiments, the nucleotide sequence II a is completely reverse complementary to the nucleotide sequence I a . In some embodiments, there is a base mismatch between the second nucleotide in the nucleotide sequence II a and the second nucleotide in the nucleotide sequence I a in the direction from the 3' end to the 5' end in the direction from the 5' end. By including this base mismatch, the target gene expression inhibition activity of the first siRNA disclosed herein can be further enhanced while maintaining a low off-target effect.
  • nucleotide sequence Ia is the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and nucleotide sequence IIa is the nucleotide sequence shown in SEQ ID NO:7:
  • Za3 is selected from A, U, G or C, and Za4 is a nucleotide complementary to Za3 .
  • Za3 is U, and Za4 is A.
  • the first sense strand and the first antisense strand are the same or different in length, the length of the first sense strand is 19-23 nucleotides, and the length of the first antisense strand is 19-26 nucleotides.
  • the length ratio of the first sense strand and the first antisense strand provided by the present disclosure can be 19/19, 19/20, 19/21, 19/22, 19/23, In some embodiments, the length ratio of the first sense strand to the first antisense strand is 19/21, 21/23 or 23/25.
  • the first sense strand further comprises a nucleotide sequence III a
  • the first antisense strand further comprises a nucleotide sequence IV a
  • Each nucleotide of the nucleotide sequence III a is independently one of non-fluorinated modified nucleotides
  • each nucleotide of the nucleotide sequence IV a is independently one of non-fluorinated modified nucleotides and is not a stabilizing modified nucleotide.
  • the length of the nucleotide sequence III a is 1, 2, 3 or 4 nucleotides.
  • the nucleotide sequence IV a and the nucleotide sequence III a are equal in length, and the nucleotide sequence IV a and the nucleotide sequence III a are substantially reverse complementary or completely reverse complementary.
  • the nucleotide sequence III a is connected to the 5' end of the nucleotide sequence I a
  • the nucleotide sequence IV a is connected to the 3' end of the nucleotide sequence II a
  • the nucleotide sequence IV a is substantially reverse complementary or completely reverse complementary to the second ANGPTL3 mRNA sequence.
  • the second ANGPTL3 mRNA sequence is a nucleotide sequence in the mRNA expressed by the ANGPTL3 gene that is adjacent to the first ANGPTL3 mRNA sequence and has the same length as the nucleotide sequence IV a .
  • the first ANGPTL3 mRNA sequence is the sequence shown in SEQ ID NO: 1 in the mRNA expressed by the ANGPTL3 gene.
  • the length of nucleotide sequence III a and IV a is 1 nucleotide, the base of nucleotide sequence III a is A, and the base of nucleotide sequence IV a is U.
  • the length of nucleotide sequence III a and IV a is 2 nucleotides, the base composition of nucleotide sequence III a is CA, and the base composition of nucleotide sequence IV a is UG.
  • the length of nucleotide sequence III a and IV a is 3 nucleotides, the base composition of nucleotide sequence III a is CCA, and the base composition of nucleotide sequence IV a is UGG.
  • the length of nucleotide sequence III a and IV a is 4 nucleotides
  • the base composition of nucleotide sequence III a is GCCA
  • the base composition of nucleotide sequence IV a is UGGC.
  • nucleotide sequence III a and nucleotide sequence IV a are completely reverse complementary, and therefore, given the base composition of nucleotide sequence III a , the base composition of nucleotide sequence IV a is also determined.
  • the first siRNA further contains a nucleotide sequence Va .
  • Each nucleotide in the nucleotide sequence Va is independently one of non-fluorinated modified nucleotides and is not a stabilized modified nucleotide.
  • the nucleotide sequence Va is 1 to 3 nucleotides in length and is connected to the 3' end of the first antisense strand to form a 3' overhang.
  • the length ratio of the first sense strand and the first antisense strand provided by the present disclosure can 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 nucleotide sequence Va is 2 nucleotides in length, thus, The length ratio of the first sense strand and the first antisense strand provided in the present disclosure may be 19/21, 21/23 or 23/25.
  • Each nucleotide in the nucleotide sequence Va can be any nucleotide.
  • the length of the nucleotide sequence Va is 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence Va is 2 consecutive thymine deoxyribonucleotides (dTdT) and 2 consecutive uracil ribonucleotides (UU); or, in order to improve the affinity of the first antisense strand to the target mRNA, the nucleotide sequence Va is completely reverse complementary to the third segment of the ANGPTL3 mRNA sequence.
  • the third segment of the ANGPTL3 mRNA sequence is a nucleotide sequence in the mRNA expressed by the ANGPTL3 gene that is adjacent to the first segment of the ANGPTL3 mRNA sequence or the second segment of the ANGPTL3 mRNA sequence and has a length equal to that of the nucleotide sequence Va . Therefore, in some embodiments, the length ratio of the first sense strand to the first antisense strand is 19/21 or 21/23. At this time, the first siRNA disclosed in the present invention has better mRNA silencing activity.
  • the first sense strand contains the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and the first antisense strand contains the nucleotide sequence shown in SEQ ID NO:8:
  • Za4 is the first nucleotide at the 5' end of the first antisense strand
  • Za3 is selected from A, U, G or C
  • Za4 is a nucleotide complementary to Za3 .
  • the first sense strand contains the nucleotide sequence shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12
  • the first antisense strand contains the nucleotide sequence shown in SEQ ID NO:13:
  • Za4 is the first nucleotide at the 5' end of the first antisense strand
  • Za3 is selected from A, U, G or C
  • Za4 is a nucleotide complementary to Za3 .
  • the nucleotide sequence 1b is the same as the nucleotide sequence shown in SEQ ID NO:46. No more than 2 nucleotide differences.
  • the nucleotide difference between the nucleotide sequence 1 b and the nucleotide sequence shown in SEQ ID NO:46 may include the difference at the Z b3 position and/or the nucleotide difference at any other nucleotide position in the nucleotide sequence 1 b .
  • the nucleotide difference between the nucleotide sequence 1 b and the nucleotide sequence shown in SEQ ID NO:46 may include the nucleotide difference at the Z b3 position and/or the nucleotide difference at the nucleotide position adjacent to Z b3 .
  • the nucleotide sequence Ib is the nucleotide sequence shown in SEQ ID NO:46.
  • nucleotide sequence II b there is no more than 1 nucleotide difference between the nucleotide sequence II b and the nucleotide sequence shown in SEQ ID NO:47.
  • the difference between the nucleotide sequence II b and the nucleotide sequence shown in SEQ ID NO:47 includes a difference at position Z b4 , and Z b4 is selected from U, G or C.
  • Z b3 is a nucleotide complementary to Z b4 .
  • the difference between the nucleotide sequence II b and the nucleotide sequence shown in SEQ ID NO:47 includes a difference at position Z b4 , and Z b4 is selected from U, G or C.
  • nucleotide sequence Ib and nucleotide sequence IIb are substantially reverse complements, essentially reverse complements, or completely reverse complements.
  • the nucleotides at positions 3-19 of the nucleotide sequence II b are completely reverse complementary to the nucleotides at positions 1-17 of the nucleotide sequence I b in the direction from the 5' end to the 3' end. In some embodiments, the nucleotide sequence II b is completely reverse complementary to the nucleotide sequence I b . In some embodiments, there is a base mismatch between the second nucleotide in the nucleotide sequence II b and the second nucleotide in the nucleotide sequence I b in the direction from the 3' end to the 5' end in the direction from the 5' end. By including this base mismatch, the target gene expression inhibition activity of the second siRNA disclosed herein can be further enhanced while maintaining a low off-target effect.
  • nucleotide sequence I b is the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51
  • nucleotide sequence II b is the nucleotide sequence shown in SEQ ID NO:52:
  • Z b3 is selected from A, U, G or C, and Z b4 is a nucleotide complementary to Z b3 .
  • Z b3 is U
  • Z b4 is A.
  • the second sense strand and the second antisense strand are the same or different in length, the second sense strand is 19-23 nucleotides in length, and the second antisense strand is 19-26 nucleotides in length.
  • the length ratio of the second sense strand and the second antisense strand provided by the present disclosure can be 19/19, 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 second sense strand to the second antisense strand is 19/21, 21
  • the second sense strand further contains nucleotide sequence III b
  • the second antisense strand further contains nucleotide sequence IV b .
  • Each nucleotide of nucleotide sequence III b is independently one of non-fluorinated modified nucleotides
  • each nucleotide of nucleotide sequence IV b is independently one of non-fluorinated modified nucleotides and is not a stabilized modified nucleotide.
  • the length of nucleotide sequence III b is 1, 2, 3 or 4 nucleotides.
  • Nucleotide sequence IV b and nucleotide sequence III b are equal in length, and nucleotide sequence IV b and nucleotide sequence III b are substantially reverse complementary or completely reverse complementary. Nucleotide sequence III b is connected to the 5' end of nucleotide sequence I b , and nucleotide sequence IV b is connected to the 3' end of nucleotide sequence II b .
  • Nucleotide sequence IV b is substantially reverse complementary or completely reverse complementary to the second APOC3 mRNA sequence
  • the second APOC3 mRNA sequence is a nucleotide sequence in the mRNA expressed by the APOC3 gene that is adjacent to the first APOC3 mRNA sequence and has the same length as nucleotide sequence IV b .
  • the first APOC3 mRNA sequence is the sequence shown in SEQ ID NO: 46 in the mRNA expressed by the APOC3 gene.
  • the length of nucleotide sequence III b and IV b is 1 nucleotide, the base of nucleotide sequence III b is C, and the base of nucleotide sequence IV b is G.
  • the length of nucleotide sequence III b and IV b is 2 nucleotides, the base composition of nucleotide sequence III b is GC, and the base composition of nucleotide sequence IV b is GC.
  • the length of nucleotide sequence III b and IV b is 3 nucleotides, the base composition of nucleotide sequence III b is UGC, and the base composition of nucleotide sequence IV b is GCA.
  • nucleotide sequence III b and IV b is 4 nucleotides
  • the base composition of nucleotide sequence III b is UUGC
  • the base composition of nucleotide sequence IV b is GCAA.
  • nucleotide sequence III b and nucleotide sequence IV b are completely reverse complementary, and therefore, given the base composition of nucleotide sequence III b , the base composition of nucleotide sequence IV b is also determined.
  • the second siRNA further comprises a nucleotide sequence V b .
  • Each nucleotide in the nucleotide sequence V b is independently one of the non-fluorinated modified nucleotides and is not a stabilizing modified nucleotide.
  • the nucleotide sequence V b has a length of 1 to 3 nucleotides and is connected to the 3' end of the second antisense strand to form a 3' overhang.
  • the length ratio of the second sense strand and the second antisense strand provided by the present disclosure can 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 nucleotide sequence V b is 2 nucleotides, thus, the length ratio of the second sense strand and the second antisense strand provided by the present disclosure can be 19/21, 21/23 or 23/25.
  • Each nucleotide in the nucleotide sequence V b can be any nucleotide.
  • the length of the nucleotide sequence V b is 2 nucleotides, and in the direction from the 5' end to the 3' end, the nucleotide sequence V b is 2 consecutive thymine deoxyribonucleotides and 2 consecutive uracil ribonucleotides; or, in order to improve the affinity of the second antisense strand to the target mRNA, the nucleotide sequence V b is completely reverse complementary to the third APOC3 mRNA sequence.
  • the third APOC3 mRNA sequence is a nucleotide sequence in the mRNA expressed by the APOC3 gene that is adjacent to the first APOC3 mRNA sequence or the second APOCL3 mRNA sequence and has a length equal to that of the nucleotide sequence V b . Therefore, in some embodiments, the ratio of the length of the second sense strand to the second antisense strand is 19/21 or 21/23. At this time, the second siRNA disclosed in the present invention has better mRNA silencing activity.
  • the second sense strand contains the nucleotide sequence shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 or SEQ ID NO:51
  • the second antisense strand contains the nucleotide sequence shown in SEQ ID NO:53:
  • Z b4 is the first nucleotide at the 5′ end of the second antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 .
  • the second sense strand contains the nucleotide sequence shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57
  • the second antisense strand contains the nucleotide sequence shown in SEQ ID NO:58:
  • Z b4 is the first nucleotide at the 5′ end of the second antisense strand
  • Z b3 is selected from A, U, G or C
  • Z b4 is a nucleotide complementary to Z b3 .
  • the nucleotides in the first siRNA and the second siRNA of the present disclosure are each independently modified or unmodified nucleotides.
  • some or all of the nucleotides in the first siRNA and the second siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups do not result in significant weakening or loss of their function when inhibiting the expression of related genes.
  • modified nucleotide refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base.
  • the modified nucleotide will not cause the function of siRNA to inhibit gene expression to be significantly weakened or lost.
  • the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
  • the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence IIa and nucleotide sequence IIb are each independently 2'-fluoro modified nucleotides.
  • the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II a and the nucleotide sequence II b are each independently a 2'-fluoro-modified nucleotide, and the other nucleotides in the nucleotide sequence II a and the nucleotide sequence II b are each independently one of the non-fluorinated modified nucleotides.
  • the 7th to 9th nucleotides of the nucleotide sequence Ia and the nucleotide sequence Ib are each independently a 2'-fluoro-modified nucleotide.
  • the 7th to 9th nucleotides of the nucleotide sequence Ia and the nucleotide sequence Ib are each independently a 2'-fluoro-modified nucleotide, and the other nucleotides in the nucleotide sequence Ia and the nucleotide sequence Ib are each independently one of the non-fluoro-modified nucleotides.
  • siRNA disclosed herein can achieve a good balance between gene expression regulatory activity and in vivo stability by having the above-mentioned modifications.
  • fluorinated 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, and has a structure shown in the following formula (7).
  • Non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from one of the 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-fluorinated group.
  • nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorinated groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'-NH 2 ) is as shown in formula (9).
  • the 2'-deoxynucleotide (DNA) is as shown in formula (10):
  • Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides.
  • the nucleotide analogs can be isonucleotides, bridged nucleotides (BNAs) or acyclic nucleotides.
  • BNA refers to a constrained or inaccessible nucleotide.
  • 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.
  • BNA may be LNA, ENA, cET BNA, etc., wherein 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 nucleotides formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is as shown in formula (15) and GNA is as shown in formula (16):
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotides are compounds formed by a change in the position of a base on the ribose ring of a nucleotide.
  • an isonucleotide may be a compound formed by a 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; and R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.
  • each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose moiety with a methoxy group.
  • fluorinated modified nucleotide refers to a compound having a structure shown in formula (7) formed by replacing the 2'-hydroxyl group of a nucleotide with fluorine;
  • methoxy-modified nucleotide refers to a compound having a structure shown in formula (8) formed by replacing the 2'-hydroxyl group of a nucleotide ribose group with a methoxy group.
  • no more than 3 non-fluorinated modified nucleotides in the antisense strand are 2'-deoxynucleotides, and each of the remaining non-fluorinated modified nucleotides is a methoxy-modified nucleotide; or, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide; the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the siRNA comprising stabilizing modified nucleotides of the present disclosure is a siRNA having the following modifications: Modified siRNA: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I a and the nucleotide sequence I b are each independently fluorinated modified nucleotides, and the nucleotides at the remaining positions in the corresponding sense chain are methoxy-modified nucleotides; in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II a and the nucleotide sequence II b are each independently fluorinated modified nucleotides, the nucleotide at position 3 or 5 in the corresponding antisense chain is a stabilizing modified nucleotide, the nucleotide at position 18 is a 2'-deoxy nucleo
  • the siRNA with the above modification is not only low in cost, but also makes it difficult for ribonucleases in the blood to cut nucleic acids, thereby increasing the stability of nucleic acids and making them more resistant to ribonuclease hydrolysis.
  • the above modification reduces the off-target effect of siRNA and does not significantly reduce the inhibitory performance of siRNA.
  • the first siRNA provided by the present disclosure is one of siANGa1-M1, siANGa1-M2, siANGa2-M1, siANGa2-M2, siANGa3-M1, siANGa3-M2, siANGa4-M1, siANGa4-M2, siANGa5-M1, siANGa5-M2, siANGa6-M1, siANGa6-M2, siANGa7-M1, siANGa7-M2, siANGa8-M1, or siANGa8-M2 listed in Table 1a.
  • the second siRNA provided by the present disclosure is one of siAPOCb1-M1, siAPOCb1-M2, siAPOCb2-M1, siAPOCb2-M2, siAPOCb3-M1, siAPOCb3-M2, siAPOCb4-M1, siAPOCb4-M2, siAPOCb5-M1, siAPOCb5-M2, siAPOCb6-M1, siAPOCb6-M2, siAPOCb7-M1, siAPOCb7-M2, siAPOCb8-M1 or siAPOCb8-M2 listed in Table 1b.
  • the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group having a modified group.
  • the phosphate group having a modified group is a thiophosphate subunit formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate subunit having a structure as shown in formula (1):
  • This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
  • the phosphorothioate subunit linkage is present in At least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the foregoing. In some embodiments, the phosphorothioate subunit linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate subunit linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate subunit linkage is present at at least one of the following positions:
  • the “sense strand” refers to the “first sense strand” and the “second sense strand”
  • the “antisense strand” refers to the “first antisense strand” and the “second antisense strand”.
  • the first siRNA of the present disclosure is one of siANGa1-M1S, siANGa1-M2S, siANGa2-M1S, siANGa2-M2S, siANGa3-M1S, siANGa3-M2S, siANGa4-M1S, siANGa4-M2S, siANGa5-M1S, siANGa5-M2S, siANGa6-M1S, siANGa6-M2S, siANGa7-M1S, siANGa7-M2S, siANGa8-M1S or siANGa8-M2S listed in Table 1a.
  • the first siRNA has a sequence as shown in siANGa1-M1S, siANGa2-M1S, siANGa3-M1S or siANGa4-M1S
  • the second siRNA of the present disclosure is one of siAPOCb1-M1S, siAPOCb1-M2S, siAPOCb2-M1S, siAPOCb2-M2S, siAPOCb3-M1S, siAPOCb3-M2S, siAPOCb4-M1S, siAPOCb4-M2S, siAPOCb5-M1S, siAPOCb5-M2S, siAPOCb6-M1S, siAPOCb6-M2S, siAPOCb7-M1S, siAPOCb7-M2S, siAPOCb8-M1S or siAPOCb8-M2S listed in Table 1b.
  • the second siRNA has a sequence shown in siAPOCb1-M1S, siAPOCb2-M1S, siAPOCb3-M1S or siAPOCb4-M1S.
  • the 5' terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are well known to those skilled in the art, such as 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 as shown in formula (2)
  • the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a nucleotide modified with a thiophosphate as shown in formula (5).
  • the first siRNA of the present disclosure is siANGa1-M1P1, siANGa1-M2P1, siANGa2-M1P1, siANGa2-M2P1, siANGa3-M1P1, siANGa3-M2P1, siANGa4-M1P1, siANGa4-M2P1, siANGa5-M1P1, siANGa5-M2P1, siANGa6-M1P1, siANGa6-M2P1, siANGa7-M1P1, siANGa7-M2P1, siANGa8-M1P1, siANGa8-M2P1, siANGa9-M1P1, siANGa9-M2P1, siANGa10-M1P1, siANGa10-M2P1, siANGa11-M1P1, siANGa11-M2P1, siANGa12-M1P1, siANGa12-M2P1, siANGa13-M1P1, siANGa13-M2P1, siANGa14-M1P1,
  • the second siRNA disclosed herein is siAPOCb1-M1P1, siAPOCb1-M2P1, siAPOCb2-M1P1, siAPOCb2-M2P1, siAPOCb3-M1P1, siAPOCb3-M2P1, siAPOCb4-M1P1, siAPOCb4-M2P1, siAPOCb5-M1P1, siAPOCb5-M2P1, siAPOCb6-M1P1, siAPOCb6-M2P1, siAPOCb7-M1P1, siAPOCb7-M2P1, siAPOCb8-M1P1, siAPOCb8-M2P1, siAPOCb1-M1SP1, siAPOCb1-M2SP1, siAPOCb2-M1SP1, One of siAPOCb2-M2SP1, siAPOCb3-M1SP1, siAPOCb3-M2SP1, siAPOCb4-M1SP1, siAPOCb4-M2SP1, siAPOCb1-M2
  • the inventors of the present disclosure unexpectedly discovered that the first siRNA and the second siRNA provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability and significantly low off-target effects, but also retain very high gene inhibition activity.
  • the first siRNA and the second siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services.
  • the modified nucleotide groups can be introduced into the first siRNA and the second siRNA of the present disclosure by using nucleoside monomers with corresponding modifications.
  • the method of preparing nucleoside monomers with corresponding modifications and the method of introducing the modified nucleotide groups into siRNA are also well known to those skilled in the art.
  • the first RNAi agent comprises one or more first siRNAs and/or one or more first siRNA conjugates, each of which comprises a first siRNA group formed by a first siRNA and a conjugated group conjugated thereto;
  • the second RNAi agent comprises one or more second siRNAs and/or one or more second siRNA conjugates, each of which comprises a second siRNA group formed by a second siRNA and a conjugated group conjugated thereto.
  • the first RNAi agent is a first siRNA conjugate and/or the second RNAi agent is a second siRNA conjugate, that is, at least one of the first RNAi agent and the second RNAi agent is a siRNA conjugate.
  • the first RNAi agent is a first siRNA conjugate
  • the second RNAi agent is a second siRNA conjugate.
  • the conjugated group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery auxiliary group.
  • the first siRNA group, linker and targeting group or delivery auxiliary group are sequentially covalently or non-covalently linked to form a first siRNA conjugate; the second siRNA group, linker and targeting group or delivery auxiliary group are sequentially covalently or non-covalently linked to form a second siRNA conjugate.
  • Each targeting group is selected from a ligand that can bind to a cell surface receptor.
  • each of the targeting groups The delivery auxiliary group is selected from a ligand capable of binding to the cell surface ASGPR; each delivery auxiliary group is selected from a group capable of increasing the biocompatibility of the siRNA conjugate in the target organ or tissue for delivery.
  • conjugation refers to the connection between two or more chemical moieties, each having a specific function, in a covalently linked manner; accordingly, “conjugate” refers to a compound formed by covalently linking the chemical moieties.
  • first siRNA conjugate and second siRNA conjugate represent compounds formed by covalently linking one or more chemical moieties having a specific function to siRNA.
  • siRNA conjugates can be understood as “first siRNA conjugate” and “second siRNA conjugate”, or a general term for multiple siRNA conjugates or a siRNA conjugate represented by a certain chemical formula.
  • conjugation group should be understood as a specific compound that can be conjugated to siRNA through reaction to ultimately form the first siRNA conjugate and the second siRNA conjugate of the present disclosure.
  • the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence.
  • the targeting group is 1-6.
  • the targeting group is 2-4.
  • the first siRNA molecule or the second siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group.
  • the conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the sense strand, also at the 5' end of the antisense strand, and can also be in the internal sequence. In some embodiments, the conjugation site of the first siRNA or the second siRNA and conjugated group is at the 3' end of the sense strand.
  • the conjugated group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugated group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond.
  • the conjugated group is usually connected to the phosphate group of the nucleotide; when the conjugated group is connected to the internal sequence of the siRNA, the conjugated group is usually connected to the ribose sugar ring or the base.
  • connection methods can be referred to in the literature: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.
  • 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.
  • linkers, the types of targeting groups, and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
  • the 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 herein by reference.
  • At least one or each of the targeting groups is selected from ligands that can bind to surface receptors of cells expressing the ANGPTL3 gene and the APOC3 gene.
  • At least one or each of the targeting groups is selected from a ligand that can bind to a mammalian hepatocyte surface receptor (ASGPR).
  • each of the targeting groups is independently a ligand that is affinity-bound to an asialoglycoprotein receptor on the surface of a mammalian hepatocyte.
  • each of the targeting groups is independently an asialoglycoprotein or sugar.
  • each of the targeting groups is independently an asialoglycoprotein, such as asialo serum globulin (ASOR) or asialo fetuin (ASF).
  • each of the targeting groups is independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-mannopyranose, ⁇ -D-glucose, ⁇ -D-glucose, , ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-
  • the linker of the present disclosure has a structure as shown in formula (301):
  • k is an integer from 1 to 3;
  • LA has a structure including an amide bond as shown in formula (302)
  • LB has a structure including an amide bond as shown in formula (303).
  • n 302 , q 302 and p 302 are each independently an integer of 2-6, and optionally, n 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer of 4-16, and optionally, n 303 is an integer of 8-12, Indicates the site to which a group is covalently attached.
  • each LA is connected to one of the targeting groups via an ether bond, and is connected to the LC portion via an ether bond formed by the oxygen atom of the hydroxyl group in the LC portion;
  • LB is connected to the nitrogen atom of the amino group in the LC portion via an amide bond formed by the carbonyl group in formula (303), and is connected to the first siRNA or the second siRNA via a phosphate bond or a phosphorothioate bond formed by the oxygen atom in formula (303).
  • the first siRNA conjugate and the second siRNA conjugate provided by the present disclosure each have a structure as shown in formula (305):
  • Nu represents the first siRNA or the second siRNA provided by the present disclosure.
  • the linker of the present disclosure has a structure shown in formula (306):
  • n 306 is an integer from 0 to 3
  • each p 306 is independently an integer from 1 to 6,
  • the linking group is connected to the targeting group by forming an ether bond through the oxygen atom marked by *
  • the linking group is connected to the first siRNA or the second siRNA by forming a phosphate bond or a phosphorothioate bond through at least one of the oxygen atoms marked by #, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form a hydroxyl group, or are connected to a C 1 -C 3 alkyl group to form a C 1 -C 3 alkoxy group
  • the first siRNA conjugate and the second siRNA conjugate of the present disclosure each have a structure as shown in formula (307):
  • Nu represents the first siRNA or the second siRNA provided by the present disclosure.
  • the first siRNA conjugate and the second siRNA of the present disclosure each have a structure shown in formula (308):
  • n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
  • Each m1, m2 or m3 is independently an integer selected from 2-10;
  • R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each 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 has the structure shown in Formula A59:
  • E 1 is OH, SH or BH 2
  • Nu represents the first siRNA or the second siRNA provided by the present disclosure
  • each M1 represents a targeting group, and its definition and selectable range are the same as above.
  • each M1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
  • L is defined as a linear alkyl for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl resulting from the above substitutions and/or replacements.
  • the length of L is the number of atoms in the chain connecting the two points of attachment.
  • a ring resulting from the replacement of a carbon atom of the linear alkylene group (such as a heterocyclylene or heteroarylene) is counted as one atom.
  • n1 can be an integer of 1-3, and n3 can be an integer of 0-4, ensuring that the number of M1 ligands in the conjugate is at least 2; in some embodiments, n1+n3 ⁇ 2, so that the number of M1 ligands is at least 3, making it easier for M1 ligands to bind to the asialoglycoprotein receptor on the surface of the liver, thereby promoting the conjugate to enter the cell through endocytosis.
  • n1 is an integer of 1-2
  • n3 is an integer of 0-1
  • n1+n3 2-3.
  • the spatial positions between multiple M1 ligands can be made suitable for the binding of M1 ligands to the asialoglycoprotein receptor on the liver surface.
  • m1, m2 and m3 are each independently an integer of 2-5.
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from one of H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 -C 10 alkoxy
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, methyl and ethyl.
  • R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are all H.
  • R 3 is a group of the structure shown in formula A59, wherein E 1 is OH, SH or BH 2 . Based on the consideration of the availability of raw materials for preparation, in some embodiments, E 1 is OH or SH.
  • R 2 is selected to achieve the connection between N and A59 on the nitrogen-containing skeleton.
  • nitrogen-containing skeleton refers to a chain structure in which carbon atoms connected to R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are connected to N. Therefore, R 2 can be any connecting group that can connect the A59 group to N on the nitrogen-containing skeleton in an appropriate manner.
  • the R 2 group needs to contain both a connecting site connected to N on the nitrogen-containing skeleton and a connecting site connected to P in R 3.
  • the site in R 2 that is connected to N on the nitrogen-containing skeleton forms an amide bond with N, and the site that is connected to P on R 3 forms a phosphate bond with P.
  • the length of R 2 is 4-15 atoms.
  • R 2 is B5, B6, B5' or B6':
  • the value range of q 2 can be an integer from 1 to 10. In some embodiments, q 2 is an integer from 1 to 5.
  • L1 is to connect the M1 ligand to the N on the nitrogen-containing skeleton, providing a targeting function for the first siRNA conjugate or the second siRNA conjugate of the present disclosure.
  • L1 is selected from a combination of one or more of the formula A1-A26 groups.
  • L1 is selected from A1, A4, A5, A6, A combination of one or more of A8, A10, A11 and A13; in some embodiments, L1 is selected from a combination of at least two of A1, A4, A8, A10 and A11; in some embodiments, L1 is selected from a combination of at least two of A1, A8, A10.
  • the length of L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms.
  • 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 atoms in length.
  • 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, and in some embodiments, j2 is an integer of 3-5.
  • R' is a C 1 -C 4 alkyl group, and in some embodiments, R' is one of methyl, ethyl and isopropyl.
  • Ra is one of A27, A28, A29, A30 and A31, and in some embodiments, Ra is A27 or A28.
  • Rb is a C 1 -C 5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl and butyl.
  • j1, j2, R', Ra, and Rb are selected in formulas A1-A26 to achieve the connection of the M 1 ligand to the N on the nitrogen-containing skeleton, and to make the spatial position between the M 1 ligands more suitable for the M 1 ligand to bind to the liver surface asialoglycoprotein receptor.
  • the first siRNA conjugate and the second siRNA conjugate of the present disclosure each independently has a structure shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):
  • Nu represents the first siRNA or the second siRNA disclosed in the present invention.
  • the first siRNA conjugate and the second siRNA conjugate each independently have a conjugate of the structure shown in formula (403) or a water-soluble salt thereof, wherein Nu is formed by siRNA shown in one or more of siANGa1-M1S, siANGa2-M1S, siANGa3-M1S, siANGa4-M1S, siAPOCb1-M1S, siAPOCb2-M1S, siAPOCb3-M1S and siAPOCb4-M1S.
  • the siRNA group, the conjugated group is connected to the 3' position of the ribose of the 3' terminal nucleotide of the sense strand of the siRNA group.
  • P in formula A59 can be connected to any possible position in the siRNA sequence, for example, P in formula A59 can be connected to any nucleotide of the sense strand or the antisense strand; in some embodiments, P in formula A59 is connected to any nucleotide of the sense strand. In some embodiments, P in formula A59 is connected to the end of the sense strand or the antisense strand; in some embodiments, P in formula A59 is connected to the end of the sense strand. The end refers to the first 4 nucleotides of the sense strand or the antisense strand from one end thereof.
  • P in formula A59 is connected to the end of the sense strand or the antisense strand; in some embodiments, P in formula A59 is connected to the 3' end of the sense strand.
  • a separate antisense strand can be released to inhibit target gene expression through the RNAi mechanism.
  • the P in formula A59 can be connected 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' position of the nucleotide or the base of the nucleotide. In some embodiments, the P in formula A59 can be connected to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond.
  • the P in formula A59 is connected to the oxygen atom formed after the 3' hydroxyl of the 3' terminal nucleotide of the sense strand is dehydrogenated, or the P in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the sense strand, or the P in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl of the 5' terminal nucleotide of the sense strand.
  • the first siRNA contained in the first siRNA conjugate of the present disclosure can be, for example, any siRNA sequence listed in Table 1a
  • the second siRNA contained in the second siRNA conjugate of the present disclosure can be, for example, any siRNA sequence listed in Table 1b.
  • the pharmaceutical composition formed by the first siRNA conjugate and the second siRNA conjugate of the present disclosure exhibits low off-target effects and high gene expression inhibition activity.
  • P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • S represents a specific stabilizing modification such as moe , wherein the underlined letter combination moe represents that the nucleotide adjacent to the left of the letter combination moe is a nucleotide with 2'-O-methoxyethyl modification.
  • each S is moe .
  • P1 is VP, Ps or P which represent specific modifications, wherein the letter combination VP represents that the nucleotide adjacent to the right side of the letter combination VP is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), the letter combination Ps represents that the nucleotide adjacent to the right side of the letter combination Ps is a nucleotide modified with phosphorothioate, and the capital letter P represents that the nucleotide adjacent to the right side of the letter P is a 5'-phosphate nucleotide.
  • each U in the sequences listed in Tables 1a-1c above can be arbitrarily replaced by T without significantly affecting the activity or off-target effects of the siRNA.
  • first siRNA conjugate and the second siRNA conjugate can be prepared by methods known in the prior art.
  • WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates
  • WO2014025805A1 records the preparation method of the structure shown in formula (305)
  • Rajeev et al. described the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908
  • Chinese patent application CN110959011A discloses in detail the method for preparing the siRNA conjugate shown in formula (308).
  • the contents of the above-mentioned documents are incorporated herein by reference in their entirety.
  • the first siRNA conjugate and the second siRNA conjugate of the present disclosure can be obtained by other methods well known to those skilled in the art.
  • the first RNAi agent and/or the The second RNAi agent also contains at least one of a solvent, a pharmaceutically acceptable carrier or an excipient.
  • a solvent e
  • the first RNAi agent, the second RNAi agent and the third RNAi agent are present in the form of various RNAi preparations commonly used in the art, for example, the RNAi preparation can be a liquid preparation, such as an injection.
  • the liquid preparation can be an injection for subcutaneous injection, an injection for intramuscular injection or an injection for intravenous injection.
  • all siRNA and siRNA conjugates in the first RNAi agent and/or the second RNAi agent and/or the third RNAi agent are present in the form of powder, for example, in the form of a lyophilized powder injection. When administering, the lyophilized powder injection is mixed with a solvent to prepare a liquid preparation.
  • the solvent can include various solvents such as common solvents such as water, deionized water, ethanol, and pH buffer.
  • the pH buffer can be a tris hydroxymethylaminomethane hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.
  • the amount of the solvent is adjusted according to the required injection concentration.
  • the solution dissolves siRNA or siRNA to prepare a solution with a concentration of 0.01mg/mL-200mg/mL siRNA, 20mg/mL-200mg/mL/, 50mg/mL-100mg/mL, 0.01mg/mL-5mg/mL siRNA, 0.1mg/mL-5mg/mL/, 0.5mg/mL-3mg/mL.
  • the required concentration those skilled in the art can easily determine the content of the solvent.
  • the first RNAi agent, the second RNAi agent and the third RNAi agent are all in the form of a preparation for subcutaneous injection.
  • the first RNAi agent, the second RNAi agent and the third RNAi agent may also be a spray formulation administered to the lungs by spray or to other organs and tissues (such as the liver) through the lungs, or an inhalation formulation inhaled through the oropharynx, or a pharmaceutical preparation administered by nasal administration, etc.
  • the first component contains at least one of siRNA and siRNA conjugate and a pharmaceutically acceptable carrier and/or excipient, and the types and contents of the carrier and/or excipient in the injection for subcutaneous injection, the injection for intramuscular injection, the injection for intravenous injection, the spray formulation administered to the lungs by spray or to other organs and tissues (such as the liver) through the lungs, the inhalation formulation inhaled through the oropharynx, or the pharmaceutical preparation administered by nasal administration, etc. are well known to those skilled in the art.
  • 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 nanoparticles,
  • the invention can be selected from the group consisting of carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic/glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), poly(2-dimethylaminoethyl methacrylate), and one or more of their derivatives.
  • PKI polyethyleneimine
  • PAMAM polyamidoamine
  • DOTAP 1,2-dioleoyl-3-
  • the pharmaceutical composition there is no particular requirement for the content of siRNA or siRNA conjugate and pharmaceutically acceptable carrier.
  • the weight ratio of siRNA or siRNA conjugate to pharmaceutically acceptable carrier can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).
  • the pharmaceutical composition may further include other pharmaceutically acceptable excipients or solvents, and the excipients may be one or more of various preparations or compounds conventionally used in the art.
  • the other pharmaceutically acceptable excipients may include a protective agent and/or an osmotic pressure regulator.
  • the solvent may include a pH buffer or water, etc.
  • the pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.
  • 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 can be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole/kilogram (mOsm/kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
  • the dosage of the preparation made of the pharmaceutical composition during administration can be adjusted due to different administration methods.
  • the pharmaceutical composition may be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient to prepare a liquid preparation during administration.
  • the liquid preparation may be, but is not limited to, administered subcutaneously, intramuscularly or intravenously, or administered by spraying into the lungs.
  • the pharmaceutical composition may be delivered to the esophagus, or to other organs (such as the liver) through the lungs by spraying, or by inhalation through the oropharynx, or by nasal administration.
  • the pharmaceutical composition is for subcutaneous injection.
  • the pharmaceutical composition can be in the form of a liposome preparation.
  • the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and/or a pegylated lipid.
  • the organic amine, auxiliary lipid and pegylated lipid can be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, auxiliary lipid and pegylated lipid described in Chinese patent application CN103380113A (incorporated herein in its entirety by reference).
  • the organic amine may be a compound described in Chinese patent application CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:
  • X101 and X102 are each independently O, S, NA or CA, wherein A is hydrogen or a C1 - C20 hydrocarbon chain;
  • Y 101 and Z 101 are each independently C ⁇ O, C ⁇ S, S ⁇ O, CH—OH or SO 2 ;
  • R101 , R102 , R103 , R104, R105 , R106 and R107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;
  • x is an integer from 1 to 10;
  • R 103 and the nitrogen in formula (201) form The structure shown in formula (202) or formula (203):
  • HCC represents a hydrocarbon chain
  • 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 linear alkyl or alkenyl group having 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 can be any one of the following formulas (204) to (213):
  • each "HCC” represents a hydrocarbon chain
  • each * shows a possible connection point between R 103 and the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve connection with the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):
  • the helper lipid is cholesterol, a cholesterol analog and/or a cholesterol derivative.
  • the PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
  • the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).
  • the pharmaceutical composition particles formed by the first siRNA and the second siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm. More typically, the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, 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 ratio (weight/weight ratio) of the first siRNA and the second siRNA to all lipids is in the range of 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, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1:6 to about 1:10.
  • the weight ratio of the first and second siRNAs of the present disclosure to the total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • each component of the pharmaceutical composition can exist independently when it is sold, and can exist in the form of a liquid preparation when it is used.
  • the pharmaceutical composition formed by the first siRNA and the second siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the first siRNA and the second siRNA provided by the present disclosure replace the existing siRNA; in some embodiments, it can be prepared according to the following method:
  • the organic amine, the auxiliary lipid and the PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, for example, 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, one or more, for example, ethanol.
  • the first siRNA or the second siRNA provided by the present disclosure is dissolved in a buffered saline solution, respectively.
  • the concentration of the buffered saline solution is 0.05-0.5M, for example, 0.1-0.2M, and the pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2, and the amount of the buffered saline solution is such that the concentration of the siRNA aqueous solution does not exceed 0.6mg/mL, for example, 0.2-0.4mg/mL.
  • the buffered salt is selected from one or more of soluble acetates and soluble citrates, 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 an incubated liposome preparation.
  • the volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), for example, 1:4.
  • the incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilized to obtain the pharmaceutical composition provided by the present disclosure, whose physicochemical parameters are pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400mOsm/kg; for example, the physicochemical parameters can be pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400mOsm/kg.
  • the concentration or dilution can be performed before, after or simultaneously with the removal of impurities.
  • the impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is a phosphate buffer solution (PBS) with a pH of 7.4.
  • the sterilization method can adopt various existing methods, such as filtration sterilization on a 0.22 ⁇ m filter.
  • the pharmaceutical composition contains only the first RNAi agent and the second RNAi agent, the first RNAi agent contains only one first siRNA conjugate or the second RNAi agent contains only one second siRNA conjugate.
  • the first RNAi agent contains only one first siRNA conjugate
  • the second RNAi agent contains only one second siRNA conjugate.
  • the first RNAi agent and the second RNAi agent are present in separate preparations for subcutaneous injection, that is, the first RNAi agent and the second RNAi agent are stored separately and can be used separately. In some embodiments, the first RNAi agent and the second RNAi agent are present in a uniformly mixed preparation for subcutaneous injection.
  • the present disclosure provides methods for treating and/or preventing diseases or symptoms associated with dyslipidemia
  • the present disclosure also provides a method for treating and/or preventing diseases or symptoms associated with dyslipidemia, the method comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof, wherein the pharmaceutical composition is selected from the pharmaceutical composition described above.
  • the first RNAi agent and the second RNAi agent in the pharmaceutical composition exist as two separate preparations during the preparation of the drug. In some embodiments, the first RNAi agent and the second RNAi agent in the pharmaceutical composition exist as one preparation during the preparation of the drug.
  • the present disclosure also provides a method for treating and/or preventing a disease or symptom associated with dyslipidemia, the method comprising administering an effective amount of a first RNAi agent and a second RNAi agent to a subject in need thereof, the first RNAi agent comprising one or more first siRNAs and/or one or more first siRNA conjugates, the first siRNA comprising a first sense strand and a first antisense strand, each nucleotide in the first siRNA being independently a modified or unmodified nucleotide, wherein the first sense strand comprises a nucleotide sequence I a , the first antisense strand comprises a nucleotide sequence II a , the nucleotide sequence I a and the nucleotide sequence II a are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I a is at least 15 consecutive nucleotides consistent with the nucleotide sequence fragment shown in any
  • the second RNAi agent comprises one or more second siRNAs and/or one or more second siRNA conjugates, the second siRNA comprises a second sense strand and a second antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the second sense strand contains a nucleotide sequence I b , the second antisense strand contains a nucleotide sequence II b , the nucleotide sequence I b and the nucleotide sequence II b are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I b is at least 15 consecutively identical to the nucleotide sequence fragment shown in SEQ ID NO: 98 or 99; each of the second siRNA conjugates comprises a second siRNA group formed by the second siRNA and a conjugated group conjugated thereto.
  • the first RNAi agent and the second RNAi agent can be administered simultaneously, and the total amount of siRNA and siRNA group can be used to administer 0.1 mg/kg to 10 mg/kg of the subject's body weight of the pharmaceutical composition to the subject.
  • the dosage can be 0.5 mg/kg to 3 mg/kg of the subject's body weight of the pharmaceutical composition.
  • the treatment method provided by the present disclosure can also be administered in the manner of separately administering the first RNAi agent and the second RNAi agent, and the subject is administered 0.1mg/kg to 10mg/kg of the first RNAi agent and 0.1mg/kg to 10mg/kg of the second RNAi agent, respectively, based on the total amount of siRNA and siRNA groups.
  • the subject is administered 0.5mg/kg to 3mg/kg of the first RNAi agent and 0.5mg/kg to 10mg/kg of the second RNAi agent, respectively.
  • the first RNAi agent is administered after the subject is administered the second RNAi agent after the subject is administered the second RNAi agent after the first RNAi agent is administered after an interval of 0.5min to 30min.
  • the first RNAi agent is administered after an interval of 1min.
  • the first RNAi agent and the second RNAi agent are usually administered to a subject in need thereof by subcutaneous injection.
  • the disease or condition associated with dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.
  • the present disclosure provides the use of the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating and/or preventing a disease or symptom associated with dyslipidemia.
  • the disease or symptom associated with dyslipidemia in the present disclosure is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.
  • the present disclosure also provides a method for inhibiting the expression levels of ANGPTL3 mRNA and APOC3 mRNA in cells, the method comprising contacting the cells with an effective dose of the pharmaceutical composition of the present disclosure.
  • the purpose of preventing and/or treating pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through the mechanism of regulating gene expression.
  • compositions provided by the present disclosure can be used to prevent and/or treat the pathological conditions or diseases, or to prepare medicaments for preventing and/or treating the pathological conditions or diseases described herein.
  • administration/administration refers to placing a pharmaceutical composition into a subject's body by a method or approach that allows the pharmaceutical composition to be at least partially positioned at a desired site to produce a desired effect.
  • Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more of the pharmaceutical composition being delivered to a specific site than to the entire body of the subject; whereas systemic administration results in the pharmaceutical composition being delivered to substantially the entire body of the subject.
  • the present disclosure is intended to provide a means for preventing and/or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, it is a mode of administration that is capable of delivering the drug to the liver.
  • the drug may be administered to a 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, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including oral administration and sublingual administration).
  • the administration frequency may be once or more every day, every week, every two weeks, every three weeks, every month, or every year.
  • the dosage of the pharmaceutical composition disclosed in the present invention may be a conventional dosage in the art, which may be determined based on various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy may be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determination of LD50 (a dose that causes 50% of the population to die) and ED50 (a dose that causes 50% of the maximum response intensity in quantitative reactions, and a dose that causes 50% of the experimental subjects to have a positive reaction in qualitative reactions).
  • the range of human dosages may be derived based on data obtained from cell culture analysis and animal studies.
  • the dosage of the pharmaceutical composition can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in further embodiments 0.05-20 mg/kg body weight, in further embodiments 0.1-15 mg/kg body weight, and in yet further embodiments 0.1-10 mg/kg body weight.
  • the above dosage may be preferred.
  • the cell is a hepatocyte.
  • the hepatocyte can be a cell selected from a hepatoma cell line such as Hep3B, HepG2, Huh7, or an isolated primary hepatocyte, and in some embodiments, a primary hepatocyte.
  • the dosage of siRNA and siRNA groups in the provided pharmaceutical composition is that those skilled in the art are easily determined according to the effect obtained by expectation.
  • the dosage of siRNA and siRNA groups in the provided pharmaceutical composition is such an amount: it is enough to reduce the expression of the target gene, and causes 1pM to 1 ⁇ M or 0.01nM to 100nM or 0.05nM to 50nM or to the extracellular concentration of about 5nM at the target cell surface.
  • the amount required for reaching the local concentration will vary with various factors, and the factors include delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, whether delivery is local or systemic, etc.
  • the concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.
  • the present disclosure provides a kit, which comprises the pharmaceutical composition provided by the present disclosure.
  • the kit described herein may provide a pharmaceutical composition in one container.
  • the kit described herein may include a container for providing a pharmaceutically acceptable excipient.
  • the kit may also include other ingredients, such as stabilizers or preservatives.
  • the kits described herein may include at least one other therapeutic agent in a container other than the container in which the pharmaceutical composition described herein is provided.
  • the kits may include instructions for mixing the siRNA or siRNA conjugate with a pharmaceutically acceptable carrier and/or excipient or other ingredients, if any.
  • the siRNA or siRNA conjugate and/or pharmaceutically acceptable carrier and adjuvant can be provided in any form, such as liquid form, dry form or lyophilized form.
  • the siRNA or siRNA conjugate and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile.
  • sterile water can be provided in the kit of the present disclosure.
  • the reagents and culture media used in the following examples are commercially available, and the nucleic acid electrophoresis, real-time PCR and other operations used are carried out in accordance with the methods described in Molecular Cloning (Cold Spring Harbor L Boratory Press (1989)).
  • conjugates 1-2 in Table 2 below were prepared, with the only difference being that the sense strand and antisense strand of siRNA contained in each siRNA conjugate were as shown in Table 2. According to the nucleic acid sequences of siRNA numbered as conjugate 1-conjugate 2 in Table 2 below, the sense strand and antisense strand of siRNA were synthesized respectively.
  • siRNA conjugates 1-2 were diluted to a concentration of 0.2 mg/mL (in terms of siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 M ⁇ *cm (25°C)), the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass SP1ectrometry, purchased from Waters, model: LCT Premier). The measured values are consistent with the theoretical values, indicating that the synthesized conjugates 1-2 are the double-stranded nucleic acid sequences of the target design.
  • LC-MS liquid chromatography-mass spectrometer
  • Each siRNA conjugate has a structure shown in formula (403), and the siRNA groups contained in the siRNA conjugates have siRNA sequences corresponding to conjugates 1-2 in Table 2, respectively, the conjugate groups are connected to the ribose 3' position of the 3' terminal nucleotide of the sense strand of the siRNA group, and the siRNA conjugates are in the form of sodium salts.
  • Conjugate 1 belongs to the aforementioned first siRNA conjugate
  • conjugate 2 belongs to the aforementioned second siRNA conjugate.
  • capital letters C, G, U, A, and T represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the underlined letter combination moe indicates that the nucleotide adjacent to the left of the letter combination moe is a ribose 2'-O-methoxyethyl-modified nucleotide; lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate subunits.
  • This experimental example investigates the effect of the pharmaceutical composition obtained by combining Conjugate 1 and Conjugate 2 on lowering blood lipids in human APOC3 transgenic mice.
  • mice in each group were randomly divided into 4 groups according to serum TG content> 2mmol/L, 5 mice in each group were male, and the drug dosage of all animals was calculated according to body weight.
  • the first group of mice was injected with 1.5mg/kg of conjugate 1
  • the second group of mice was injected with 1.5mg/kg of conjugate 2
  • the third group of mice was injected with a single dose mixture containing 0.75mg/kg conjugate 1 and 0.75mg/kg conjugate 2 (i.e., the drug composition group)
  • the fourth group of mice was injected with PBS solution.
  • the dosage of the three groups of mice was calculated based on the amount of siRNA contained therein, and the volume was 5mL/kg.
  • each conjugate was provided in a PBS aqueous solution, and according to the dosage and dosage volume, the concentration of 0.75mg/kg conjugate to be configured was converted to 0.15mg/mL, and the concentration of 1.5mg/kg conjugate to be configured was 0.3mg/mL.
  • the day of administration was recorded as day 1, and blood was collected from the orbital venous plexus of mice on days 8, 15, 22, 29, and 36 after administration, and serum lipid levels were detected at each time point.
  • the amount of blood collected from the orbital venous plexus was about 100 ⁇ L each time, and the supernatant was separated from the plasma after centrifugation at 1800 g centrifugal force and 2°C-8°C for 15 minutes, and the contents of triglycerides (TG) and total cholesterol (TC) in the serum were further detected using PM1P000/3 fully automatic serum biochemical analyzer (SABA, Italy).
  • Normalized blood lipid level (blood lipid content after administration/blood lipid content before administration) ⁇ 100%.
  • the inhibition rate of blood lipid level (1-blood lipid content after administration/blood lipid content before administration) ⁇ 100%.
  • the blood lipid level here refers to the levels of triglycerides and total cholesterol.
  • FIG1 is a line graph showing relative serum TG levels at different time points after administration of PBS, conjugates or pharmaceutical compositions in human APOC3 transgenic mice.
  • FIG2 is a line graph showing the relative levels of serum TC at different time points in human APOC3 transgenic mice after administration of PBS, conjugate or pharmaceutical composition.
  • the maximum inhibition rate of mouse plasma TG level by 1.5 mg/kg dose of conjugate 2 and the pharmaceutical composition containing only 0.75 mg/kg conjugate 1 and 0.75 mg/kg conjugate 2 is comparable, both of which are about 86%.
  • the TG level in mice given only 1.5 mg/kg dose of conjugate 2 recovered faster, indicating that the inhibition rate has decreased, and it is difficult to maintain the inhibition effect during the entire test period.
  • the experimental results show that the pharmaceutical composition of the present invention has better TG long-term inhibition than conjugate 2; on the other hand, the inhibition of TG level by 1.5 mg/kg dose of conjugate 1 and the pharmaceutical composition containing only 0.75 mg/kg conjugate 1 and 0.75 mg/kg conjugate 2 maintains a comparable inhibition rate for a long time, however, the maximum inhibition rate of TG level by 1.5 mg/kg dose of conjugate 1 is 74.17%, which is significantly lower than the maximum inhibition rate of TG level by the pharmaceutical composition. This indicates that at the same dose, the pharmaceutical composition of the present disclosure can reach the maximum inhibitory effect faster and can inhibit blood lipid levels more quickly and effectively than conjugate 1.
  • the maximum inhibition rate of 1.5 mg/kg of conjugate 1 and 1.5 mg/kg of conjugate 2 on mouse plasma TC levels is 59.72%, while the maximum inhibition rate of TC levels of the pharmaceutical composition containing only 0.75 mg/kg of conjugate 1 and 0.75 mg/kg of conjugate 2 can reach 66.51%, which is unexpectedly higher than the maximum inhibition rate of 1.5 mg/kg of conjugate 1 and 1.5 mg/kg of conjugate 2 administered alone, and has the long-term effect of inhibiting TC levels as 1.5 mg/kg of conjugate 1, and is significantly better than 1.5 mg/kg of conjugate 2.
  • the pharmaceutical composition of the present invention can achieve a good balance between a higher maximum blood lipid inhibition effect and a longer-lasting blood lipid inhibition level at the same total siRNA dose, and thus shows a good potential for preventing and/or treating symptoms or diseases associated with dyslipidemia.

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Abstract

药物组合物，包含第一RNAi剂和第二RNAi剂，第一RNAi剂包含第一siRNA和/或第一siRNA缀合物；第二RNAi剂包含第二siRNA和/或第二siRNA缀合物；第一siRNA包含第一正义链和第一反义链，该第一正义链含有与SEQ ID NO:93-97中任意一项有至少15个核苷酸连续一致的核苷酸序列I a；第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，该第二siRNA包含第二正义链和第二反义链，所述第二正义链含有与SEQ ID NO:98或99有至少15个核苷酸连续一致的核苷酸序列I b。药物组合物和治疗方法可以有效治疗和/或预防与血脂异常相关的疾病或症状。

Description

一种核酸、含有该核酸的组合物与缀合物及其用途 技术领域
本公开涉及一种药物组合物，该药物组合物包括可靶向ANGPTL3 mRNA的第一RNAi剂和可靶向APOC3 mRNA的第二RNAi剂，以及该药物组合物用来治疗和/或预防与血脂异常相关的疾病或症状的方法。
背景技术
高脂血症为血脂异常的一种，是脂肪代谢或运转异常，使血浆脂质高于正常值的一种全身性疾病，正严重威胁着全球患者的健康。现有的治疗血脂异常的药物主要有他汀类、胆固醇吸收抑制剂、树脂类、普罗步考、贝特类和烟酸及其衍生物。
血管生成素样蛋白3(ANGPTL3)是一种主要在肝脏表达的分泌蛋白，因其基因结构与血管生成素相似得名。现有研究证实血脂异常与ANGPTL3的高表达量是有相关性的，ANGPTL3通过与脂肪组织结合，抑制脂蛋白脂肪酶的活性来调节脂质代谢。ANGPTL3的低表达可以减缓血脂异常引起的动脉粥样硬化。载脂蛋白C3(APOC3)在脂质代谢中具有重要的作用，携带有APOC3突变基因的人血液循环中APOC3的表达量下降46％，血浆内甘油三酯水平较普通人下降39％。
小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以序列特异性的方式抑制或阻断任何感兴趣的目的基因的表达，从而达到治疗疾病的目的。因此，若能从基因水平沉默基因表达，阻断ANGPTL3和APOC3的生成，无疑将是最为理想的治疗手段。然而，在目前研究中，发现单独使用抑制APOC3或ANGPTL3 mRNA的siRNA，难以同时获得较高的最大抑制效率和较长的持续抑制时间的良好平衡。
发明内容
为了提供一种能够显著降低血脂水平的药物组合物，本发明的发明人意外发现，通过采用将能够靶向ANGPTL3 mRNA和靶向APOC3 mRNA的两种siRNA联合给药的方式，能够降低血浆中的血脂，尤其是能够显著降低血浆中的甘油三酯含量。并且能够同时获得较高的最大抑制效率和较长的持续抑制时间的良好平衡。
在一方面，本公开提供了一种药物组合物，所述药物组合物包含第一RNAi剂和第二RNAi剂，所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，所述第一siRNA包含第一正义链和第一反义链，所述第一siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，所述核苷酸序列Ia和所述核苷酸序列IIa至少部分反向互补形成双链区，其中，所述核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列有至少15个核苷酸连续一致；每种所述第一siRNA缀合物包含一种所述第一siRNA形成的第一siRNA基团和与其缀合的缀合基团；
所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列有至少15个核苷酸连续一致；所述第二siRNA缀合物包含一种所述第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
在另一方面，本公开还提供了一种本公开所述的药物组合物在制备用作治疗和/或预防与血脂异常相关的疾病或症状的药物中的用途。
在又一方面，本公开还提供了一种治疗和/或预防与血脂异常相关的疾病或症状的方法，所述方法包括向有需要的受试者给予上述第一RNAi剂和第二RNAi剂。
在又一方面，本公开还提供了一种抑制细胞中ANGPTL3 mRNA和APOC3mRNA表达水平的方法，所述方法包括将有效量的本公开所述的药物组合物与所述细胞接触。
以引用的方式并入
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本文的程度相同。
有益效果
本公提供的药物缀合物具有较好的降血脂作用和较长的长效性，例如，在人 APOC3转基因小鼠中，在siRNA总剂量相同的给药剂量下，与单一种类的siRNA缀合物相比，本公开的药物组合物与缀合物2具有相当的最大抑制率，显著高于缀合物1，并且本公开药物组合物具有与缀合物1相当的长效性，也显著高于缀合物2的持续抑制效果；对于对小鼠血浆中TC水平的抑制，药物组合物具有比单独给予缀合物1或缀合物2更高的最大抑制率，且药物组合物具有和缀合物1相当的长效性，比缀合物2的持续抑制效果更优。可见，与单独给予抑制ANGPTL3或APOC3 mRNA的siRNA相比，本公开的药物组合物在相同的总siRNA剂量下，能够达到更高的血脂最大抑制效果和更长效的血脂抑制水平的良好平衡，因此显示出具有预防和/或治疗与血脂异常相关的症状或疾病的良好潜力。
附图说明
图1为在人APOC3转基因小鼠中，给予PBS、缀合物或药物组合物后，不同时间点时的血清TG相对水平的折线图。
图2为人APOC3转基因小鼠中，给予PBS、缀合物或药物组合物后，不同时间点时的血清TC相对水平的折线图。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
在本公开中，如无其他说明，ANGPTL3 mRNA或“ANGPTL3基因表达的mRNA”是指具有如Genbank注册号NM_014495.4所示序列的mRNA，“ANGPTL3靶基因”即ANGPTL3基因是指转录上述ANGPTL3 mRNA的基因。APOC3 mRNA或“APOC3基因表达的mRNA”是指具有如Genbank注册号NM_000040.3所示序列的mRNA，“APOC3靶基因”即APOC3基因是指转录上述APOC3 mRNA的基因。
定义
在上文及下文中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸，在一 些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。
在上文及下文中，所述“氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。“核苷酸类似物”指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。所述“甲氧基修饰的核苷酸”指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在本文的上下文中，表述“互补”或“反向互补”可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基各自与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
在上文及下文中，如无特别说明，“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。
在上文及下文中，特别是在描述本公开的siRNA、药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)是指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNA phosphoramidites也称为Nucleoside phosphoramidites)。 亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。
在上文或下文中，“经取代的”或“被取代的”基团，如经取代的烷基、经取代的烷氧基、经取代的氨基、经取代的脂族基团、经取代的杂脂族基团、经取代的酰基、经取代的芳基或经取代的杂芳基。其中，如无其他说明，“经取代的”或“被取代的”基团是指该基团中的氢原子被一个或多个取代基所替代而形成的基团。例如，“经取代的烷氧基”是指烷氧基中的一个或多个氢原子被取代基所替代而形成的基团。本领域技术人员能够理解，可用于本公开应用的化合物中可以包含各种取代基，只要是该取代基的引入不会影响本公开的功能，能够实现本公开的目的，就可用于本公开。在一些实施方式中，所述取代基选自于由以下基团所组成的组：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、-CN、-NO2、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤代烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。在一些实施方式中，所述取代基是C1-C3烷基、C6-C8芳基、-OC1-C3烷基、-OC1-C3烷基苯基、卤素、-OH、-NH2、-CN或-NO2中的一种。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C1-C6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥连接的碳原子。
如本文所使用的，“芳基”是指通过从环碳原子中除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本文所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的至少一个环是完全不饱和的，即， 包含根据Hückel理论的环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。在一些实施方式中，杂芳基中的杂原子是氧化的杂原子。在一些实施方式中，杂芳基中包含一个或多个氮原子。在一些实施方式中，杂芳基中的氮原子中的一个或多个是季铵化的氮原子。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1,3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1,4]二噁庚英基(benzo[b][1,4]dioxepinyl)、苯并[b][1,4]噁嗪基(benzo[b][1,4]oxazinyl)、1,4-苯并二噁烷基(1,4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3,2-d]嘧啶基、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6,7-二氢-5H-环戊烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6-二氢苯并[h]喹唑啉基(5,6-dihydrobenzo[h]quinazolinyl)、5,6-二氢苯并[h]噌啉基(5,6dihydrobenzo[h]cinnolinyl)、6,7-二氢-5H-苯并[6,7]环庚烷并[1,2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3,2-c]吡啶基、5,6,7,8,9,10-六氢环辛烷并[d]嘧啶基、5,6,7,8,9,10-六氢环辛烷并[d]哒嗪基、5,6,7,8,9,10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5,8-甲醇-5,6,7,8-四氢喹唑啉基(5,8-methano-5,6,7,8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1,6-萘啶酮基(1,6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5,6,6a,7,8,9,10,10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3,4-d]嘧啶基、吡啶基、吡啶并[3,2-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5,6,7,8-四氢喹唑啉基、5,6,7,8-四氢苯并[4,5]噻吩并[2,3-d]嘧啶基、6,7,8,9-四氢-5H-环庚烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6,7,8-四氢吡啶并[4,5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2,3-d]嘧啶基、噻吩并[3,2-d]嘧啶基、噻吩并[2,3-c]吡啶基(thieno[2,3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。
如本文所使用的，“卤素取代基”或“卤代”指氟代、氯代、溴代和碘代，术 语“卤素”包括氟、氯、溴和碘。
如本文所使用的，“卤代烷基”是指指定数量的氢原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能度对特定的反应条件不敏感，并且可以在分子中的该官能度上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-二甲氧基三苯甲基)和TMTr(4,4',4”-三甲氧基三苯甲基)。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、兔、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗”指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的，“预防”指的是获得有益的或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将双链siRNA、药物组合物或siRNA缀合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种生理症状的受试者，即便可能该疾病的诊断尚未作出。如本文所述的，“预防与高血脂相关的疾病或症状”是指当所述受试者开始出现血脂升高倾向时，通过给予受试者本公开的药物组合物避免受试者体内血脂升高至异常水平，所述异常水平指人类受试者体内总胆固醇(TC)水平超过6.2mmol/L，甘油三酯(TG)水平超过2.3mmol/L，和/或低密度脂蛋白胆固醇(LDL-C)水平超过4.1mmol/L。
本公开所述的siRNA、药物组合物或siRNA缀合物中，所述siRNA或siRNA缀合物可以以成盐的形式或未成盐的形式存在。本公开所述的siRNA、药物组合物或siRNA缀合物中，所述siRNA或siRNA缀合物可以部分或全部的水可溶性盐的形式存在，所述水可溶性盐可以是其胺盐或碱金属盐，在一些实施方式中，所述碱金属盐可以是钾盐或钠盐。在一些实施方式中，所述胺盐可以是铵盐、甲胺盐或三乙胺盐。在一些实施方式中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH4 +，有机铵阳离子中的一种。有机铵阳离子可以是甲胺形成的阳离子、三乙胺形成的阳离子或季铵阳离子。出于提高溶解性考虑，在一种实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K+和/或Na+，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N,N-二异丙基乙胺形成的铵离子。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。
本公开的药物组合物
在一方面，本公开提供了一种能够降低血脂水平，尤其是能够降低血浆中甘油三酯含量的药物组合物。
本公开的药物组合物包含第一RNAi剂和第二RNAi剂，所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，所述第一siRNA包含第一正义链和第一反义链，所述第一siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，所述核苷酸序列Ia和所述核苷酸序列IIa至少部分反向互补形成双链区，其中，所述核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列有至少15个核苷酸连续一致；每种所述第一siRNA缀合物包含所述第一siRNA形成的第一siRNA基团和与其缀合的缀合基团；
所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核 苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列有至少15个核苷酸连续一致；所述第二siRNA缀合物包含所述第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
本公开提供的药物组合物中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，第一RNAi剂和第二RNAi剂的重量比为1:(0.1-10)。在一些实施方式中，第一RNAi剂和第二RNAi剂的重量比为1:(0.5-5)。在一些实施方式中，第一RNAi剂和第二RNAi剂的重量比为1:(0.5-2)。在一些实施方式中，第一RNAi剂和第二RNAi剂的重量比为1:1、1:2或2:1。
在一些实施方式中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，所述药物组合物中第一RNAi剂的重量为1mg–1000mg，例如可以为1mg–800mg、1mg–600mg、1mg–500mg、1mg–300mg、1mg–250mg、1mg-100mg。在一些实施方式中，所述第一RNAi剂的重量为5mg-500mg。在一些实施方式中，所述第二RNAi剂的重量为1mg–1000mg，例如可以为1mg–800mg、1mg–600mg、1mg–500mg、1mg–300mg、1mg–250mg、1mg-100mg。在一些实施方式中，所述药物组合物中第一RNAi剂的重量为5mg-500mg。在一些实施方式中，所述第一RNAi剂的重量为5mg-500mg，所述第二RNAi剂的重量为5mg-500mg。
第一RNAi剂与第二RNAi剂
如前所述，本公开提供的药物组合物包含第一RNAi剂和第二RNAi剂。所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物。第一siRNA包含第一正义链和第一反义链，第一siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，核苷酸序列Ia和核苷酸序列IIa至少部分反向互补形成双链区，其中，核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列有至少15个核苷酸连续一致。
AGCCAAAAUCAAGAUUUGCUAUGUUAGACGAUGUAAAA  (SEQ ID NO:93)
UCAACAUAUUUGAUCAGUCUUUUUAUGAU  (SEQ ID NO:94)
CCAAGCCAAGAGCACCAAGAACUACUCCC  (SEQ ID NO:95)
GUAGUCCAUGGACAUUAAUUCAACAUCGA  (SEQ ID NO:96)
GUGUGGAGAAAACAACCUAAAUGGUAAAU  (SEQ ID NO:97)
在一些实施方式中，核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-AAAUCAAGAUUUGCUAUGZa1-3'(SEQ ID NO:1)；
5'-Za2CAUAGCAAAUCUUGAUUU-3'(SEQ ID NO:2),
其中，Za1为U，Za2为A，核苷酸序列Ia中包含位置对应于Za1的核苷酸Za3，核苷酸序列IIa中包含位置对应于Za2的核苷酸Za4，Za4是反义链5'末端的第一个核苷酸。
在本公开的上文与下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置，例如，核苷酸序列Ia的3'端第一个核苷酸是位置对应于SEQ ID NO:1的第1个核苷酸的核苷酸。
所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列有至少15个核苷酸连续一致：
GUUGCUUAAAAGGGACAGUAUUCUCAGUGCUCUCCUACCCCA  (SEQID NO:98)
CCUCCCAAUAAAGCUGGACAAGAAGCUGC  (SEQ ID NO:99)
在一些实施方式中，核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列长度相等，且不多于3个核苷酸差异，核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
5'-UUAAAAGGGACAGUAUUCZb1-3'(SEQ ID NO:46)；
5'-Zb2GAAUACUGUCCCUUUUAA-3'(SEQ ID NO:47),
其中，Zb1为U，Zb2为A，核苷酸序列Ib中包含位置对应于Zb1的核苷酸Zb3，核苷酸序列IIb中包含位置对应于Zb2的核苷酸Zb4，Zb4是反义链5'末端的第一个核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIa和核苷酸序列IIb的第3-6个核苷酸中的至少1个为稳定化修饰核苷酸，所述稳定化修饰核苷 酸指核苷酸的核糖2'位羟基被稳定化修饰基团取代的核苷酸，与相应位置的核苷酸为未修饰的核苷酸的siRNA相比，包含所述稳定化修饰核苷酸的第一siRNA和第二siRNA的热稳定性增加，并且所述稳定化修饰基团的空间位阻大于2'-O-甲基。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第3个和/或第5个核苷酸为稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第3个核苷酸为所述稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第5个核苷酸为所述稳定化修饰核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第3-9个核苷酸中不超过2个核苷酸为所述稳定化修饰核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第3-9个核苷酸之外的核苷酸均不是稳定化修饰核苷酸。若核苷酸序列II中在第3-6个核苷酸中的至少1个为稳定化修饰的核苷酸的同时，在第3-9个核苷酸之外包含稳定化修饰核苷酸，可能会显著影响该siRNA的目标序列表达水平的调节能力。
通过对特定位置处稳定化修饰核苷酸的个数进行限定，本公开的第一siRNA和第二siRNA可获得较佳的药学活性与低毒性的平衡，同时还具有优异的稳定性。
在一些实施方式中，本公开的上下文中“热稳定性增加”是指siRNA的双链热解离温度(Tm)升高。在一些实施方式中，“热稳定性增加”是指siRNA的Tm升高至少0.05℃，在一些实施方式中指siRNA的Tm升高0.1-6℃。在一些实施方式中指siRNA的Tm升高0.5-4℃。不受理论解释限制地，通过在特定位置包含稳定化修饰核苷酸，本公开的第一siRNA中第一反义链与ANGPTL3基因表达的mRNA或第二siRNA中第二反义链与APOC3基因表达的mRNA结合能力基本不受影响，而与脱靶目标mRNA之间的结合显著降低，从而降低甚至消除脱靶效应。
在一些实施方式中，每个所述稳定化修饰基团独立地具有-X-R所示的结构，其中，X为O、NR'、S或SiR'2；R为C2-C6烷基、取代的C2-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，每个R'独立地为H、C1-C6烷基、取代的C1-C6烷基、C6-C8芳基、取代的C6-C8芳基中的一种，所述取代的C2-C6烷基、取代的 C6-C8芳基或取代的C1-C6烷基是指C2-C6烷基、C6-C8芳基或C1-C6烷基中的一个或多个氢原子被取代基取代而形成的基团，所述取代基选自以下取代基中的一种或多种：C1-C3烷基、C6-C8芳基、C1-C3烷氧基、卤素、氧亚基和硫亚基。注意的是，本公开并非旨在涵盖全部符合上述结构的修饰基团，而仅涉及那些能够实现siRNA热稳定性增加的稳定化修饰基团。在一些实施方式中，每个所述稳定化修饰基团独立地选自2'-O-甲氧基乙基、2'-O-烯丙基、2'-烯丙基、2'-O-2-N-甲基氨基-2-氧亚基乙基、2'-O-2-N,N-二甲基氨基乙基、2'-O-3-氨基丙基和2'-O-2,4-二硝基苯基中的一种。在一些实施方式中，每个所述稳定化修饰基团为2'-O-甲氧基乙基。
在一些实施方式中，本公开的第一siRNA和第二siRNA中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中的第3个或第5个核苷酸为2'-O-甲氧基乙基修饰的核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb中第3-9个核苷酸中不超过2个核苷酸为2'-O-甲氧基乙基修饰的核苷酸。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb的第2、6、14、16个核苷酸，如果不是2'-O-甲氧基乙基修饰的核苷酸的话，为2'-氟代修饰的核苷酸。在一些实施方式中，所述核苷酸序列IIa和核苷酸序列IIb中的全部核苷酸均为修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列IIa和核苷酸序列IIb的第2、6、14、16个核苷酸，如果不是2'-O-甲氧基乙基修饰的核苷酸的话，为2'-氟代修饰的核苷酸，所述核苷酸序列IIa和核苷酸序列IIb中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
在一些实施方式中，核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列之间不多于2个核苷酸差异。其中，所述核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列之间的核苷酸差异可包括Za3位置处的差异和/或核苷酸序列Ia中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列之间的核苷酸差异可包括Za3位置处和/或与Za3相邻核苷酸位置处的核苷酸差异。
在一些实施方式中，核苷酸序列Ia是如SEQ ID NO:1所示的核苷酸序列。
在一些实施方式中，核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。在一些实施方式中，核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列之间的差异包括Za4位置处的差异，且Za4选自U、G或C。并且， Za3是与Za4互补的核苷酸。在一些实施方式中，核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列之间的差异包为Za4位置处的差异，且Za4选自U、G或C。在一些实施方式中，本公开第一siRNA和第二siRNA的核苷酸序列中的每个U可任意地被T替换。这些核苷酸差异并不会显著降低siRNA的靶基因抑制能力或者提高siRNA的脱靶效应。而这些包含核苷酸差异的siRNA也在本公开的保护范围之内。
在一些实施方式中，核苷酸序列Ia和核苷酸序列IIa基本上反向互补、实质上反向互补或完全反向互补。基本上反向互补是指两个核苷酸序列之间存在不多于3个碱基的错配；实质上反向互补是指两个核苷酸序列之间存在不多于1个碱基的错配；完全反向互补是指两个核苷酸序列之间没有错配。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIa的第3-19位的核苷酸与核苷酸序列Ia的第1-17位的核苷酸完全反向互补。在一些实施方式中，核苷酸序列IIa与核苷酸序列Ia完全反向互补。在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIa中的第2个核苷酸与按照3'末端到5'末端的方向所述核苷酸序列Ia中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，可在保持低的脱靶效应的同时，进一步提升本公开的第一siRNA的目标基因表达抑制活性。
在一些实施方式中，核苷酸序列Ia是SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，核苷酸序列IIa是SEQ ID NO:7所示的核苷酸序列：
5'-AAAUCAAGAUUUGCUAUGZa3-3'(SEQ ID NO:3)；
5'-AAAUCAAGAUUUGCUAUCZa3-3'(SEQ ID NO:4)；
5'-AAAUCAAGAUUUGCUAUAZa3-3'(SEQ ID NO:5)；
5'-AAAUCAAGAUUUGCUAUUZa3-3'(SEQ ID NO:6)；
5'-Za4CAUAGCAAAUCUUGAUUU-3'(SEQ ID NO:7),
其中，Za3选自A、U、G或C，Za4是与Za3互补的核苷酸。在一些具体实施方式中，Za3为U，Za4为A。
在一些实施方式中，第一正义链和第一反义链长度相同或不同，第一正义链的长度为19-23个核苷酸，第一反义链的长度为19-26个核苷酸。这样，本公开提供的第一正义链和第一反义链的长度比可以是19/19、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或23/26。在一些实施方式中，第一正义链和第一反义链的长度比为19/21、21/23或23/25。
在一些实施方式中，第一正义链还含有核苷酸序列IIIa，第一反义链还含有核苷酸序列IVa。核苷酸序列IIIa的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，核苷酸序列IVa的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是稳定化修饰核苷酸。核苷酸序列IIIa的长度为1个、2个、3个或4个核苷酸。核苷酸序列IVa和核苷酸序列IIIa长度相等，并且核苷酸序列IVa和核苷酸序列IIIa实质上反向互补或完全反向互补。核苷酸序列IIIa连接在核苷酸序列Ia的5'末端，核苷酸序列IVa连接在核苷酸序列IIa的3'末端。核苷酸序列IVa与第二段ANGPTL3 mRNA序列实质上反向互补或完全反向互补。第二段ANGPTL3 mRNA序列为ANGPTL3基因表达的mRNA中与第一段ANGPTL3 mRNA序列相邻、且长度与核苷酸序列IVa相同的核苷酸序列。其中，第一段ANGPTL3 mRNA序列为ANGPTL3基因表达的mRNA中SEQ ID NO:1所示的序列。
在一些实施方式中，核苷酸序列IIIa和IVa的长度均为1个核苷酸，核苷酸序列IIIa的碱基为A，核苷酸序列IVa的碱基为U。或者，核苷酸序列IIIa和IVa的长度均为2个核苷酸，核苷酸序列IIIa的碱基组成为CA，核苷酸序列IVa的碱基组成为UG。或者，核苷酸序列IIIa和IVa的长度均为3个核苷酸，核苷酸序列IIIa的碱基组成为CCA，核苷酸序列IVa的碱基组成为UGG。或者，核苷酸序列IIIa和IVa的长度均为4个核苷酸，核苷酸序列IIIa的碱基组成为GCCA，核苷酸序列IVa的碱基组成为UGGC。
在一些实施方式中，核苷酸序列IIIa和核苷酸序列IVa完全反向互补，因此，给出了核苷酸序列IIIa的碱基组成，核苷酸碱基IVa的碱基组成也就确定了。
在一些实施方式中，第一siRNA还含有核苷酸序列Va。核苷酸序列Va中的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是稳定化修饰核苷酸。核苷酸序列Va的长度为1至3个核苷酸，连接在第一反义链的3'末端构成3'突出端。由此，本公开提供的第一正义链和第一反义链的长度比可以是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或23/26。在一些实施方式中，核苷酸序列Va的长度为2个核苷酸，由此， 本公开提供的第一正义链和第一反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列Va中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约成本，核苷酸序列Va的长度为2个核苷酸，并且按照5'末端到3'末端的方向，核苷酸序列Va为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)、连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高第一反义链与靶mRNA的亲和力，核苷酸序列Va与第三段ANGPTL3 mRNA序列完全反向互补。其中，第三段ANGPTL3 mRNA序列为ANGPTL3基因表达的mRNA中与第一段ANGPTL3 mRNA序列或第二段ANGPTL3 mRNA序列相邻、并且长度与核苷酸序列Va相等的核苷酸序列。因此，在一些实施方式中，第一正义链和第一反义链的长度之比为19/21或21/23，此时，本公开的第一siRNA具有更好的mRNA沉默活性。
在一些实施方式中，第一正义链含有如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，第一反义链含有如SEQ ID NO:8所示的核苷酸序列：
5'-AAAUCAAGAUUUGCUAUGZa3-3'(SEQ ID NO:3)；
5'-AAAUCAAGAUUUGCUAUCZa3-3'(SEQ ID NO:4)；
5'-AAAUCAAGAUUUGCUAUAZa3-3'(SEQ ID NO:5)；
5'-AAAUCAAGAUUUGCUAUUZa3-3'(SEQ ID NO:6)；
5'-Za4CAUAGCAAAUCUUGAUUUUG-3'(SEQ ID NO:8),
其中，Za4是第一反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za4是与Za3互补的核苷酸。
在一些实施方式中，第一正义链含有如SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12所示的核苷酸序列，第一反义链含有如SEQ ID NO:13所示的核苷酸序列：
5'-CAAAAUCAAGAUUUGCUAUGZa3-3'(SEQ ID NO:9)；
5'-CAAAAUCAAGAUUUGCUAUCZa3-3'(SEQ ID NO:10)；
5'-CAAAAUCAAGAUUUGCUAUAZa3-3'(SEQ ID NO:11)；
5'-CAAAAUCAAGAUUUGCUAUUZa3-3'(SEQ ID NO:12)；
5'-Za4CAUAGCAAAUCUUGAUUUUGGC-3'(SEQ ID NO:13),
其中，Za4是第一反义链5'末端的第一个核苷酸，Za3选自A、U、G或C，并且Za4是与Za3互补的核苷酸。
在一些实施方式中，核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列之间 不多于2个核苷酸差异。其中，所述核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列之间的核苷酸差异可包括Zb3位置处的差异和/或核苷酸序列Ib中任意一个其它核苷酸位置处的核苷酸差异。在一些实施方式中，核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列之间的核苷酸差异可包括Zb3位置处和/或与Zb3相邻核苷酸位置处的核苷酸差异。
在一些实施方式中，核苷酸序列Ib是如SEQ ID NO:46所示的核苷酸序列。
在一些实施方式中，核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列之间不多于1个核苷酸差异。在一些实施方式中，核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列之间的差异包括Zb4位置处的差异，且Zb4选自U、G或C。并且，Zb3是与Zb4互补的核苷酸。在一些实施方式中，核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列之间的差异包为Zb4位置处的差异，且Zb4选自U、G或C。
在一些实施方式中，核苷酸序列Ib和核苷酸序列IIb基本上反向互补、实质上反向互补或完全反向互补。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIb的第3-19位的核苷酸与核苷酸序列Ib的第1-17位的核苷酸完全反向互补。在一些实施方式中，核苷酸序列IIb与核苷酸序列Ib完全反向互补。在一些实施方式中，按照5'末端到3'末端的方向，所述核苷酸序列IIb中的第2个核苷酸与按照3'末端到5'末端的方向所述核苷酸序列Ib中的第2个核苷酸之间存在碱基错配。通过包含该碱基错配，可在保持低的脱靶效应的同时，进一步提升本公开的第二siRNA的目标基因表达抑制活性。
在一些实施方式中，核苷酸序列Ib是SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，核苷酸序列IIb是SEQ ID NO:52所示的核苷酸序列：
5'-UUAAAAGGGACAGUAUUCZb3-3'(SEQ ID NO:48)；
5'-UUAAAAGGGACAGUAUUGZb3-3'(SEQ ID NO:49)；
5'-UUAAAAGGGACAGUAUUAZb3-3'(SEQ ID NO:50)；
5'-UUAAAAGGGACAGUAUUUZb3-3'(SEQ ID NO:51)；
5'-Zb4GAAUACUGUCCCUUUUAA-3'(SEQ ID NO:52),
其中，Zb3选自A、U、G或C，Zb4是与Zb3互补的核苷酸。在一些具体实施方式中，Zb3为U，Zb4为A。
在一些实施方式中，第二正义链和第二反义链长度相同或不同，第二正义链的长度为19-23个核苷酸，第二反义链的长度为19-26个核苷酸。这样，本公开提供的第二正义链和第二反义链的长度比可以是19/19、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或23/26。在一些实施方式中，第二正义链和第二反义链的长度比为19/21、21/23或23/25。
在一些实施方式中，第二正义链还含有核苷酸序列IIIb，第二反义链还含有核苷酸序列IVb。核苷酸序列IIIb的每个核苷酸独立地为非氟代修饰的核苷酸中的一种，核苷酸序列IVb的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是稳定化修饰核苷酸。核苷酸序列IIIb的长度为1个、2个、3个或4个核苷酸。核苷酸序列IVb和核苷酸序列IIIb长度相等，并且核苷酸序列IVb和核苷酸序列IIIb实质上反向互补或完全反向互补。核苷酸序列IIIb连接在核苷酸序列Ib的5'末端，核苷酸序列IVb连接在核苷酸序列IIb的3'末端。核苷酸序列IVb与第二段APOC3 mRNA序列实质上反向互补或完全反向互补，第二段APOC3 mRNA序列为APOC3基因表达的mRNA中与第一段APOC3 mRNA序列相邻、且长度与核苷酸序列IVb相同的核苷酸序列。其中，第一段APOC3 mRNA序列为APOC3基因表达的mRNA中SEQ ID NO:46所示的序列。
在一些实施方式中，核苷酸序列IIIb和IVb的长度均为1个核苷酸，核苷酸序列IIIb的碱基为C，核苷酸序列IVb的碱基为G。或者，核苷酸序列IIIb和IVb的长度均为2个核苷酸，核苷酸序列IIIb的碱基组成为GC，核苷酸序列IVb的碱基组成为GC。或者，核苷酸序列IIIb和IVb的长度均为3个核苷酸，核苷酸序列IIIb的碱基组成为UGC，核苷酸序列IVb的碱基组成为GCA。或者，核苷酸序列IIIb和IVb的长度均为4个核苷酸，核苷酸序列IIIb的碱基组成为UUGC，核苷酸序列IVb的碱基组成为GCAA。
在一些实施方式中，核苷酸序列IIIb和核苷酸序列IVb完全反向互补，因此，给出了核苷酸序列IIIb的碱基组成，核苷酸碱基IVb的碱基组成也就确定了。
在一些实施方式中，第二siRNA还含有核苷酸序列Vb。核苷酸序列Vb中的每个核苷酸独立地为非氟代修饰的核苷酸中的一种且不是稳定化修饰核苷酸。核苷酸序列Vb的长度为1至3个核苷酸，连接在第二反义链的3'末端构成3'突出 端。由此，本公开提供的第二正义链和第二反义链的长度比可以是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或23/26。在一些实施方式中，核苷酸序列Vb的长度为2个核苷酸，由此，本公开提供的第二正义链和第二反义链的长度比可以是19/21、21/23或23/25。
所述核苷酸序列Vb中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约成本，核苷酸序列Vb的长度为2个核苷酸，并且按照5'末端到3'末端的方向，核苷酸序列Vb为连续的2个胸腺嘧啶脱氧核糖核苷酸、连续的2个尿嘧啶核糖核苷酸；或者，为了提高第二反义链与靶mRNA的亲和力，核苷酸序列Vb与第三段APOC3 mRNA序列完全反向互补。其中，第三段APOC3 mRNA序列为APOC3基因表达的mRNA中与第一段APOC3 mRNA序列或第二段APOCL3 mRNA序列相邻、并且长度与核苷酸序列Vb相等的核苷酸序列。因此，在一些实施方式中，第二正义链和第二反义链的长度之比为19/21或21/23，此时，本公开的第二siRNA具有更好的mRNA沉默活性。
在一些实施方式中，第二正义链含有如SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，第二反义链含有如SEQ ID NO:53所示的核苷酸序列：
5'-UUAAAAGGGACAGUAUUCZb3-3'(SEQ ID NO:48)；
5'-UUAAAAGGGACAGUAUUGZb3-3'(SEQ ID NO:49)；
5'-UUAAAAGGGACAGUAUUAZb3-3'(SEQ ID NO:50)；
5'-UUAAAAGGGACAGUAUUUZb3-3'(SEQ ID NO:51)；
5'-Zb4GAAUACUGUCCCUUUUAAGC-3'(SEQ ID NO:53),
其中，Zb4是第二反义链5′末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸。
在一些实施方式中，第二正义链含有如SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57所示的核苷酸序列，第二反义链含有如SEQ ID NO:58所示的核苷酸序列：
5'-GCUUAAAAGGGACAGUAUUCZb3-3'(SEQ ID NO:54)；
5'-GCUUAAAAGGGACAGUAUUGZb3-3'(SEQ ID NO:55)；
5'-GCUUAAAAGGGACAGUAUUAZb3-3'(SEQ ID NO:56)；
5'-GCUUAAAAGGGACAGUAUUUZb3-3'(SEQ ID NO:57)；
5'-Zb4GAAUACUGUCCCUUUUAAGCAA-3'(SEQ ID NO:58),
其中，Zb4是第二反义链5′末端的第一个核苷酸，Zb3选自A、U、G或C，并且Zb4是与Zb3互补的核苷酸。
如前所述，本公开的第一siRNA和第二siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本公开的第一siRNA和第二siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致其抑制相关基因表达时功能明显削弱或丧失。
在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIa和核苷酸序列IIb的第2、6、14、16个核苷酸，如果不是稳定化修饰核苷酸的话，各自独立地为2'-氟代修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列IIa和核苷酸序列IIb的第2、6、14、16个核苷酸，如果不是稳定化修饰核苷酸的话，各自独立地为2'-氟代修饰的核苷酸，核苷酸序列IIa和核苷酸序列IIb中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列Ia和核苷酸序列Ib的第7-9个核苷酸各自独立地为2'-氟代修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，核苷酸序列Ia和核苷酸序列Ib的第7-9个核苷酸各自独立地为2'-氟代修饰的核苷酸，核苷酸序列Ia和核苷酸序列Ib中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
本公开的siRNA通过具有上述修饰，能够实现基因表达调节活性和体内稳定性的良好平衡。
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。
在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。
这些核糖基2'位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2'-烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(8)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH2)如式(9)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(10)所示：
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2'-、4'-位处以提供一个2',4'-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中， 无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(17)或(18)所示。
上述式(17)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET BNA、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在上文及下文中，“氟代修饰的核苷酸”指核苷酸的2'-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”指核苷酸核糖基团的2'-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。
在一些实施方式中，其中，反义链中不多于3个非氟代修饰的核苷酸为2'-脱氧核苷酸，其余每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸；或者，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸；所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在一些实施方式中，本公开的包含稳定化修饰核苷酸的siRNA是具有以下修 饰的siRNA：按照5'末端到3'末端的方向，核苷酸序列Ia和核苷酸序列Ib的第7、8、9位或者第5、7、8、9位的核苷酸各自独立地为氟代修饰的核苷酸，对应正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；按照5'末端到3'末端的方向，核苷酸序列IIa和核苷酸序列IIb的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸各自独立地为氟代修饰的核苷酸，对应反义链中第3位或第5位的核苷酸为稳定化修饰核苷酸，第18位的核苷酸为2'-脱氧核苷酸或甲氧基修饰的核苷酸，其余位置的核苷酸为甲氧基修饰的核苷酸。
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核糖核酸酶水解的性能。同时，上述修饰降低了siRNA的脱靶效应并未显著降低siRNA的抑制性能。
在一些实施方式中，本公开提供的第一siRNA为表1a中列出的siANGa1-M1、siANGa1-M2、siANGa2-M1、siANGa2-M2、siANGa3-M1、siANGa3-M2、siANGa4-M1、siANGa4-M2、siANGa5-M1、siANGa5-M2、siANGa6-M1、siANGa6-M2、siANGa7-M1、siANGa7-M2、siANGa8-M1或siANGa8-M2中的一种。
在一些实施方式中，本公开提供的第二siRNA为表1b中列出的siAPOCb1-M1、siAPOCb1-M2、siAPOCb2-M1、siAPOCb2-M2、siAPOCb3-M1、siAPOCb3-M2、siAPOCb4-M1、siAPOCb4-M2、siAPOCb5-M1、siAPOCb5-M2、siAPOCb6-M1、siAPOCb6-M2、siAPOCb7-M1、siAPOCb7-M2、siAPOCb8-M1或siAPOCb8-M2中的一种。
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分为具有修饰基团的磷酸酯基。在一些实施方式中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯亚基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯亚基：
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯亚基连接存在于由 以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯亚基连接存在于以下位置中的至少一处：
正义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
正义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
正义链的3'末端端部第1个核苷酸和第2个核苷酸之间；
正义链的3'末端端部第2个核苷酸和第3个核苷酸之间；
反义链的5'末端端部第1个核苷酸和第2个核苷酸之间；
反义链的5'末端端部第2个核苷酸和第3个核苷酸之间；
反义链的3'末端端部第1个核苷酸和第2个核苷酸之间；以及
反义链的3'末端端部第2个核苷酸和第3个核苷酸之间。
其中，在本公开的上下文中，为了叙述简便，以“正义链”指“第一正义链”和“第二正义链”，以“反义链”指“第一反义链”和“第二反义链”。
在一些实施方式中，本公开的第一siRNA为表1a中列出的siANGa1-M1S、siANGa1-M2S、siANGa2-M1S、siANGa2-M2S、siANGa3-M1S、siANGa3-M2S、siANGa4-M1S、siANGa4-M2S、siANGa5-M1S、siANGa5-M2S、siANGa6-M1S、siANGa6-M2S、siANGa7-M1S、siANGa7-M2S、siANGa8-M1S或siANGa8-M2S中的一种。在一些实施方式中，第一siRNA具有如siANGa1-M1S、siANGa2-M1S、siANGa3-M1S或siANGa4-M1S所示的序列
在一些实施方式中，本公开的第二siRNA为表1b中列出的siAPOCb1-M1S、siAPOCb1-M2S、siAPOCb2-M1S、siAPOCb2-M2S、siAPOCb3-M1S、siAPOCb3-M2S、siAPOCb4-M1S、siAPOCb4-M2S、siAPOCb5-M1S、siAPOCb5-M2S、siAPOCb6-M1S、siAPOCb6-M2S、siAPOCb7-M1S、siAPOCb7-M2S、siAPOCb8-M1S或siAPOCb8-M2S中的一种。在一些实施方式中，第二siRNA具有siAPOCb1-M1S、siAPOCb2-M1S、siAPOCb3-M1S或siAPOCb4-M1S所示的序列。
在一些实施方式中，反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。
常用的所述5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5'-磷酸核苷酸可具有如下结构：
再如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开了如下4种5'-磷酸类似物修饰的核苷酸：
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。
在一些实施方式中，5'-磷酸核苷酸为式(2)所示的含有5'-磷酸修饰的核苷酸，5'-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。
在一些实施方式中，本公开的第一siRNA为下表1a中列出的siANGa1-M1P1、siANGa1-M2P1、siANGa2-M1P1、siANGa2-M2P1、siANGa3-M1P1、siANGa3-M2P1、siANGa4-M1P1、siANGa4-M2P1、siANGa5-M1P1、siANGa5-M2P1、siANGa6-M1P1、siANGa6-M2P1、siANGa7-M1P1、siANGa7-M2P1、siANGa8-M1P1、siANGa8-M2P1、siANGa1-M1SP1、siANGa1-M2SP1、siANGa2-M1SP1、siANGa2-M2SP1、siANGa3-M1SP1、siANGa3-M2SP1、siANGa4-M1SP1、siANGa4-M2SP1、siANGa5-M1SP1、siANGa5-M2SP1、siANGa6-M1SP1、siANGa6-M2SP1、siANGa7-M1SP1、siANGa7-M2SP1、siANGa8-M1SP1和siANGa8-M2SP1中的一种。
在一些实施方式中，本公开的第二siRNA为下表1b中列出的siAPOCb1-M1P1、siAPOCb1-M2P1、siAPOCb2-M1P1、siAPOCb2-M2P1、siAPOCb3-M1P1、 siAPOCb3-M2P1、siAPOCb4-M1P1、siAPOCb4-M2P1、siAPOCb5-M1P1、siAPOCb5-M2P1、siAPOCb6-M1P1、siAPOCb6-M2P1、siAPOCb7-M1P1、siAPOCb7-M2P1、siAPOCb8-M1P1、siAPOCb8-M2P1、siAPOCb1-M1SP1、siAPOCb1-M2SP1、siAPOCb2-M1SP1、siAPOCb2-M2SP1、siAPOCb3-M1SP1、siAPOCb3-M2SP1、siAPOCb4-M1SP1、siAPOCb4-M2SP1、siAPOCb5-M1SP1、siAPOCb5-M2SP1、siAPOCb6-M1SP1、siAPOCb6-M2SP1、siAPOCb7-M1SP1、siAPOCb7-M2SP1、siAPOCb8-M1SP1或siAPOCb8-M2SP1中的一种。
本公开的发明人意外发现，本公开提供的第一siRNA和第二siRNA不仅具有显著增强的血浆和溶酶体稳定性、显著低的脱靶效应，还保留很高的基因抑制活性。
本公开提供的第一siRNA和第二siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开的第一siRNA和第二siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。
第一siRNA缀合物和第二siRNA缀合物
如前所示，第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，每种所述第一siRNA缀合物包含一种第一siRNA形成的第一siRNA基团和与其缀合的缀合基团；第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，每种所述第二siRNA缀合物包含一种第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
在一些实施方式中，所述第一RNAi剂为第一siRNA缀合物和/或所述第二RNAi剂为第二siRNA缀合物，即第一RNAi剂和第二RNAi剂中至少一个为siRNA缀合物。在一些实施方式中，第一RNAi剂为第一siRNA缀合物，并且第二RNAi剂为第二siRNA缀合物。
在一些实施方式中，缀合基团包含接头和药学上可接受的靶向基团和/或递送辅助基团。其中，第一siRNA基团、接头和靶向基团或者递送辅助基团依次共价连接或非共价连接形成第一siRNA缀合物；第二siRNA基团、接头和靶向基团或者递送辅助基团依次共价连接或非共价连接形成第二siRNA缀合物。每个靶向基团选自能够和细胞表面受体结合的配体，在一些实施方式中，每个所述靶向基团 选自能够和细胞表面ASGPR结合的配体；每个递送辅助基团选自能够增加siRNA缀合物在递送目标器官或组织中的生物相容性的基团。
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“第一siRNA缀合物”和“第二siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。siRNA缀合物应根据上下文，可理解为“第一siRNA缀合物”和“第二siRNA缀合物”，或多个siRNA缀合物的总称或者某个化学式所表示的siRNA缀合物。在本公开的上下文中，“缀合基团”应当理解为可通过反应缀合至siRNA，最终形成本公开的第一siRNA缀合和第二siRNA缀合物的特定化合物。
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团和任选的接头(linker)，并且所述siRNA、所述接头和所述靶向基团依次连接。在一些实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述第一siRNA分子或第二siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在正义链的3'端或5'端，也可在反义链的5'端，还可以在内部序列中。在一些实施方式中，第一siRNA或第二siRNA与缀合基团的缀合位点在正义链的3'末端。
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2'-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3'-位羟基上，此时核苷酸之间采用2'-5'磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7。
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。
在一些实施方式中，所述靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。
在一些实施方式中，至少一个或每个所述靶向基团选自能够和表达ANGPTL3基因和APOC3基因的细胞表面受体结合的配体。
在一些实施方式中，至少一个或每个所述靶向基团选自能够和哺乳动物肝实质细胞表面受体(ASGPR)结合的配体。在一些实施方式中，每个所述靶向基团独立地为与哺乳动物肝细胞表面的去唾液酸糖蛋白受体亲和的配体。在一些实施方式中，每个所述靶向基团独立地为去唾液酸糖蛋白或糖。在一些实施方式中，每个所述靶向基团独立地为去唾液酸糖蛋白，例如去唾液酸血清类枯蛋白(asialoorosomucoid,ASOR)或去唾液酸始球蛋白(asialofetuin，ASF)。在一些实施方式中，每个所述靶向基团独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳糖胺、N-异丁酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖、L-4-硫代核糖中的一种。在一些实施方式中，至少一个或每个所述靶向基团为半乳糖胺或N-乙酰半乳糖胺。
在一些实施方式中，本公开的接头具有如式(301)所示的结构：
其中，k为1-3的整数；
LA具有如式(302)所示的包含酰胺键的结构，LB具有如式(303)所示的包 含N-酰基吡咯烷的结构，含有羰基和氧原子，LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的连接基团；
其中，n302、q302和p302各自独立地为2-6的整数，可选地，n302、q302和p302各自独立地为2或3；n303为4-16的整数，可选地，n303为8-12的整数，表示基团共价连接的位点。
所述接头中，每个LA分别与一个所述靶向基团通过醚键连接，并通过LC部分中羟基的氧原子与LC部分形成醚键而连接；LB通过式(303)中的羰基与LC部分中氨基的氮原子形成酰胺键而连接，并通过式(303)中的氧原子与所述第一siRNA或第二siRNA通过氧原子形成磷酸酯键或硫代磷酸酯键相连接。
在一些实施方式中，本公开提供的第一siRNA缀合物和第二siRNA缀合物各自具有如式(305)所示的结构：
其中，Nu表示本公开提供的第一siRNA或第二siRNA。
在一些实施方式中，本公开的接头具有式(306)所示的结构：
其中，n306为0-3的整数，每个p306独立地为1-6的整数，表示基团共价连接的位点；所述连接基团通过由*标出的氧原子与所述靶向基团形成醚键连接；所述连接基团由#标出的氧原子中的至少一个与所述第一siRNA或第二siRNA形成磷酸酯键或硫代磷酸酯键而连接，其余由#标出的氧原子与氢原子连接形成羟基，或者与C1-C3烷基连接形成C1-C3烷氧基；
在一些实施方式中，本公开的第一siRNA缀合物和第二siRNA缀合物各自具有如式(307)所示的结构：
其中，Nu表示本公开提供的第一siRNA或第二siRNA。
在一些实施方式中，本公开的第一siRNA缀合物和第二siRNA各自具有式(308)所示的结构：
其中，
n1为选自1-3的整数，n3为选自0-4的整数；
每个m1、m2或m3各自独立地为选自2-10的整数；
R10、R11、R12、R13、R14或R15各自独立地为H，或选自于由以下基团所组成的组：C1-C10烷基、C1-C10卤代烷基以及C1-C10烷氧基；
R3具有式A59所示的结构：
其中，E1为OH、SH或BH2，Nu表示本公开提供的第一siRNA或第二siRNA；
R2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，R2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2、-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
每个L1独立地是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，L1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1- C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；
表示基团共价连接的位点；
M1表示靶向基团，其定义和可选择的范围与上述相同。在一些实施方式中，每个M1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。
技术人员会理解的是，尽管为了方便起见，L1被定义为线性烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或置换而产生的胺或烯基。为了本公开内容的目的，L1的长度是连接两个附着点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。
当M1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M1配体的个数至少为2；在一些实施方式中，n1+n3≥2，这样可以使得M1配体的个数至少为3，使得M1配体与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述缀合物通过内吞作用进入细胞。实验表明，当M1配体的个数大于3个时，M1配体与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方式中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。
在一些实施方式中，m1、m2和m3独立地选自2-10的整数时，可以使多个M1配体之间的空间位置适合M1配体与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的缀合物更为简单，更容易合成和/或降低成本，在一些实施方式中，m1、m2和m3各自独立地为2-5的整数，在一些实施方式中，m1＝m2＝m3。
本领域技术人员可以理解，当R10、R11、R12、R13、R14和R15各自独立地选自H、C1-C10烷基、C1-C10卤代烷基、以及C1-C10烷氧基中的一种时，不会改变 本文公开的缀合物的性质，均可以实现本公开的目的。在一些实施方式中，R10、R11、R12、R13、R14和R15各自独立地选自H、甲基和乙基。在一些实施方式中，R10、R11、R12、R13、R14和R15均为H。
根据本公开提供的第一siRNA缀合物或第二siRNA缀合物，R3为式A59所示结构的基团，其中，E1为OH、SH或BH2，基于制备原料易获取性的考虑，在一些实施方式中，E1为OH或SH。
在一些实施方式中，R2的选择是为了实现含氮骨架上的N与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R10、R11、R12、R13、R14和R15的碳原子与N互相连接的链状结构。因此，R2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N的连接基团。在一些实施方式中，在通过固相合成的工艺制备本公开的第一siRNA缀合物或第二siRNA缀合物的情况下，R2基团中需要同时含有与含氮骨架上的N连接的连接位点和与R3中的P相连接的连接位点。在一些实施方式中，R2中所述与含氮骨架上的N连接的位点与N形成酰胺键，所述与R3上的P连接的位点与P形成磷酸酯键。在一些实施方式中，R2的长度为4-15个原子。在一些实施方式中，R2是B5、B6、B5’或B6’：
其中，表示基团共价键连接的位点。
q2的取值范围可以是1-10的整数，在一些实施方式中，q2为1-5的整数。
L1的作用是将M1配体与含氮骨架上的N连接，为本公开的第一siRNA缀合物或第二siRNA缀合物提供靶向功能。在一些实施方式中，L1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方式中，L1选自A1、A4、A5、A6、 A8、A10、A11和A13中的一种或多种的连接组合；在一些实施方式中，L1选自A1、A4、A8、A10和A11中至少2个的连接组合；在一些实施方式中，L1选自A1、A8、A10中至少2个的连接组合。
在一些实施方式中，L1的长度可以为3-25个原子，3-20个原子、4-15个原 子或5-12个原子。在一些实施方式中是，L1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。
在一些实施方式中，j1为2-10的整数，在一些实施方式中，j1为3-5的整数。在一些实施方式中，j2为2-10的整数，在一些实施方式中，j2为3-5的整数。R’为C1-C4的烷基，在一些实施方式中，R’为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方式中，Ra为A27或A28。Rb为C1-C5的烷基，在一些实施方式中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方式中，在式A1-A26中各自对j1、j2、R’、Ra、Rb进行选择，以实现M1配体与含氮骨架上的N连接，并使M1配体之间的空间位置更适合M1配体与肝表面去唾液酸糖蛋白受体结合。
在一些实施方式中，本公开的第一siRNA缀合物和第二siRNA缀合物各自独立地具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：





其中，Nu表示本公开的第一siRNA或第二siRNA。
在本公开的一些实施方式中，第一siRNA缀合物和第二siRNA缀合物各自独立地具有式(403)所示结构的缀合物或其水可溶性盐，其中Nu是siANGa1-M1S、siANGa2-M1S、siANGa3-M1S、siANGa4-M1S、siAPOCb1-M1S、siAPOCb2-M1S、siAPOCb3-M1S和siAPOCb4-M1S中的一种或多种所示的siRNA形成的 siRNA基团，所述缀合基团连接至所述siRNA基团正义链3'末端核苷酸的核糖3'位。
在一些实施方式中，式A59中的P可以连接到siRNA序列中任何可能的位置，例如，式A59中的P可以连接到正义链或反义链的任何一个核苷酸上；在一些实施方式中，式A59中的P连接到正义链的任何一个核苷酸上。在一些实施方式中，式A59中的P连接到正义链或反义链的端部；在一些实施方式中，式A59中的P连接到正义链的端部。所述端部指所述正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方式中，式A59中的P连接到正义链或反义链的末端；在一些实施方式中，式A59中的P连接到正义链的3'末端。在连接至正义链的上述位置的情况下，本公开提供的缀合物进入细胞后，在解旋时，可以释放出单独的反义链，以通过RNAi机制抑制靶基因表达。
式A59中的P可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5'位、核苷酸的2'位、核苷酸的3'位或核苷酸的碱基上。在一些实施方式中，式A59中的P可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。在一些实施方式中，式A59中的P连接在正义链3'末端核苷酸的3'羟基脱氢后形成的氧原子上，或者式A59中的P通过取代正义链中的一个核苷酸的2'-羟基中的氢与核苷酸连接，或者式A59中的P通过取代正义链5'末端核苷酸的5'羟基中的氢与核苷酸连接。
在一些实施方式中，本公开的第一siRNA缀合物包含的第一siRNA可以是例如表1a中列出的任何一种siRNA序列，本公开的第二siRNA缀合物包含的第二siRNA可以是例如表1b中列出的任何一种siRNA序列。由本公开的第一siRNA缀合物和第二siRNA缀合物形成的药物组合物表现出低的脱靶效应和高的基因表达抑制活性。
表1a第一种siRNA序列




表1b第二种siRNA序列



其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母d表示该字母d右侧相邻的一个核苷酸为2'-脱氧核苷酸；下划线标出的大写字母S表示该字母S左侧相邻的一个核苷酸为稳定化修饰核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯亚基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中，S是表示具体的稳定化修饰例如moe，其中，下划线标出的字母组合moe表示在该字母组合moe左侧相邻的一个核苷酸为具有2'-O-甲氧基乙基修饰的核苷酸。在一些实施方式中，每个S均是moe。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。另外，上述表1a-1c中所列的序列中的每个U可任意地被T替换，不会对siRNA的活性或脱靶效应产生明显影响。
本公开siRNA缀合物的制备
上述第一siRNA缀合物和第二siRNA缀合物可以通过现有技术中已知的方法制备。例如，WO2015006740A2中详细描述了多种siRNA缀合物的制备方法，WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015,16,903-908中描述了式(307)所示结构的制备方法，中国专利申请CN110959011A详细公开了制备式(308)所示的siRNA缀合物的方法。以引用的方式将上述文献内容整体并入本文。此外，可以通过本领域技术人员熟知的其它方式，获得本公开的第一siRNA缀合物和第二siRNA缀合物。
溶剂、药学上可接受的载体和辅料
在一些实施方式中，本公开提供的药物组合物中，所述第一RNAi剂和/或所 述第二RNAi剂中还含有溶剂、药学上可接受的载体或辅料中的至少一种。下述对溶剂、药学上可接受的载体以及辅料的定义和选择范围适用于上述第一RNAi剂和第二RNAi剂。
在一些实施方式中，为了使用方便，所述第一RNAi剂、第二RNAi剂和第三RNAi剂以各种本领域常用的RNAi制剂形式存在，例如，所述RNAi制剂可以为液体制剂，例如注射液。所述液体制剂可以是用于皮下注射的注射液、用于肌肉注射的注射液或用于静脉注射的注射液，在一些实施方式中，为了有利于运输和/或储存，所述第一RNAi剂和/或第二RNAi剂和/或第三RNAi剂中的全部siRNA和siRNA缀合物以粉末的形式存在，例如，以冻干粉针剂存在，实施给药时，将冻干粉针剂与溶剂混合，配制成液体制剂。在一些实施方式中，所述溶剂可以包括各类溶剂例如水、去离子水、乙醇、pH缓冲液等常见溶剂。所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述溶剂的用量根据所需要的的注射液浓度进行调整，以siRNA或siRNA基团计，所述溶液剂溶解siRNA或siRNA配制成浓度为0.01mg/mL-200mg/mL siRNA、20mg/mL-200mg/mL/、50mg/mL-100mg/mL、0.01mg/mL-5mg/mLsiRNA、0.1mg/mL-5mg/mL/、0.5mg/mL-3mg/mL的溶液。根据所需浓度，本领域技术人员可以容易地确定所述溶剂的含量。
为了获得更好的抑制效果，在一些实施方式中，所述第一RNAi剂、第二RNAi剂和第三RNAi剂均为用于皮下注射的制剂形式存在。
在一些实施方式中，所述第一RNAi剂、第二RNAi剂和第三RNAi剂，也可以是通过喷雾给药到肺部或通过喷雾经肺部给药到其它脏器组织(如肝脏)的喷雾制剂、或通过口咽吸入的吸入制剂、或鼻腔给药等方式的药物制剂。因此，所述第一组分含有siRNA和siRNA缀合物中的至少一种和药学上可接受的载体和/或辅料，所述用于皮下注射的注射液、用于肌肉注射的注射液、用于静脉注射的注射液，通过喷雾给药到肺部或通过喷雾经肺部给药到其它脏器组织(如肝脏)的喷雾制剂、通过口咽吸入的吸入制剂或鼻腔给药等方式给药的药物制剂中的载体和/或辅料的种类和含量为本领域技术人员所公知。
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳 米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。
在一些实施方式中，所述药物组合物中，对siRNA或siRNA缀合物和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA或siRNA缀合物与药学上可接受的载体的重量比可以为1:(1-500)。在一些的实施方式中，上述重量比为1:(1-50)。
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料或溶剂，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括保护剂和/或渗透压调节剂。溶剂可以包括pH缓冲液或水等。
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。在一些实施方式中，所述药物组合物所制成的制剂在给药过程中的剂量会因给药方式的不同而发生调整。
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺 部、或通过喷雾经肺部给药到其它脏器组织(如肝脏)、或通过口咽吸入、或鼻腔给药等方式递送所述药物组合物。在一些实施方式中，所述药物组合物用于皮下注射给药。
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于中国专利申请CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。
在一些实施方式中，所述有机胺可为中国专利申请CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：
其中：
X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；
Y101和Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；
R101、R102、R103、R104、R105、R106和R107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；
x是1-10的整数；
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如 式(202)或式(203)所示的结构：
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。
在一些实施方式中，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：

其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。
其中，式(201)所示化合物可以根据中国专利申请CN103380113A中的描述制备。
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：
所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物。
所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80):(19.7-80):(0.3-50)，例如可以为(50-70):(20-40):(3-20)。
在一些实施方式中，由本公开的第一siRNA和第二siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm。更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。
在一些实施方式中，由本公开的第一siRNA和第二siRNA与上述含胺的转染试剂形成的药物组合物中，第一siRNA和第二siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内。例如，本公开的第一siRNA和第二siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的第一siRNA和第二siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的第一siRNA和第二siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。
将本公开提供的第一siRNA或第二siRNA分别溶解于缓冲盐溶液中，分别 得到siRNA水溶液；或者将第一siRNA和第二siRNA共同溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA水溶液的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1：(2-5)，例如可以为1:4。
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH＝7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。
在一些实施方式中，所述药物组合物仅含有所述第一RNAi剂和所述第二RNAi剂，所述第一RNAi剂仅含有一种第一siRNA缀合物或所述第二RNAi剂仅含有一种第二siRNA缀合物。或者，所述第一RNAi剂仅含有一种第一siRNA缀合物，且所述第二RNAi剂仅含有一种第二siRNA缀合物。
在一些实施方式中，所述第一RNAi剂和第二RNAi剂分别以单独的用于皮下注射的制剂形式存在，即，所述第一RNAi剂和第二RNAi剂是分开存放的，可以单独使用。在一些实施方式中，所述第一RNAi剂和所述第二RNAi剂以混合均匀的用于皮下注射的制剂形式存在。
本公开提供的治疗和/或预防与血脂异常相关的疾病或症状的方法
本公开还提供了一种治疗和/或预防与血脂异常相关的疾病或症状的方法，所述方法包括向有需要的受试者给予有效量的药物组合物，所述药物组合物选自本公开上述的药物组合物。
在一些实施方式中，所述药物组合物中的第一RNAi剂和第二RNAi剂在制备药物的过程中作为两种单独的制剂存在。在一些实施方式中，所述药物组合物中的第一RNAi剂和第二RNAi剂在制备药物的过程中作为一种制剂存在。
在另一方面，本公开还提供了一种治疗和/或预防与血脂异常相关的疾病或症状的方法，所述方法包括向有需要的受试者给予有效量的第一RNAi剂和第二RNAi剂，所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，所述第一siRNA包含第一正义链和第一反义链，所述第一siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，所述核苷酸序列Ia和所述核苷酸序列IIa至少部分反向互补形成双链区，其中，所述核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列片段至少15个核苷酸连续一致；每种所述第一siRNA缀合物包含所述第一siRNA形成的第一siRNA基团和与其缀合的缀合基团。所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列片段至少15个连续一致；每种所述第二siRNA缀合物包含所述第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
本公开提供的治疗方法中，可以采用第一RNAi剂和第二RNAi剂同时给药的方式，以siRNA和siRNA基团的总量计，向受试者给予0.1mg/kg～10mg/kg受试者体重的药物组合物。在一些实施方式中，给药量可以为0.5mg/kg～3mg/kg受试者体重的药物组合物。
本公开提供的治疗方法还可以采用第一RNAi剂和第二RNAi剂分别给药的方式，以siRNA和siRNA基团的总量计，分别向受试者给予0.1mg/kg～10mg/kg的第一RNAi剂和0.1mg/kg～10mg/kg的第二RNAi剂。在一些实施方式中，分别向受试者给予0.5mg/kg～3mg/kg的第一RNAi剂和0.5mg/kg～10mg/kg的第二RNAi剂。在一些实施方式中，向受试者给予第二RNAi剂后，间隔0.5min～30min后给予第一RNAi剂。例如可以为，向受试者给予第二RNAi剂后，间隔1min后给予第一RNAi剂。
本公开中，通常以皮下注射的方式向有需要的受试者给予第一RNAi剂和第二RNAi剂。
在一些实施方式中，所述与血脂异常相关的疾病或症状为高胆固醇血症、高甘油三酯血症或动脉粥样硬化。
本公开的药物组合物的应用
在一些实施方式中，本公开提供了本公开的药物组合物在制备用于治疗和/或预防与血脂异常相关的疾病或者症状的药物中的用途。在一些实施方式中，本公开中与血脂异常相关的疾病或症状为高胆固醇血症、高甘油三酯血症或动脉粥样硬化。
在一些实施方式中，本公开还提供了一种抑制细胞中ANGPTL3 mRNA和APOC3 mRNA表达水平的方法，所述方法包括将有效剂量的本公开的药物组合物与所述细胞接触。
通过将本公开提供的药物组合物给予有需要的受试者，可以通过对基因表达进行调控的机制达到预防和/或治疗由细胞中特定基因的表达而引起的病理状况或疾病的目的。
因此，本公开提供的药物组合物可用于预防和/或治疗所述病理状况或疾病、或用于制备用于预防和/或治疗本文所述病理状况或疾病的药物。
本文所使用的术语“给药/给予”是指通过使得至少部分地将药物组合物定位于期望的位点以产生期望效果的方法或途径，将药物组合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者整个身体相比将更多药物组合物递送至特定位点；而全身给药导致将药物组合物递送至受试者的基本整个身体。考虑到本公开旨在提供预防和/或治疗由肝细胞中特定基因的表达而引起的病理状况或疾病的手段，在一些实施方式中为能够将药物递送至肝脏的给药方式。
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月或每年1次或多次。
本公开所述的药物组合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体致死的剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中，指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。
在给予本公开所述的药物组合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或C3H/HeNCrlVr小鼠，以药物组合物中的siRNA和siRNA基团的总量计：药物组合物的用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在进一步的实施方式中为0.05-20mg/kg体重，在更进一步的实施方式中为0.1-15mg/kg体重，在又进一步的实施方式中为0.1-10mg/kg体重。在给予本公开的药物组合物时，可优选上述用量。
另外，通过将本公开的药物组合物导入特定基因异常表达的细胞，还可以通过基因表达调控的机制达到抑制细胞中该特定基因的表达这一目的。在一些实施方式中，所述细胞为肝细胞。在一些实施方式中，所述肝细胞可以是选自Hep3B、HepG2、Huh7等肝癌细胞系的细胞或分离的原代肝细胞，在一些实施方式中为原代肝细胞。
采用本公开提供的方法抑制特定基因在细胞中表达，所提供的药物组合物中的siRNA和siRNA基团的用量是本领域技术人员根据期望获得的效果容易确定的。例如，在一些实施方式中，所提供的药物组合物中siRNA和siRNA基团用量是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送是局部还是全身等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓度。
试剂盒
本公开提供了一种试剂盒，所述试剂盒包含本公开提供的药物组合物。
在一些实施方式中，本文所述的试剂盒可在一个容器中提供药物组合物。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。 在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述药物组合物的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将siRNA或siRNA缀合物与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。
在本公开的试剂盒中，所述siRNA或siRNA缀合物和/或药学上可接受的载体和辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述siRNA或siRNA缀合物和药学上可接受的载体和/或辅料以及所述药物组合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LBboratory Press(1989))所记载的方法进行。
制备例1-2本公开提供的siRNA缀合物的合成
按照CN110959011A制备例14所述的制备方法，制备获得了以下表2中的缀合物1-2，区别仅在于，各siRNA缀合物中含有的siRNA的正义链和反义链分别如表2中所示。按照以下表2中编号为缀合物1-缀合物2的siRNA的核酸序列，分别合成siRNA的正义链和反义链。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将各siRNA缀合物稀释至浓度为0.2mg/mL(以siRNA计)后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass SP1ectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。实测值与理论值一致，说明所合成的缀合物1-2是目标设计的双链核酸序列。各个siRNA缀合物分别具有式(403)所示的结构，并且该siRNA缀合物包含的siRNA基团分别具有表2中缀合物1-2所对应的siRNA序列，缀合基团连接至siRNA基团正义链3'末端核苷酸的核糖3'位，并且所述siRNA缀合物处于钠盐形式。缀合物1属于前述的第一siRNA缀合物，缀合物2属于前述的第二siRNA缀合物。
表2 siRNA缀合物中的siRNA序列

其中，大写字母C、G、U、A、T表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；下划线标出的字母组合moe表示该字母组合moe左侧相邻的一个核苷酸为核糖2'-O-甲氧基乙基修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯亚基连接。
实验例1本公开的药物组合物在人APOC3转基因小鼠体内对降低血脂的作用
本实验例考察由缀合物1和缀合物2组合得到的药物组合物在人APOC3转基因小鼠体内对降低血脂的作用。
将等级为SPF级、年龄≥6周龄的B6；CBA-Tg(APOC3)3707Bre/J模型小鼠按照血清TG含量>2mmol/L随机分为4组，每组5只均为雄性，所有动物根据体重计算药量，采用皮下注射单次给药的方式，向第一组小鼠注射1.5mg/kg的缀合物1、向第二组小鼠注射1.5mg/kg的缀合物2、向第三组小鼠注射包含0.75mg/kg缀合物1和0.75mg/kg缀合物2的单一剂量混合物(即为给予药物组合物组)、向第四组小鼠注射PBS溶液。其中，三组小鼠的给药剂量均以其中含有的siRNA的量计，体积为5mL/kg。其中，各缀合物分别以PBS水溶液提供，根据给药剂量和给药体积，换算出0.75mg/kg缀合物应配置的浓度为0.15mg/mL、1.5mg/kg缀合物应配置的浓度为0.3mg/mL。以给药当天记为第1天，在给药后第8、15、22、29、36天对小鼠眼眶静脉丛取血，在各时间点检测血清血脂水平。其中，眼眶静脉采血量每次约100μL，1800g离心力、2℃-8℃条件下离心15min后取上清分离出血浆，进一步使用PM1P000/3全自动血清生化仪(SABA，意大利)检测血清中甘油三酯(TG)和总胆固醇(TC)的含量。
标准化的血脂水平＝(给药后血脂含量/给药前血脂含量)×100％。
血脂水平的抑制率＝(1-给药后血脂含量/给药前血脂含量)×100％。
该处血脂水平指甘油三酯和总胆固醇的水平。
图1为在人APOC3转基因小鼠中，给予PBS、缀合物或药物组合物后，不同时间点时的血清TG相对水平的折线图。
图2为人APOC3转基因小鼠中，给予PBS、缀合物或药物组合物后，不同时间点时的血清TC相对水平的折线图。
由图1结果可知，一方面，以siRNA计，1.5mg/kg剂量的缀合物2和仅含0.75mg/kg缀合物1和0.75mg/kg缀合物2的药物组合物对小鼠血浆TG水平的最大抑制率相当，均约为86％，然而在测试时间内，仅给予1.5mg/kg剂量的缀合物2的小鼠中TG水平更快回升，表明抑制率有所下降，难以在整个测试期间内维持抑制效果。因此，实验结果表明，本公开的药物组合物具有比缀合物2更好的TG长效抑制性；另一方面，1.5mg/kg剂量的缀合物1和仅含0.75mg/kg缀合物1和0.75mg/kg缀合物2的药物组合物对TG水平的抑制在长时间内保持抑制率相当，然而1.5mg/kg剂量的缀合物1对TG水平的最大抑制率为74.17％，显著低于药物组合物对TG水平的最大抑制率。这表明，在相同剂量下，本公开的药物组合物能够更快地达到最大抑制效果，比缀合物1能够更加迅速有效地抑制血脂水平。
进一步地，由图2结果可知，以siRNA计，1.5mg/kg剂量的缀合物1和1.5mg/kg剂量的缀合物2对小鼠血浆TC水平的最大抑制率为59.72％，而仅含0.75mg/kg缀合物1和0.75mg/kg缀合物2的药物组合物对TC水平的最大抑制率可以达到66.51％，意外地，均高于单独给予1.5mg/kg剂量的缀合物1和1.5mg/kg剂量的缀合物2的最大抑制率，且具有和1.5mg/kg剂量的缀合物1的对TC水平抑制的长效性，并且显著优于1.5mg/kg剂量的缀合物2。
综上所述，与单独给予抑制ANGPTL3或APOC3的siRNA相比，本公开的药物组合物在相同的总siRNA剂量下，能够达到更高的血脂最大抑制效果和更长效的血脂抑制水平的良好平衡，因此显示出具有预防和/或治疗与血脂异常相关的症状或疾病的良好潜力。
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在 不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (28)

  1. 一种药物组合物，所述药物组合物包含第一RNAi剂和第二RNAi剂，所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，所述第一siRNA包含第一正义链和第一反义链，所述第一siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，所述核苷酸序列Ia和所述核苷酸序列IIa至少部分反向互补形成双链区，其中，所述核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列有至少15个核苷酸连续一致；每种所述第一siRNA缀合物包含一种所述第一siRNA形成的第一siRNA基团和与其缀合的缀合基团；
    所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列有至少15个核苷酸连续一致；所述第二siRNA缀合物包含一种所述第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
  2. 如权利要求1所述的药物组合物，其中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，所述第一RNAi剂和第二RNAi剂的重量比为1:(0.1-10)。
  3. 如权利要求1或2所述的药物组合物，其中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，所述第一RNAi剂和第二RNAi剂的重量比为1:(0.5-5)。
  4. 如权利要求1所述的药物组合物，其中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，每份所述药物组合物中，第一RNAi剂的含量为5mg-500mg，第二RNAi剂的含量为5mg-800mg。
  5. 如权利要求1-4中任意一项所述的药物组合物，其中，所述第一RNAi剂和/或所述第二RNAi剂中还含有溶剂、药学上可接受的载体或辅料中的至少一种。
  6. 如权利要求1-5中任意一项所述的药物组合物，所述核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-AAAUCAAGAUUUGCUAUGZa1-3'(SEQ ID NO:1)；
    5'-Za2CAUAGCAAAUCUUGAUUU-3'(SEQ ID NO:2),
    其中，所述Za1为U，Za2为A，所述核苷酸序列Ia中包含位置对应于Za1的核苷酸Za3，所述核苷酸序列IIa中包含位置对应于Za2的核苷酸Za4，所述Za4是所述反义链5'末端的第一个核苷酸；
    和/或，所述核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-UUAAAAGGGACAGUAUUCZb1-3'(SEQ ID NO:46)；
    5'-Zb2GAAUACUGUCCCUUUUAA-3'(SEQ ID NO:47),
    其中，Zb1为U，Zb2为A，所述核苷酸序列Ib中包含位置对应于Zb1的核苷酸Zb3，所述核苷酸序列IIb中包含位置对应于Zb2的核苷酸Zb4，所述Zb4是所述反义链5'末端的第一个核苷酸。
  7. 如权利要求6所述的药物组合物，其中，所述第一正义链和第一反义链长度相同或不同，所述第一正义链的长度为19-23个核苷酸，所述第一反义链的长度为21-26个核苷酸；所述核苷酸序列Ia是SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述核苷酸序列IIa是SEQ ID NO:8所示的核苷酸序列：
    5'-AAAUCAAGAUUUGCUAUGZa3-3'(SEQ ID NO:3)；
    5'-AAAUCAAGAUUUGCUAUCZa3-3'(SEQ ID NO:4)；
    5'-AAAUCAAGAUUUGCUAUAZa3-3'(SEQ ID NO:5)；
    5'-AAAUCAAGAUUUGCUAUUZa3-3'(SEQ ID NO:6)；
    5'-Za4CAUAGCAAAUCUUGAUUUUG-3'(SEQ ID NO:8),
    其中，Za3选自A、U、G或C，Za4是与Za3互补的核苷酸；
    和/或，所述第二正义链和第二反义链长度相同或不同，所述第二正义链的长度为19-23个核苷酸，所述第二反义链的长度为21-26个核苷酸；所述核苷酸序列Ib是SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述核苷酸序列IIb是SEQ ID NO:53所示的核苷酸序列：
    5'-UUAAAAGGGACAGUAUUCZb3-3'(SEQ ID NO:48)；
    5'-UUAAAAGGGACAGUAUUGZb3-3'(SEQ ID NO:49)；
    5'-UUAAAAGGGACAGUAUUAZb3-3'(SEQ ID NO:50)；
    5'-UUAAAAGGGACAGUAUUUZb3-3'(SEQ ID NO:51)；
    5'-Zb4GAAUACUGUCCCUUUUAAGC-3'(SEQ ID NO:53),
    其中，Zb3选自A、U、G或C，Zb4是与Zb3互补的核苷酸。
  8. 如权利要求1-7中任意一项所述的药物组合物，其中，按照5'末端到3'末端的方向，所述核苷酸序列IIa和所述核苷酸序列IIb的第3-6个核苷酸中的至少1个各自独立地为稳定化修饰核苷酸，所述稳定化修饰核苷酸指核苷酸的核糖2'位羟基被稳定化修饰基团取代的核苷酸，每个所述稳定化修饰基团独立地选自2'-O-甲氧基乙基、2'-O-烯丙基、2'-烯丙基、2'-O-2-N-甲基氨基-2-氧亚基乙基、2'-O-2-N,N-二甲基氨基乙基、2'-O-3-氨基丙基和2'-O-2,4-二硝基苯基中的一种。
  9. 如权利要求8所述的药物组合物，其中，按照5'末端到3'末端的方向，所述核苷酸序列Ia和所述核苷酸序列Ib的第7-9个核苷酸各自独立地为2'-氟代修饰的核苷酸，所述核苷酸序列Ia和所述核苷酸序列Ib中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种，所述核苷酸序列IIa和所述核苷酸序列IIb的第2、6、14、16个核苷酸，如果不是所述稳定化修饰核苷酸的话，各自独立地为2'-氟代修饰的核苷酸，所述核苷酸序列IIa和所述核苷酸序列IIb中的其它核苷酸各自独立地为非氟代修饰的核苷酸中的一种。
  10. 如权利要求1-9中任意一项所述的药物组合物，其中，所述第一siRNA具有如siANGa1-M1S、siANGa2-M1S、siANGa3-M1S或siANGa4-M1S所示的序列，所述第二siRNA具有如siAPOCb1-M1S、siAPOCb2-M1S、siAPOCb3-M1S或siAPOCb4-M1S所示的序列。
  11. 如权利要求1-10中任意一项所述的药物组合物，其中，每种所述第一siRNA缀合物和第二siRNA缀合物中的所述缀合基团包含接头和药学上可接受的靶向基团，所述第一siRNA基团、所述接头和所述靶向基团依次共价连接或非共价连接形成所述第一siRNA缀合物，所述第二siRNA基团、所述接头和所述靶向基团依次共价连接或非共价连接形成所述第二siRNA缀合物，每个所述靶向基团选自能够和细胞表面ASGPR结合的配体。
  12. 如权利要求11所述的药物组合物，其中，所述第一siRNA缀合物和所述第二siRNA缀合物各自独立地是具有式(403)所示结构的缀合物或其水可溶性盐，其中Nu是siANGa1-M1S、siANGa2-M1S、siANGa3-M1S、siANGa4-M1S、siAPOCb1-M1S、siAPOCb2-M1S、siAPOCb3-M1S和siAPOCb4-M1S中的一种所示的siRNA形成的siRNA基团，所述缀合基团连接至所述siRNA基团正义链3'末端核苷酸的核糖3'位。
  13. 如权利要求1-12中任意一项所述的药物组合物，其中，所述第一RNAi剂为第一siRNA缀合物，和/或所述第二RNAi剂为第二siRNA缀合物。
  14. 如权利要求1-13中任意一项所述的药物组合物，其中，所述第一RNAi剂和第二RNAi剂分别以单独的用于皮下注射的制剂形式存在；或者，所述第一RNAi剂和第二RNAi剂以混合均匀的用于皮下注射的形式存在。
  15. 如权利要求1-14中任意一项所述的药物组合物在制备用作治疗和/或预防与血脂异常相关的疾病或症状的药物中的用途。
  16. 如权利要求15所述的用途，其中，所述血脂异常相关的疾病或症状为高胆固醇血症、高甘油三酯血症或动脉粥样硬化。
  17. 一种治疗和/或预防与血脂异常相关的疾病或症状的方法，所述方法包括向有需要的受试者给予有效量的第一RNAi剂和第二RNAi剂，所述第一RNAi剂包含一种或多种第一siRNA和/或一种或多种第一siRNA缀合物，所述第一siRNA包含第一正义链和第一反义链，所述第一siRNA中的每个核苷酸各自独立 地为修饰或未修饰的核苷酸，其中，所述第一正义链含有一段核苷酸序列Ia，第一反义链含有一段核苷酸序列IIa，所述核苷酸序列Ia和所述核苷酸序列IIa至少部分反向互补形成双链区，其中，所述核苷酸序列Ia与SEQ ID NO:93-97中任意一项所示的核苷酸序列片段至少15个核苷酸连续一致；每种所述第一siRNA缀合物包含一种所述第一siRNA形成的第一siRNA基团和与其缀合的缀合基团；
    所述第二RNAi剂包含一种或多种第二siRNA和/或一种或多种第二siRNA缀合物，所述第二siRNA包含第二正义链和第二反义链，所述第二siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述第二正义链含有一段核苷酸序列Ib，第二反义链含有一段核苷酸序列IIb，所述核苷酸序列Ib和所述核苷酸序列IIb至少部分反向互补形成双链区，其中，所述核苷酸序列Ib与SEQ ID NO:98或99所示的核苷酸序列片段至少15个连续一致；每种所述第二siRNA缀合物包含一种所述第二siRNA形成的第二siRNA基团和与其缀合的缀合基团。
  18. 如权利要求17所述的方法，其中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，向受试者给予所述第一RNAi剂和第二RNAi剂的重量比为1:(0.1-10)。
  19. 如权利要求18所述的方法，其中，以所述第一RNAi剂和第二RNAi剂中各自含有的siRNA和siRNA基团的总量计，向受试者给予所述第一RNAi剂和第二RNAi剂的重量比为1:(0.5-5)。
  20. 如权利要求17-19中任意一项所述的方法，其中，以siRNA和siRNA基团的总量计，向受试者给予0.1mg/kg～10mg/kg受试者体重的所述药物组合物；或者，以siRNA和siRNA基团的总量计，分别向受试者给予0.1mg/kg～10mg/kg的第一RNAi剂和0.1mg/kg～10mg/kg的第二RNAi剂。
  21. 如权利要求20所述的方法，其中，以siRNA和siRNA基团的总量计，向受试者给予0.5mg/kg～3mg/kg受试者体重的所述药物组合物；或者，以siRNA和siRNA基团的总量计，分别给予0.5mg/kg～3mg/kg的第一RNAi剂和0.5mg/kg～10mg/kg的第二RNAi剂。
  22. 如权利要求20或21所述的方法，其中，所述方法包括向有需要的受试者同时给予所述第一RNAi剂和第二RNAi剂；或者，
    向受试者给予所述第二RNAi剂后，间隔0.5min～30min后给予所述第一RNAi剂。
  23. 如权利要求22所述的方法，其中，所述方法包括：以皮下注射的方式向有需要的受试者给予所述第一RNAi剂和第二RNAi剂。
  24. 如权利要求17-23中任意一项所述的方法，其中，所述与血脂异常相关的疾病或症状为高胆固醇血症、高甘油三酯血症或动脉粥样硬化。
  25. 如权利要求17-24中任意一项所述的方法，其中，所述核苷酸序列Ia与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列IIa与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-AAAUCAAGAUUUGCUAUGZa1-3'(SEQ ID NO:1)；
    5'-Za2CAUAGCAAAUCUUGAUUU-3'(SEQ ID NO:2),
    其中，所述Za1为U，Za2为A，所述核苷酸序列Ia中包含位置对应于Za1的核苷酸Za3，所述核苷酸序列IIa中包含位置对应于Za2的核苷酸Za4，所述Za4是所述反义链5'末端的第一个核苷酸；
    和/或，所述核苷酸序列Ib与SEQ ID NO:46所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列IIb与SEQ ID NO:47所示的核苷酸序列长度相等，且不多于3个核苷酸差异：
    5'-UUAAAAGGGACAGUAUUCZb1-3'(SEQ ID NO:46)；
    5'-Zb2GAAUACUGUCCCUUUUAA-3'(SEQ ID NO:47),
    其中，Zb1为U，Zb2为A，所述核苷酸序列Ib中包含位置对应于Zb1的核苷酸Zb3，所述核苷酸序列IIb中包含位置对应于Zb2的核苷酸Zb4，所述Zb4是所述反义链5'末端的第一个核苷酸。
  26. 如权利要求17-25中任意一项所述的方法，其中，所述第一正义链和第一反义链长度相同或不同，所述第一正义链的长度为19-23个核苷酸，所述第一 反义链的长度为21-26个核苷酸；所述核苷酸序列Ia是SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列，所述核苷酸序列IIa是SEQ ID NO:7所示的核苷酸序列：
    5'-AAAUCAAGAUUUGCUAUGZa3-3'(SEQ ID NO:3)；
    5'-AAAUCAAGAUUUGCUAUCZa3-3'(SEQ ID NO:4)；
    5'-AAAUCAAGAUUUGCUAUAZa3-3'(SEQ ID NO:5)；
    5'-AAAUCAAGAUUUGCUAUUZa3-3'(SEQ ID NO:6)；
    5'-Za4CAUAGCAAAUCUUGAUUU-3'(SEQ ID NO:7),
    其中，Za3选自A、U、G或C，Za4是与Za3互补的核苷酸；
    和/或，所述第二正义链和第二反义链长度相同或不同，所述第二正义链的长度为19-23个核苷酸，所述第二反义链的长度为21-26个核苷酸；所述核苷酸序列Ib是SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的核苷酸序列，所述核苷酸序列IIb是SEQ ID NO:52所示的核苷酸序列：
    5'-UUAAAAGGGACAGUAUUCZb3-3'(SEQ ID NO:48)；
    5'-UUAAAAGGGACAGUAUUGZb3-3'(SEQ ID NO:49)；
    5'-UUAAAAGGGACAGUAUUAZb3-3'(SEQ ID NO:50)；
    5'-UUAAAAGGGACAGUAUUUZb3-3'(SEQ ID NO:51)；
    5'-Zb4GAAUACUGUCCCUUUUAA-3'(SEQ ID NO:52),
    其中，Zb3选自A、U、G或C，Zb4是与Zb3互补的核苷酸。
  27. 如权利要求17-26中任意一项所述的方法，其中，所述第一siRNA具有如siANGa1-M1S、siANGa2-M1S、siANGa3-M1S或siANGa4-M1S所示的序列，所述第二siRNA具有如siAPOCb1-M1S、siAPOCb2-M1S、siAPOCb3-M1S或siAPOCb4-M1S所示的序列。
  28. 一种抑制细胞中ANGPTL3 mRNA和APOC3 mRNA表达水平的方法，所述方法包括将有效量的权利要求1-14中任意一项所述的药物组合物与所述细胞接触。
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