WO2023185946A1 - 一种寡核苷酸缀合物、含有该寡核苷酸缀合物的组合物及制备方法和用途 - Google Patents

一种寡核苷酸缀合物、含有该寡核苷酸缀合物的组合物及制备方法和用途 Download PDF

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WO2023185946A1
WO2023185946A1 PCT/CN2023/084791 CN2023084791W WO2023185946A1 WO 2023185946 A1 WO2023185946 A1 WO 2023185946A1 CN 2023084791 W CN2023084791 W CN 2023084791W WO 2023185946 A1 WO2023185946 A1 WO 2023185946A1
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group
nucleotide
alkyl
oligonucleotide
conjugate
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French (fr)
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高山
丁学锋
曹力强
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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous 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 an oligonucleotide conjugate capable of binding to transferrin receptor (TfR).
  • TfR transferrin receptor
  • the present disclosure also relates to compositions containing these oligonucleotide conjugates and their preparation methods and uses.
  • Oligonucleotides include, but are not limited to, small interfering RNA (siRNA), small activating RNA (saRNA), single-stranded oligonucleotides, etc.
  • siRNA small interfering RNA
  • siRNA small activating RNA
  • Single-stranded oligonucleotides etc.
  • oligonucleotides, especially double-stranded oligonucleotides have become known to the public as active pharmaceutical ingredients, and considerable progress has been made in the development of double-stranded oligonucleotide drugs.
  • oligonucleotides that have shown excellent pharmaceutical activity in preclinical pharmaceutical research centers are difficult to effectively reach specific target organs or target tissues due to the lack of effective delivery carriers, and therefore are difficult to use in actual drug development, especially For some neurological diseases, including central nervous system diseases and peripheral nervous system diseases, muscle diseases. Therefore, oligonucleotides that can be effectively delivered to specific target organs or target tissues, especially such as the brain, spinal cord, optic nerve tissue, olfactory nerve tissue, ear nerves and other peripheral nerve tissue, neuromuscular junction or muscle tissue, and tumor tissue There remains a significant practical need in the field for acid drugs.
  • the present invention provides an oligonucleotide conjugate with high delivery efficiency, which can effectively deliver the oligonucleotide to the target organ or target tissue, such as the central nervous system, and exhibits high pharmaceutical activity.
  • an oligonucleotide conjugate comprising:
  • the functional double-stranded oligonucleotide includes a sense strand and an antisense strand, the sense strand and the antisense strand respectively include 15-25 nucleotides, each The nucleotides are modified or unmodified nucleotides;
  • n 0 polypeptide ligands each of which is composed of 5-12 modified or unmodified amino acids, wherein n 0 is an integer of 2-6, and the polypeptide ligands are in contact with transferrin
  • the receptor has affinity;
  • each of the polypeptide ligands is connected to the functional double-stranded oligonucleotide through a covalent bond or through a linking group R I , and each of the polypeptide ligands is connected through the N-terminus or C-terminus of the polypeptide ligand. Attached to the functional double-stranded oligonucleotide.
  • the present disclosure also provides a pharmaceutical composition containing an oligonucleotide conjugate of the present disclosure and a pharmaceutically acceptable carrier thereof.
  • the present disclosure also provides use of the oligonucleotide conjugates and/or pharmaceutical compositions of the present disclosure in the preparation of a medicament for inhibiting target mRNA expression of target gene expression in a cell.
  • the present disclosure also provides a method for inhibiting target gene expression in a cell, the method comprising combining an effective amount of an oligonucleotide conjugate of the present disclosure and/or a pharmaceutical composition of the present disclosure with said Cell contact.
  • the present disclosure also provides a kit comprising the oligonucleotide conjugate of the present disclosure. compounds and/or pharmaceutical compositions.
  • polypeptide-conjugated oligonucleotide conjugates and/or pharmaceutical compositions provided by the present disclosure have good stability, high target gene expression regulation activity, high delivery efficiency in the central nervous system, and exhibit Very high pharmaceutical activity. Furthermore, if a lipophilic group is conjugated to the aforementioned oligonucleotide conjugate, the delivery efficiency of the aforementioned oligonucleotide conjugate can be further improved and generally exhibit higher drug activity.
  • Specific instructions are as follows.
  • the double-stranded oligonucleotides and/or pharmaceutical compositions of the present disclosure exhibit excellent inhibitory effects on mRNA expression of target genes in the central nervous system in vivo.
  • the oligonucleotide conjugate of the present disclosure conjugated with two polypeptide ligands basically inhibited SOD1 mRNA in different parts of the central nervous system by 50%, especially in the cortex area.
  • the rate can be as high as 80.02%.
  • the oligonucleotide conjugates of the present disclosure conjugated with lipophilic groups have an inhibitory rate of more than 60% on SOD1 mRNA in different parts of the central nervous system, especially in the cortex, hippocampus and cerebellum. rates are higher than 70%.
  • the inhibition rate in the cortex is as high as 88.33%. Displays excellent SOD1 mRNA inhibitory activity.
  • the oligonucleotide conjugate of the present disclosure containing 2 polypeptide ligands showed an inhibitory activity higher than 55% in various regions of the brain, and the inhibition rate of SOD1 mRNA in the cortical region was Can be as high as 81.55%.
  • the inhibition rate of SOD1 mRNA in different parts of the central nervous system by the oligonucleotide conjugate of the present disclosure conjugated with a lipophilic group was higher than 75%.
  • the inhibition rate of SOD1 mRNA was higher than 85%.
  • the inhibition rate in the cerebellum is as high as 89.11%.
  • the conjugate of the present disclosure with a lipophilic group conjugated at the 5' end of the sense chain and three polypeptide ligands conjugated at the 3' end also showed up to 80.00% and 75.90% in the cortical area and hippocampus area, respectively.
  • Inhibition rate of SOD1 mRNA This shows that the conjugates of the present disclosure containing different linking groups and containing different numbers of lipophilic groups and/or polypeptide ligands at different positions all show excellent SOD1 mRNA inhibitory activity and show good pharmaceutical activity. .
  • the conjugates containing multiple polypeptide ligands of the present disclosure exhibit significantly higher inhibition rates than the reference conjugates containing only one polypeptide ligand, and contain different polypeptide ligands, multiple peptide ligands, etc.
  • the conjugates provided by the present disclosure which are linked at different positions in the oligonucleotide, all exhibit similar effects.
  • the conjugates provided by the present disclosure showed significantly higher inhibition of target mRNA in different regions within the central nervous system compared to a comparative conjugate conjugated with only 1 polypeptide ligand. Rate.
  • the inhibition rate of the conjugate provided by the present disclosure is as high as 48.18%, which is 18.68% higher than that of the comparative conjugate.
  • the inhibitory rate of the conjugate provided by the present disclosure is as high as 65.11%, which is 29.36% higher than that of the comparative conjugate, showing a significantly better inhibitory effect.
  • the inhibition rates of the conjugates of the present disclosure are all higher than 60%, and the highest is as high as 74.45%, which can reach 2.5 times the inhibition rate of the comparison conjugates.
  • the inhibition rate of the conjugate of the present disclosure was also as high as 75.37%, More than twice the inhibition rate of the control conjugate.
  • oligonucleotide conjugates and pharmaceutical compositions provided by the present disclosure can effectively regulate the expression level of target genes in vivo and in vitro, and have excellent regulation of TfR-expressing tissues or cells, such as various tissues in the central nervous system.
  • the function of targeting the mRNA level of target gene expression can effectively treat and/or prevent disease symptoms related to the mRNA level of target gene expression, and has good application prospects.
  • SOD1 mRNA refers to the mRNA with Genbank registration number NM_011434.2 or NM_000454.5;
  • RPTOR mRNA refers to the sequence shown with Genbank registration number NM_020761.3.
  • the capital letters C, G, U, and A represent the base composition of nucleotides; the small letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy group.
  • P1 indicates that the nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • P1 is Represents specifically modified VP, Ps or P, where the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) Nucleotide, the letter combination Ps indicates that the nucleotide adjacent to the right side of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right side of the letter P is 5 '-Phosphonucleotide.
  • V-VP vinyl phosphate
  • E-VP vinyl phosphate
  • the letter combination Ps indicates that the nucleotide adjacent to the right side of the letter combination Ps is a phosphorothioate modified nucleotide
  • the capital letter P indicates that the nucleotide adjacent to the right side of the letter P is 5 '-Phosphonucleotide.
  • fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine
  • non-fluorinated modified nucleotide refers to Nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of a nucleotide with a non-fluorine group.
  • Nucleotide analogue refers to a nucleotide that can replace a nucleotide in a nucleic acid, but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymus Pyrimidine deoxyribonucleotide group. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleotides.
  • the "methoxy-modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the expressions "complementary” or “reverse complementary” are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are each associated with the other strand. The bases on them are paired in a complementary manner.
  • the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (G) always pairs with the pyrimidine base Pair with cytosine (C).
  • Each base pair contains a purine and a pyrimidine.
  • mismatch in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.
  • each letter represents an amino acid, where G represents glycine, A represents alanine, V represents valine, L represents leucine, and I stands for isoleucine, P stands for proline, F stands for phenylalanine, Y stands for tyrosine, W stands for tryptophan, S stands for serine, T stands for threonine, C stands for cysteine, and M stands for Methionine, N represents asparagine, Q represents glutamine, D represents aspartic acid, E represents glutamic acid, K represents lysine, R represents arginine, and H represents histidine.
  • polypeptide sequence refers to the polypeptide sequence formed by the dehydration condensation reaction of multiple amino acid monomers at the carboxyl position and the amino position.
  • N-terminus of the polypeptide sequence refers to the polypeptide sequence formed by the condensation reaction.
  • the end of the polypeptide containing the unreacted amino group, and the C-terminus of the polypeptide sequence refers to the end of the polypeptide formed by condensation containing the unreacted carboxyl group.
  • 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 3 base mismatches between the two nucleotide sequences involved; “ means that there is no more than one base mismatch between the two nucleotide sequences; “complete reverse complementarity” means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed between the former and the latter. For example, when one nucleotide base in the latter is A, and when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is regarded as one of the two nucleotide sequences. There are nucleotide differences at this position. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference is generated at that position.
  • nucleoside monomer Refers to modified or unmodified nucleoside phosphoramidites used in solid-phase synthesis of phosphoramidites, depending on the type and order of nucleotides in the double-stranded oligonucleotide or double-stranded oligonucleotide conjugate to be prepared.
  • Monomers unmodified or modified RNAphosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites).
  • Phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in this disclosure are all commercially available.
  • conjugated means that two or more chemical moieties, each having a specific function, are covalently linked to each other; accordingly, a “conjugate” is Refers to a compound formed by covalent connections between various chemical parts.
  • oligonucleotide conjugate or siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to an oligonucleotide or siRNA. Oligonucleotide conjugate should be understood as a general term for multiple oligonucleotide conjugates or siRNA conjugates or an oligonucleotide conjugate or siRNA conjugate represented by a certain chemical formula, depending on the context.
  • conjugated molecule shall be understood to mean a molecule that can be The reaction conjugates to the specific compound of the oligonucleotide, ultimately forming an oligonucleotide conjugate or siRNA conjugate of the present disclosure.
  • substituted or substituted groups include, but are not limited to, substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic, Substituted heteroaliphatic, substituted acyl, substituted aryl or substituted heteroaryl.
  • a “substituted” or “substituted” group refers to a group formed by replacing the hydrogen atom in the group with one or more substituents.
  • 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
  • the substituent is C 1 -C 3 alkyl, C 6 -C 8 aryl, -OC 1 -C 3 alkyl, -OC 1 -C 3 alkylphenyl, halogen, - One of OH, -NH 2 , -CN or -NO 2 . It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible and/or inherently unstable. .
  • alkyl refers to straight and branched chains having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms.
  • alkyl residue having a particular 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 the same residue as alkyl but with two points of attachment.
  • saturated alkyl refers to an alkyl group in which all carbon atoms are connected by single bonds and does not contain carbon-carbon double bonds and/or carbon-carbon triple bonds.
  • alkenyl refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond obtained by removing it from an adjacent carbon atom of the parent alkyl group. Obtained by removing a molecule of hydrogen. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl base, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • the alkenyl group has 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with 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 adjacent carbon atoms from the parent alkyl group. Obtained by removing two molecules of hydrogen.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.
  • alkoxy refers to an alkyl group with the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentyloxy etc.
  • Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected through an oxygen bridge.
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms of carbon, wherein at least one ring in the ring system is completely unsaturated, i.e., contains a cyclic structure according to Hückel's theory , delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups.
  • Arylene is a subset of aryl and refers to the same residue as aryl but with two points of attachment.
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring free radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., contains cyclic delocalization according to Hückel's theory (4n +2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems.
  • the heteroatoms in the heteroaryl group are oxidized heteroatoms.
  • the heteroaryl group contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group are quaternized nitrogen atoms. A heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl examples include, but are not limited to: 1,2,3-triazolyl, azepantrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzene Benzobisoxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxinyl (benzo[b ][1,4]dioxepinyl), benzo[b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4- benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxinyl), benzopyranyl,
  • halogen substituent or “halo” refers to fluoro, chlorine, bromo and iodo, and the term “halogen” includes fluorine, chlorine, bromine and iodine.
  • haloalkyl refers to an alkyl group as defined above in which the specified number of carbon atoms is substituted with one or more halogen atoms, up to the maximum allowed number.
  • haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.
  • hydroxyl protecting groups can be used in this disclosure.
  • protecting groups render a chemical functional group insensitive to specific reaction conditions and can be added to and removed from that functional group in the molecule without materially 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, cited Each of the above-mentioned documents is incorporated into this article in its entirety.
  • the protecting group is stable under basic conditions but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups useful herein include dimethoxytrityl (DMTr), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanth-9-yl (Mox).
  • non-exclusive examples of hydroxyl protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4′′-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any species of poultry.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any species of poultry.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • treat Therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the subject may still be suffering from the underlying disorder.
  • each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond.
  • the non-bridging oxygen atom or sulfur atom in the ester bond has a negative charge, and it can exist in the form of a hydroxyl or mercapto group.
  • the hydrogen ions in the hydroxyl or mercapto group can also be partially or fully replaced by cations.
  • the cation can be any cation, such as metal cation, ammonium ion NH 4 + , or one of organic ammonium cations.
  • the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations.
  • the alkali metal ion may be K + and/or Na +
  • the cation formed by the tertiary amine may be ammonium ion formed by triethylamine and/or ammonium ion formed by N,N-diisopropylethylamine.
  • the oligonucleotides or oligonucleotide conjugates described in the present disclosure may exist at least partially in salt form.
  • the non-bridging oxygen or sulfur atoms in the phosphodiester linkage or phosphorothioate diester linkage are at least partially bound to sodium ions
  • the oligonucleotides or oligonucleotide conjugates of the present disclosure are Exists as sodium salt or partial sodium salt. Therefore, any reference to an oligonucleotide or oligonucleotide conjugate described in the present disclosure, including but not limited to an oligonucleotide conjugate represented by any structural formula described in the present disclosure, is intended to encompass such Sodium salt or partial sodium salt form of an oligonucleotide or oligonucleotide conjugate.
  • Transferrin receptor refers to transferrin receptor 1 (TfR1) and/or transferrin receptor 2 (TfR2).
  • Transferrin receptor (TfR) mediates iron-containing ferritin from outside the cell into the cell. It is present on the surface of many cells and in many target tissues or organs, such as skeletal muscle, smooth muscle, brain, eye, It is expressed in tumor tissues and so on.
  • Polypeptide ligand refers to a binding peptide composed of a certain number of amino acids that is bonded to an oligonucleotide through a covalent bond or linking group.
  • the "polypeptide ligand has affinity for the transferrin receptor” mentioned in the present disclosure means that the polypeptide ligand of the present disclosure can specifically bind to the transferrin receptor, and the sequence of the polypeptide ligand of the present disclosure does not contain Sequences of the transferrin receptor and the binding domain of transferrin.
  • “Functional double-stranded oligonucleotide” as used in this disclosure refers to a double-stranded oligonucleotide with the function of up-regulating or down-regulating the target mRNA level of target gene expression, including small activators that can up-regulate the target mRNA level of target gene expression.
  • RNA saRNA
  • siRNA small interfering RNA
  • an oligonucleotide conjugate comprising:
  • the functional double-stranded oligonucleotide includes a sense strand and an antisense strand, the sense strand and the antisense strand respectively include 15-25 nucleotides, each The nucleotides are modified or unmodified nucleotides;
  • n 0 polypeptide ligands each of which is composed of 5-12 modified or unmodified amino acids, wherein n 0 is an integer of 2-6, and the polypeptide ligands are in contact with transferrin
  • the receptor has affinity;
  • each of the polypeptide ligands is connected to the functional double-stranded oligonucleotide through a covalent bond or through a linking group R I , and each of the polypeptide ligands is connected through the N-terminus or C-terminus of the polypeptide ligand. Attached to the functional double-stranded oligonucleotide.
  • each of the polypeptide ligands is linked to the sense or antisense strand of the functional double-stranded oligonucleotide.
  • each polypeptide ligand can be connected to any position in the functional double-stranded oligonucleotide, that is, in the presence of multiple polypeptide ligands, Multiple ligands can be linked to the same nucleotide in the double-stranded oligonucleotide or to different nucleotides.
  • each of the covalent bonds and/or linking groups R I connecting the polypeptide ligand are separately connected to the double Nucleotides at different positions in a chain of oligonucleotides are linked together.
  • the nucleotides to which the covalent bonds and/or linking groups R1 of the polypeptide ligand are connected are adjacent nucleotides. In some embodiments, the nucleotides to which they are connected are separated by at least 1 nucleotide. In some embodiments, the nucleotides to which they are connected are separated by at least 3 nucleotides. In some embodiments, the nucleotides to which they are connected are separated by at least 5 nucleotides.
  • a portion of the polypeptide ligand is linked to the sense strand of the functional double-stranded oligonucleotide, and a portion of the polypeptide ligand is linked to the antisense strand of the functional double-stranded oligonucleotide.
  • all polypeptide ligands are linked to the antisense strand.
  • all polypeptide ligands are linked to the sense strand.
  • At least one of the polypeptide ligands is linked to the first nucleotide at the 3' end or the 5' end of the sense or antisense strand. In some embodiments, at least two of the polypeptide ligands are linked to the first nucleotide at the 3' end or the 5' end of the antisense strand, respectively. In order to obtain better pharmaceutical activity, in some embodiments, at least one polypeptide ligand is connected to the 3' end of the sense chain, and at least one polypeptide ligand is connected to the 5' end of the sense chain.
  • the number n 0 of polypeptide ligands in the oligonucleotide conjugate is an integer from 2 to 4.
  • n 0 2
  • one of the polypeptide ligands is connected to the first nucleotide at the 3' end or 5' end of the sense strand
  • the other polypeptide ligand is connected to the sense strand.
  • a nucleotide refer to the nucleotides in the sense strand except the first nucleotide at the 3' end or the 5' end.
  • each of the polypeptide ligands is linked to the ribose ring of the nucleotide in the functional double-stranded oligonucleotide at the 2', 3' or 5' position of the ribose ring; or, Each of the polypeptide ligands is linked to a base of a nucleotide in the functional double-stranded oligonucleotide.
  • each of the polypeptide ligands is connected to the ribose ring of the nucleotide in the functional double-stranded oligonucleotide, and the connection position is 2', 3' or 5' of the ribose ring. 'Location.
  • each of the polypeptide ligands is linked to the polypeptide ligand by a covalent bond.
  • Functional double-stranded oligonucleotide alternatively, each of the polypeptide ligands is connected to the functional double-stranded oligonucleotide through one of the linking groups RI .
  • all polypeptide ligands are connected to the functional double-stranded oligonucleotide through the linking group R I , wherein any 2, 3, 4, Five or six of the polypeptide ligands are connected to the functional double-stranded oligonucleotide through the same linking group R I.
  • the linking group R I includes a backbone moiety, 1-6 side chain moieties, and 1 conjugation linker. are respectively connected to the conjugation connection part and the side chain part, the side chain part is respectively connected to the main chain part and the polypeptide ligand, and the conjugation connection part is respectively connected to the main chain part and the The double-stranded oligonucleotides are ligated.
  • the linking group R I has a structure represented by formula (101):
  • L A is the side chain part
  • k is an integer from 1 to 6
  • LB is the conjugation linker
  • LC is the main chain part
  • Nu represents that the linking group R I is connected to the double-stranded oligonucleotide The site of the acid
  • PP represents the site where the linking group R I is connected to the polypeptide ligand
  • the length of LC is 5-30 atoms, wherein the length of LC refers to the longest atomic chain in LC formed from the atoms directly connected to LA to the atoms directly connected to LB The number of atoms in the chain. To simplify the structure, in some embodiments, the length of LC is 8-25 atoms.
  • the conjugated linker LB is a connection combination of one or more of the following 1-5 linkages: phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, and disulfide bond.
  • k is an integer of 1-3;
  • L C contains any one of the groups represented by formula (L1)-(L3), through the group represented by formula (L1)-(L3) The ether bond in the group is connected to the L A part:
  • L B is a phosphate bond or disulfide bond
  • Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and their connecting combinations:
  • each j1 is an integer from 1 to 10;
  • Each R' is C 1 -C 10 alkyl
  • Each Ra is a hydrogen atom, a C 1 -C 10 alkyl group, or selected from the group consisting of groups (L24) - (L37):
  • each LA is a connected combination of at least 2 of the groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each LA is a linked combination of groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).
  • the linking group R I has a structure shown in formula (201):
  • n 201 and m 201 are integers from 1 to 10;
  • P 201 is an integer from 1 to 3;
  • L A has a structure as shown in formula (202), in which the imino end of L A is connected to the PP in formula (201):
  • n 202 , m 202 , p 202 , and q 202 are integers from 1 to 5, and i 202 is an integer from 0 to 5. In some embodiments, n 202 , m 202 , p 202 and q 202 are 2 or 3, and i 202 is 3 or 4.
  • the linking group R I has a structure shown in formula (103):
  • Nu represents the site where the linking group R I is connected to the double-stranded oligonucleotide
  • PP represents the site where the linking group R I is connected to the polypeptide ligand
  • n 103 and m 103 are integers from 1 to 10. In some embodiments, n 103 is an integer from 1 to 3, and m 103 is an integer from 3 to 6.
  • natural cysteine in the process of synthesizing the linking group R I as shown in formula (103), natural cysteine can be used for easy availability of raw materials. In this case, as shown in formula (103) The amino group in the linking group R I becomes a hydroxyl group, that is, it has a structure shown in formula (104):
  • the linking group R I has a structure represented by formula (301):
  • Nu represents the site where the linking group R I is connected to the double-stranded oligonucleotide
  • PP represents the site where the linking group R I is connected to the polypeptide ligand
  • p301 is 1 or 0, n 301 and m 301 are integers from 0 to 10.
  • n 301 and m 301 are integers from 0 to 5. number. In some embodiments, where n 301 and m 301 are integers from 0 to 3
  • the linking group RI may also be a linking group as disclosed in WO2019128611A1, which is incorporated herein by reference in its entirety.
  • the inventors of the present disclosure unexpectedly discovered that if the above-mentioned oligonucleotide conjugate of the present disclosure further contains a lipophilic group, the delivery efficiency of the aforementioned oligonucleotide conjugate can be further improved to achieve universal results. Higher pharmaceutical activity.
  • the oligonucleotide conjugates of the present disclosure further contain a lipophilic group via a covalent bond or linking group R II to the double-stranded oligonucleotide. Nucleotides are linked.
  • the conjugate contains h 0 lipophilic groups, h 0 being an integer selected from 1-5.
  • the lipophilic group is attached to the sense or antisense strand of the functional double-stranded oligonucleotide that contains at least one polypeptide ligand.
  • the polypeptide ligand and the lipophilic group are connected to the same nucleotide.
  • the nucleotide connected to the lipophilic group is a nucleotide connected to a polypeptide ligand. adjacent nucleotides.
  • the nucleotide connected to the lipophilic group and the nucleotide connected to the polypeptide ligand are separated by 1-21 nucleotides.
  • the nucleotide connected to the lipophilic group and the nucleotide connected to the polypeptide ligand are separated by at least 1 nucleotide. In some embodiments, the nucleotide to which the lipophilic group is attached is separated from the nucleotide to which the polypeptide ligand is attached by at least 5 nucleotides.
  • the number of nucleotides spaced between the nucleotide and the nucleotide to which the polypeptide ligand is attached does not exceed 80% of the total number of nucleotides in a single strand of nucleotides.
  • the length of a single strand of nucleotides is 19, 21, 23, or 25 nucleotides, correspondingly, 80% of the total number of nucleotides is 15, 17, 18, or 20 nucleotides, respectively.
  • two polypeptide ligands are respectively connected to the first nucleotide at the 3' end of the sense strand and the first nucleotide at the 5' end, the lipophilic group and the nucleoside of one of the sense strands.
  • the distance between the nucleotide and the first nucleotide at the 5' end of the sense strand and the first nucleotide at the 3' end of the sense strand does not exceed a single nucleotide. 80% of the total nucleotides in the chain.
  • the lipophilic group is linked to the functional double-stranded oligonucleotide via a linking group R II .
  • the linking group R II is the aforementioned linking group R I , that is, the polypeptide ligand and the lipophilic group are connected to the same linking group at the same time. In some embodiments, only the polypeptide ligand or the lipophilic group is connected to the same linking group R I or R II .
  • the lipophilic group is connected to the functional double-stranded oligonucleotide through a covalent bond.
  • the attachment position is the 2', 3', or 5' position of the ribose ring in the nucleotide.
  • the connection position is the 2' position of the ribose ring.
  • each of the polypeptide ligands is linked to the double-stranded oligonucleotide through its N-terminus, i.e., through the N-terminus of the polypeptide during synthesis of the oligonucleotide conjugate.
  • the amino group is conjugated to the linking group.
  • Common conjugation methods include conjugation through dehydration condensation reaction, click chemical reaction and other chemical reactions well known to those skilled in the art.
  • each polypeptide ligand is a polypeptide ligand containing one of the sequences shown in SEQ ID NO:169-SEQ ID NO:183. In some embodiments, each polypeptide ligand is one of the polypeptide ligands shown in SEQ ID NO: 169-SEQ ID NO: 173. In some embodiments, each polypeptide ligand is a polypeptide ligand containing the sequence set forth in SEQ ID NO: 169 or SEQ ID NO: 170. In some embodiments, each polypeptide ligand is a polypeptide ligand set forth in SEQ ID NO: 169 or SEQ ID NO: 170.
  • each of the polypeptide ligands in the oligonucleotide conjugate is a polypeptide ligand as shown in SEQ ID NO: 169; or, each of the polypeptide ligands
  • the peptide ligands are all peptide ligands shown in SEQ ID NO:170.
  • polypeptide ligands described in the present disclosure include linear or cyclic forms of the above-mentioned polypeptides, and it is well known to those skilled in the art that for the polypeptide sequence shown in Table 1, when both ends are truncated or extended , some truncated or extended polypeptide sequences can still specifically bind to TfR receptors. Therefore, the aforementioned polypeptide ligands containing one of the sequences shown in SEQ ID NO:169-SEQ ID NO:183 also include Such truncated or extended polypeptide sequences are obtained.
  • At least one amino acid in the polypeptide ligand is a modified amino acid. In some embodiments, at least 50% of the amino acids in the polypeptide ligand are modified amino acids. In some embodiments, all amino acids in the polypeptide ligand are modified amino acids.
  • modified amino acids include constructing polypeptide ligands in this disclosure by using D-amino acids in place of natural L-amino acids.
  • the natural L-amino acid may be substituted by using an amino acid mimetic, which includes a structural analog of an amino acid, such as a salt or ester of a natural amino acid.
  • the C-terminus of the polypeptide may be a carboxyl group or an amide group obtained by amidation of a carboxyl group, or other substances resulting from the incorporation of one of the above-mentioned amino acid mimetic.
  • one or more natural peptide bonds in the above-mentioned peptides can be replaced by any one of the following groups, including but not limited to: sulfonamide, reverse amide, aminooxy-containing bond, ester, alkane base ketone, ⁇ , ⁇ -difluoroketone, ⁇ -fluoroketone, peptoid bond (N-alkylated glycyl amide bond).
  • the side chain of the natural amino acid in the polypeptide ligand can be a substituted amino acid, for example, it can be substituted by 4-fluorophenylalanine, 4-suspended lysine, 3-aminoproline, 2-nitro A substitution of tyrosine or N-alkyl histidine; or a ⁇ -branched amino acid or a ⁇ -branched amino acid mimetic having a chirality at the ⁇ -side chain carbon atom opposite to the native chirality, For example, allo-threonine, allo-isoleucine and their derivatives.
  • Representative modified amino acids are disclosed in Baran et al., Biochemistry, 2017, 56(30): 3863-3873. and Mehta et al., Tetrahedron Letters, 2017, 58(14): 1357-1372, incorporated by reference. The above documents are each incorporated herein in their entirety.
  • the lipophilic group is a saturated or unsaturated, linear or branched hydrocarbon group with a length of 10-30 carbon atoms; one or more of the carbon atoms may be replaced by a hydroxyl group, an amino group, or a carboxyl group. , one or more substitutions of sulfonyl or phosphoryl.
  • the lipophilic group is a linear or branched saturated hydrocarbon group with a length of 15-25 carbon atoms.
  • the lipophilic group in order to simplify the synthesis process, is connected to the functional double-stranded oligonucleotide by replacing the hydrogen atom in the 2' hydroxyl group of the ribose ring in the nucleotide.
  • the sense strand contains a nucleotide sequence I
  • the antisense strand contains a nucleotide sequence I.
  • the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region
  • the nucleotide sequence II is at least partially reverse complementary to the target mRNA
  • the target mRNA is The mRNA expressed by the target gene in the target cells in the target tissue or target organ, the target cells being cells with TfR present on the cell surface.
  • the nucleotide sequence I and the nucleotide sequence II are each composed of 19 nucleotides, and the nucleotide sequence II and the nucleotide sequence I are substantially reverse complementary, substantially Reverse complementarity, or complete reverse complementarity; substantially reverse complementarity means that there is no more than 3 base mismatches between the two nucleotide sequences; substantially reverse complementarity means that the two nucleotide sequences are substantially reverse complementary. There is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no base mismatch between the two nucleotide sequences.
  • the nucleotide sequence II to The nucleotides at positions 2-19 are completely reverse complementary to the nucleotides at positions 1-18 of the nucleotide sequence I.
  • the nucleotide sequence II is completely reverse complementary to the nucleotide sequence I, or in the direction from the 5' end to the 3' end, the second one in the nucleotide sequence II
  • the functional double-stranded oligonucleotide in the oligonucleotide conjugate of the present disclosure contains a sense strand and an antisense strand, the length of the sense strand and the antisense strand is the same or different, and the length of the sense strand is 19- 23 nucleotides, the length of the antisense strand is 19-26 nucleotides.
  • the length ratio of the sense strand and the antisense strand of the functional double-stranded oligonucleotide 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 sense strand and the antisense strand in the functional double-stranded oligonucleotide is 19/21, 21/23 or 23/25.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the length of nucleotide sequence III and nucleotide sequence IV is 1-4 nuclei.
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the The 3' end of nucleotide sequence II.
  • the nucleotide sequence III and the nucleotide sequence IV are equal in length, substantially reverse complementary, or completely reverse complementary.
  • nucleotide sequence III and nucleotide sequence IV are completely reverse complementary. Therefore, given the base composition of nucleotide sequence III, the base composition of nucleotide sequence IV is also determined. .
  • the sense strand and the antisense strand are different in length, and the antisense strand also contains a nucleotide sequence V.
  • the length of the nucleotide sequence V is 1 to 3 nucleotides and is connected to The 3' end of the antisense strand constitutes the 3' overhang of the antisense strand.
  • the sense strand also contains a nucleotide sequence VI.
  • the length of the nucleotide sequence VI is 1 to 3 nucleotides and is connected to the 3' end of the sense strand to form the 3' end of the sense strand. 'Protruding end.
  • the functional double-stranded oligonucleotide includes nucleotide sequence V, but does not include nucleotide sequence VI. Therefore, the length ratio of the sense strand and the antisense strand in the functional double-stranded oligonucleotide 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. In some embodiments, the present disclosure provides functional double-stranded oligonucleotides including nucleotide sequences V and VI.
  • the length of nucleotide sequence V is the same as or different from the length of nucleotide sequence VI. Therefore, the length ratio of the sense strand and the antisense strand in the functional double-stranded oligonucleotide provided by the present disclosure can be (19-26): (19-26). In some embodiments, the length of the nucleotide sequence V and/or VI is 2 nucleotides, whereby the length of the sense strand and the antisense strand in the functional double-stranded oligonucleotide provided by the present disclosure The ratio can be 19/21, 21/21, 21/23, 23/23, 23/25 or 25/25.
  • each nucleotide in the nucleotide sequence V can be any nucleotide.
  • the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or,
  • the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the lengths of the sense strand and the antisense strand is 19/21 or 21/23, in which case the functional double-stranded oligonucleotide has better mRNA silencing activity.
  • each nucleotide in the nucleotide sequence VI can be any nucleotide.
  • the nucleotide sequence VI is two consecutive thymines. deoxyribonucleotide (dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity of the sense strand and the antisense strand in a functional double-stranded oligonucleotide, the nucleotide sequence VI The same nucleotide as the corresponding position in the target mRNA.
  • the functional double-stranded oligonucleotide includes the nucleotide sequences V and VI, and the ratio of the lengths of the sense strand and the antisense strand is 21/21 or 23/23. At this time, the The functional double-stranded oligonucleotide has better mRNA silencing activity.
  • the nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to the 5' end of a nucleotide sequence of the target mRNA.
  • the nucleotide sequence of the target mRNA is the same as the nucleotide sequence.
  • Sequence II is substantially reverse complementary or completely reverse complementary, or that nucleotide sequence is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of nucleotide sequence II and nucleotide sequence IV.
  • the nucleotides in the functional double-stranded oligonucleotide are modified or unmodified nucleotides.
  • the nucleotides in the functional double-stranded oligonucleotide are unmodified nucleotides; in some embodiments, part of the functional double-stranded oligonucleotide or All nucleotides are modified nucleotides, and these modifications on the nucleotide groups will not cause the functional double-stranded oligonucleotide to significantly weaken or lose the regulatory function of the B target mRNA expressed by the target gene.
  • At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analogue formed by replacing the 2' hydroxyl group of the ribose group of a nucleotide with another group, or a nucleoside
  • the base on the acid is the modified base of the nucleotide.
  • the modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression.
  • modified nucleotides 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.
  • At least one nucleotide in the sense strand or antisense strand of the functional double-stranded oligonucleotide provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group has a modified The phosphate group of the group; that is, at least a part of the phosphate group and/or the ribose group in the phosphate-sugar backbone of at least one single chain in the sense chain and the antisense chain is a phosphate group with a modifying group and /or ribosyl with modifying groups.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
  • each nucleotide in the sense strand and the antisense strand in the functional oligonucleotide provided by the present disclosure is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide. .
  • the inventors of the present disclosure surprisingly found that the oligonucleotide conjugates provided by the present disclosure achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.
  • the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and there are no more than 5 fluorinated modified nucleotides in the nucleotide sequence I, According to the direction from the 5' end to the 3' end, the 7th, 8th, and 9th nucleotides in the nucleotide sequence I are fluorinated modified nucleotides; the fluorinated modification in the nucleotide sequence II There are no more than 7 nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides.
  • the core in the direction from the 5' end to the 3' end, in the sense strand, the core at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I
  • the nucleotides are fluorinated modified nucleotides, and the remaining nucleotides in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand , the nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the antisense strand The remaining nucleotides are non-fluorinated modified nucleotides.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is selected from one of nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group.
  • Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.
  • the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-substituted alkoxy modified nucleotide for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), such as formula (9 ) shown.
  • the 2'-amino modified nucleotide (2'- NH2 ) is represented by formula (10).
  • the 2'-deoxynucleotide (DNA) is represented by formula (11):
  • Nucleotide analogues are those that can replace nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides Ribonucleotide group.
  • nucleotide analogs may be isonucleotides, bridged nucleic acid (BNA), or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. The bridge is typically incorporated into the 2', 4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., wherein LNA is as shown in formula (12) and ENA is as shown in formula (13) As shown, cET BNA is shown in formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):
  • R is selected from H, OH or alkoxy (-O-alkyl).
  • Isonucleotides are compounds formed by changing the positions of the bases on the ribose ring in nucleotides.
  • the isonucleotide can be a compound formed by moving the base from the 1'-position of the ribose ring to the 2'-position or 3'-position, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' of the ribosyl group -Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • the functional double-stranded oligonucleotide is a double-stranded oligonucleotide with the following modifications: in the sense strand, in the direction from the 5' end to the 3' end, the nucleoside
  • the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of the acid sequence I are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified.
  • nucleotides in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 or at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorine modified nucleotides, and the remaining nucleotides in the antisense strand are methoxy-modified nucleotides.
  • the functional double-stranded oligonucleotide is a double-stranded oligonucleotide with the following modifications: in the direction from the 5' end to the 3' end, the functional double-stranded oligonucleotide has The nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides, according to From the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II in the antisense strand of the double-stranded oligonucleotide are fluorinated. Nucleotides, the nucleotides at the remaining positions of the antisense strand are methoxy-modified nucleotides;
  • the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 5, 7, 8 and 9 of the sense strand are The nucleotides at the remaining positions are methoxy-modified nucleotides.
  • the nucleosides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand are The acid is a fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are methoxy-modified nucleotides;
  • the 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand are fluorinated modified nucleotides, and the remaining positions of the sense strand
  • the nucleotides are methoxy-modified nucleotides.
  • the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand are Fluoro-modified nucleotides, and the remaining nucleotides in the antisense strand are methoxy-modified nucleotides.
  • Double-stranded oligonucleotides with the above modifications can make it difficult for ribonucleases in blood to cleave nucleic acids, thereby increasing the stability of nucleic acids and making nucleic acids more resistant to nuclease hydrolysis.
  • functional double-stranded oligonucleotides with the above modifications still have high regulatory function of target mRNA.
  • At least part of the front phosphate groups in the phosphate-sugar backbone of at least one single-stranded sense strand and antisense strand of the functional double-stranded oligonucleotide provided by the present disclosure has a modifying group.
  • the phosphate group with the modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the The phosphate group having a modified group is a phosphorothioate group having a structure shown in formula (1):
  • This modification can stabilize the double-stranded structure of double-stranded oligonucleotides and maintain high specificity and high affinity of base pairing.
  • the phosphorothioate group linkage is present at at least one of the group consisting of: the first position at either end of the sense strand or the antisense strand. Between the first and second nucleotides; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:
  • the 5' terminal nucleotide of the antisense strand in the functional double-stranded oligonucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • 5'-phosphate nucleotide can 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 5'-phosphate modified core represented by formula (2)
  • the nucleotides modified by 5'-phosphate analogues are nucleotides modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), as shown in formula (3), or as Phosphorothioate modified nucleotides, as shown in formula (5).
  • the functional double-stranded oligonucleotide in the oligonucleotide conjugates of the present disclosure, is saRNA or siRNA. In some embodiments, the functional double-stranded oligonucleotide is siRNA.
  • the target cells of the oligonucleotide conjugates of the present disclosure are cells in which transferrin receptors are present on the cell surface. Therefore, the oligonucleotide conjugates of the present disclosure can target any target tissue or target organ in a subject where the above-mentioned target cells are present.
  • the function of regulating target genes present in these target tissues or target organs can be exerted by using a double-stranded oligonucleotide that expresses a target mRNA different from that of a target gene.
  • the target tissue or target organ is selected from skeletal muscle, smooth muscle, cardiac muscle, eye, brain, spinal cord, ear, nose, heart, retina, muscle tissue, and tumor tissue.
  • the target gene is selected from, in some embodiments, the target gene is APP, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR or TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B or RHO.
  • the target gene is SOD1 or RPTOR.
  • the functional double-stranded oligonucleotide is siRNA.
  • the inventors of the present disclosure unexpectedly discovered that the siRNA and the oligonucleotide conjugates containing these siRNA exhibit significantly improved stability in plasma, low off-target effects, and at the same time, exhibit high target mRNA silencing activity. . Therefore, in some embodiments, the siRNA may be one of the siRNAs shown in Table 11.
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m means that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter f means The adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group;
  • P1 indicates that the right side of P1
  • the adjacent nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
  • the functional double-stranded oligonucleotide in the oligonucleotide conjugates of the present disclosure is an siRNA targeting mouse SOD1 mRNA. In some embodiments, the functional double-stranded oligonucleotide in the oligonucleotide conjugates of the present disclosure has the sequence of the siRNA listed in Table 11.
  • the synthesis method of the oligonucleotide conjugate of the present disclosure includes providing a sense strand and an antisense strand of a double-stranded oligonucleotide, and annealing the sense strand and the antisense strand to obtain the double-stranded oligonucleotide provided by the present disclosure.
  • Oligonucleotide wherein the sense strand and the antisense strand respectively comprise 15-25 nucleotides, each of the nucleotides is a modified or unmodified nucleotide, and the sense strand or the antisense strand At least one of them is a sense chain or an antisense chain connected to the polypeptide ligand, the total number of the polypeptide ligands connected to the sense chain and the antisense chain is n 0 , and n 0 is an integer of 2-6,
  • the polypeptide ligand is a ligand with affinity to the transferrin receptor, and each polypeptide ligand is connected to the sense chain and/or the antisense chain through a covalent bond or through a linking group R I.
  • Each of the polypeptide ligands is composed of 5-12 modified or unmodified amino acids, and each of the polypeptide ligands is connected to the functional double-stranded oligonucleotide through the N-terminus or C-terminus of the polypeptide ligand.
  • the number of polypeptide ligands, the location and manner of attachment to the sense strand and/or antisense strand are as described above.
  • the synthesis method further includes isolation and purification of double-stranded oligonucleotides.
  • a single nucleotide chain connected with n 0 polypeptide ligands can be prepared in the following manner: in a solvent, under coupling reaction conditions, an oligonucleotide with an active group R x1 The oligonucleotide single chain is contacted with the polypeptide ligand with the active group R The molar ratio of the polypeptide ligand of the active group R x2 is 1:1-1:n 0 .
  • the polypeptide ligand is connected to the oligonucleotide single chain through a linking group R I , and the reactive group R x1 and the reactive group R x2 are capable of being generated by a coupling reaction. The group connecting the group R I.
  • the oligonucleotide single chain with the reactive group R Obtained through liquid phase synthesis by acid triester method.
  • the oligonucleotide single chain with the reactive group R The sequence of nucleotides in the acid single chain connects the nucleoside monomers at a time, wherein at least one nucleoside monomer is a nucleoside monomer with an active group R x1 .
  • the solid phase synthesis method of phosphoramidite is well known to those skilled in the art, and its process and conditions are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, P17-P31, all of which are incorporated by reference. The content is incorporated into this article in its entirety.
  • the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.
  • the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions or click chemical reaction conditions, and the reactive group R x1 and the reactive group R x2 is a group capable of undergoing the aforementioned condensation reaction.
  • the condensation reaction conditions are conditions for an acylation condensation reaction, and the active groups R x1 and R x2 are groups capable of undergoing an acylation condensation reaction to form R I .
  • the condensation reaction conditions are conditions for dehydration condensation reaction, one of the active groups R x1 and R x2 is a group containing an acid halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. group.
  • the condensation reaction conditions are click chemistry conditions, one of the reactive groups R x1 and R x2 is a group containing an alkynyl group, and the other is a group containing an azide group.
  • the single-stranded oligonucleotide with the active group R x1 is obtained by contacting the single - stranded oligonucleotide with the active group R Prepared, the cross-linking agent contains click chemical active groups and acylation groups.
  • the reactive group R is an active ester group, for example, it can be one of an NHS ester group, an imide ester group, and a pentafluorophenyl ester group.
  • the cross-linking agent can be done as follows: stergaard, Michael E., et al.
  • the reactive group RxO is amino.
  • the coupling conditions are basic conditions.
  • the alkaline condition is a condition in which a weak alkali aqueous solution is present, such as a sodium bicarbonate aqueous solution.
  • the single-stranded oligonucleotide with the active group Rx0 is obtained by It is prepared by using phosphoramidite monomers containing active groups at corresponding positions during the synthesis of oligonucleotide single chains. Those skilled in the art can obtain phosphoramidite monomers containing reactive groups in various ways.
  • the reactive group R x0 is an amino group
  • the phosphoramidite monomer containing R x0 can be purchased commercially through methods well known to those skilled in the art Obtained or prepared, for example, the phosphoramidite monomer containing R (base)-phosphoramidite monomer , wherein the reactive group R x0 is an amino group, and the reactive group R After the chain, it is obtained by removing the trifluoroacetyl protecting group through a deprotection reaction (such as ammonolysis of concentrated ammonia water) that is easily implemented by those skilled in the art.
  • a deprotection reaction such as ammonolysis of concentrated ammonia water
  • the coupling reaction condition is one of sulfhydryl-disulfide bond exchange reactions, one of the reactive groups R x1 and R Sulfur bonded leaving group.
  • R x1 in the above-mentioned phosphoramidite monomer containing reactive group R x1 exists in the form of protected R x1 ', and the preparation method also includes deprotection reaction conditions Next, the step of isolating the single strand of the oligonucleotide containing R x1 by contacting the prepared single strand of the oligonucleotide containing R x1 ' with a deprotecting reagent.
  • the disulfide bond activator is a disulfide pyridine.
  • phosphorus acid amide monomer containing the reactive group R Amide monomers are commercially available, for example, phosphoramidite monomers represented by formula (105) can be obtained commercially.
  • n 105 is an integer from 1 to 10
  • m 105 m 103 ;
  • the coupling reaction conditions are thiol-disulfide bond exchange reaction conditions
  • the reactive group R x2 is an reactive group containing a thiol group
  • the coupling reaction conditions are click chemistry reaction conditions
  • the reactive group R x2 is a polypeptide ligand containing an azide group
  • the polypeptide ligand with the reactive group R x2 can be Commercial customization available.
  • the R I is a linking group as shown in formula (103), and the oligonucleotide conjugate of the present disclosure can be prepared by the following method: in a solvent, at a thiol-disulfide bond Under exchange reaction conditions, a single-stranded oligonucleotide having a thiol exchange group, i.e.
  • R Separate and obtain the polypeptide ligand-single-stranded oligonucleotide conjugate connected through the linking group R I ; combine the polypeptide ligand-single-stranded oligonucleotide conjugate with the oligonucleotide conjugate of the present disclosure The other single strand is annealed to form a double-stranded oligonucleotide, and the oligonucleotide conjugate of the present disclosure is obtained by separation.
  • n 107 n 103
  • pp represents the polypeptide ligand
  • Nu' represents an oligonucleotide single chain
  • m 106 m 103
  • R 106 is a sulfhydryl exchanger residue.
  • R 106 is C 7 -C 12 aryl or heteroaryl.
  • R 106 is 2-pyridyl.
  • the solvent and sulfhydryl-disulfide bond exchange reaction conditions are commonly used solvents and reaction conditions in the art for sulfhydryl-disulfide bond exchange reactions, for example, in the presence of an ammonium acetate aqueous solution with a concentration of 0.05-1M, at room temperature. The reaction is suppressed for 2-10h, for example 4-8h.
  • the dosage ratio of the solvent to the compound of formula (106) can be 100:1-2000:1L/mol; the molar ratio of the compound of formula (107) to the compound of formula (106) can be 1:1-15:1, For example, 4:1-10:1.
  • the polypeptide-single-stranded oligonucleotide conjugate can be isolated from the reaction mixture using any suitable isolation method.
  • the solvent can be removed by evaporation and the polypeptide-single-stranded oligonucleotide conjugate can be separated by chromatography.
  • the solvent can be directly removed to obtain a crude product of the polypeptide-single-chain oligonucleotide conjugate, which can be directly used in subsequent reactions.
  • polypeptide ligand represented by formula (107) can be prepared by methods well known to those skilled in the art. In some embodiments, the polypeptide ligand represented by formula (107) can be easily obtained through commercial ordering.
  • the compound represented by formula (106) can be prepared by the following method: in a solvent, under thiol-disulfide bond exchange reaction conditions, the compound represented by formula (108) and the compound represented by formula (111)
  • the compound represented by formula (106) is obtained by contacting with the thiol-disulfide bond exchanger shown.
  • m 106 , Nu' and R 106 are the same as mentioned above; the selection of the solvent and the thiol-disulfide bond exchange reaction conditions are the same as above.
  • the molar ratio of the compound of formula (111) to the compound of formula (108) is a large excess, for example, it can be 10:1-1000:1, and in some embodiments, it is 50:1-200:1.
  • the compound of formula (106) can be isolated from the reaction mixture using any suitable isolation method.
  • the solvent can be removed by evaporation, followed by isolation of the compound of formula (106) by chromatography.
  • the following conditions can be used for isolation: 20% (V/V) ethanol aqueous solution as the mobile phase, in a purifier Gel desalting purification was performed, and the product eluate was collected at a wavelength of 280 nm.
  • the solvent can be directly removed after filtration to obtain a crude product of the compound of formula (106), which can be directly used in subsequent reactions.
  • the thiol-disulfide bond exchange reagent represented by formula (111) can be prepared by methods well known to those skilled in the art.
  • the compound represented by formula (111) is a commercially available 2-2'-dithiodipyridine.
  • the single-stranded oligonucleotide represented by formula (108) can be prepared by the following method: in an aqueous solution of a reducing agent, under the conditions of the reduction reaction, the single-stranded oligonucleotide represented by formula (109) The single-stranded oligonucleotide is contacted with a reducing agent to separate and obtain the single-stranded oligonucleotide represented by formula (108).
  • the reducing agent is a reducing agent that reduces disulfide bonds into sulfhydryl groups
  • the reducing agent is TCEP
  • the conditions for the reduction reaction are to react in an aqueous solution of TCEP under normal temperature and pressure conditions.
  • the reducing agent and reduction reaction conditions are the reducing agents and reaction conditions commonly used in the art for reduction reactions.
  • the reduction The agent is TCEP.
  • the reaction conditions are 1 to 5 hours at normal temperature and pressure.
  • the reaction time is 2 to 3 hours, and the reducing agent is in a large excess.
  • the "large excess" means that the mass ratio of the reducing agent and the single-stranded oligonucleotide may be 3:1-
  • the mass ratio of the reducing agent and the single-stranded oligonucleotide can be
  • the compound of formula (108) may be isolated from the reaction mixture using any suitable isolation method.
  • the compound of formula (108) can be obtained by diluting the reaction solution with purified water and filtering, then repeating ultrafiltration and centrifugation until the conductivity of the ultrafiltration liquid is measured to be below 100 s, and collecting the products in the filter membrane.
  • the single-stranded nucleotide represented by formula (109) is prepared by using a phosphoramidite monomer represented by formula (105) during the solid-phase synthesis of single-stranded oligonucleotides.
  • the phosphoramidite monomer represented by formula (105) is easily available to those skilled in the art.
  • the phosphoramidite monomer represented by formula (105) is commercially available.
  • n 105 is an integer from 1 to 10
  • m 105 m 103 ;
  • modified nucleotide groups can also be introduced into the functional double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications to prepare methods for nucleoside monomers with corresponding modifications. And methods of introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.
  • the thiol-containing polypeptide ligand represented by formula (107) is prepared by the following method : Under the reaction conditions of amino acid dehydration and condensation, the polypeptide ligand is contacted with natural or modified cysteine or cysteine analogues, and the cysteine or cysteine analogues have the formula The structure shown in (110) is separated to obtain the thiol-containing polypeptide ligand shown in formula (107).
  • the N-terminus or C-terminus of the polypeptide ligand is connected to a group containing a sulfhydryl group, so that the prepared polypeptide ligand is connected to The single strand of oligonucleotide is connected to the N-terminus or C-terminus of the polypeptide ligand.
  • the preparation method includes protecting the N-terminal amino group or the C-terminal carboxyl group of the polypeptide ligand to prepare a reaction in which only the N-terminal or C-terminal is connected to the thiol group. Methods for protecting and deprotecting amino or carboxyl groups are well known to those skilled in the art.
  • the reaction conditions for the dehydration and condensation of amino acids are the conditions for the dehydration and condensation of amino acids commonly used in this field.
  • the synthesis method of the polypeptide ligand itself that is, the synthesis method of the polypeptide containing a specific amino acid sequence, including the synthesis method of the polypeptide ligand containing modified amino acids, is already well known to those skilled in the art, and there are currently mature open commercial methods. Customized services.
  • polypeptide sequences as shown in Table 1 of the present disclosure are available through commercial customization.
  • the cysteine or cysteine analog represented by formula (110) is easily available to those in the art, and in some embodiments, is commercially available.
  • the thiol-containing polypeptide ligand of formula (107) is commercially available.
  • the amino or carboxyl group in the polypeptide sequence of the present disclosure is a protected amino or carboxyl group.
  • the carboxyl group can be converted to an amide group for protection by amidation.
  • each polypeptide ligand is linked to one single strand of the double-stranded oligonucleotide through a linking group as shown in formula (201) , at this time, the oligonucleotide conjugate of the present disclosure can be prepared by the following method: in a solvent, under the conditions of click chemical reaction, combine an oligonucleotide single chain containing a carbon-carbon triple bond with an oligonucleotide containing -N
  • the 3- group click peptide (Click Peptide) introduces the peptide ligand into the oligonucleotide single chain containing the active group through a click chemical reaction, and separates and obtains the peptide ligand-single-chain oligonucleotide connected through the linking group R I A nucleotide conjugate, wherein the click polypeptide already has commercial customization services; the polypeptide ligand-single-
  • the click chemistry reaction conditions are commonly used click chemistry reaction conditions in the field.
  • the click chemistry reaction conditions are to react the click polypeptide with the oligonucleotide under heating conditions and in the presence of a catalyst. single strand contact.
  • the catalyst is an aqueous solution containing copper catalyst.
  • the catalyst is an aqueous solution of copper sulfate, and the molar ratio of the copper sulfate to the click polypeptide is 1:6-1:1.
  • the catalyst is a mixed solution of copper sulfate, TBTA and sodium ascorbate, wherein the molar ratio of copper sulfate and TBTA is 3:1-1:3, and the molar ratio of copper sulfate and sodium ascorbate is 1: 5-1:10.
  • the molar ratio of copper sulfate and TBTA is 1:1, and the molar ratio of copper sulfate and sodium ascorbate is 1:7.5.
  • the heating condition refers to reaction in a water bath at 30-60 degrees Celsius. In some embodiments, the heating condition refers to reaction in a water bath at 30-50 degrees Celsius.
  • the molar ratio of the click polypeptide to the single strand of the oligonucleotide is 10:1-3:1. In some embodiments, the molar ratio of the click polypeptide to the single strand of the oligonucleotide is 10:1-3:1. The molar ratio is 5:1-4:1. In some embodiments, the reaction endpoint is determined by HPLC monitoring the levels of reactants and/or products in the reaction mixture. In some embodiments, the click chemistry reaction conditions are such as Zengmin Li; Tae Seok Seo; Jingyue Ju (2004). 1,3-Dipolar cycloaddition of azides with electron-deficient alkyne under mild condition in water., 45(15 ), 3143–3146, the entire contents of which are incorporated herein by reference.
  • the polypeptide-single-stranded oligonucleotide conjugate can be isolated from the reaction mixture using any suitable isolation method.
  • the solvent can be directly removed to obtain a crude product of the polypeptide-single-chain oligonucleotide conjugate, which can be directly used in subsequent reactions.
  • the linking group R I is a linking group represented by formula (203).
  • the oligonucleotide single chain connected to the polypeptide ligand can be prepared in the following manner:
  • the click polypeptide containing the active group R x2 as shown in the formula (204) and the oligonucleotide containing the active group R A polypeptide-single-chain oligonucleotide conjugate as shown in formula (203) is obtained, wherein R x1 and R x2 are azide groups.
  • the conditions for the click chemical reaction are the same as mentioned above.
  • polypeptide ligands containing azide groups as represented by formula (204) can obtain polypeptide ligands containing azide groups as represented by formula (204) through various methods.
  • polypeptide ligands containing azide groups as represented by formula (204) Body was obtained by commercial order.
  • the single-stranded oligonucleotide represented by formula (205) can be obtained through various methods.
  • the single-stranded oligonucleotide represented by formula (206) can be obtained under normal pressure and temperature under alkaline conditions.
  • the single-stranded oligonucleotide is contacted with the active ester represented by formula (207), and the oligonucleotide represented by formula (205) is separated.
  • the alkaline conditions refer to conditions in which sodium bicarbonate aqueous solution exists.
  • the active ester as represented by formula (207) is in a large excess compared to the single-stranded oligonucleotide as represented by formula (205); in some embodiments, as represented by formula (207)
  • the molar ratio of the active ester shown and the single-stranded oligonucleotide shown in formula (206) is 200:1-50:1; in some embodiments, as shown in formula (206)
  • the molar ratio of the active ester represented by (207) and the single-stranded oligonucleotide represented by formula (206) is 150:1-
  • a mixed solvent of acetonitrile and PBS can be added to the reaction solution, centrifuged, and a nucleic acid precipitation solution (for example, 10% trichloroacetic acid, which is easily commercially available) can be added to the resulting supernatant. aqueous solution), and the precipitate is separated to obtain the single-stranded oligonucleotide represented by formula (205).
  • a nucleic acid precipitation solution for example, 10% trichloroacetic acid, which is easily commercially available
  • the active ester represented by formula (207) can obtain the active ester represented by formula (207) through various methods.
  • the active ester of formula (207) is produced by using, e.g. stergaard, Michael E., et al. "Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides.”
  • the initial reactant RCO(CH 2 ) 3 COOH in Scheme 1a(A) is replaced by a cross-linker compound represented by formula (208), the entire content of which is incorporated herein by reference in its entirety.
  • the compound represented by formula (208) is obtained in the following manner: in a solvent, under the hydrolysis conditions of the ester Under this method, the ester represented by formula (209) is hydrolyzed, and the compound represented by formula (208) is isolated.
  • the hydrolysis conditions of the ester refer to conditions in the presence of a catalyst in an alkaline aqueous solution.
  • the alkaline aqueous solution is an aqueous solution of sodium hydroxide
  • the catalyst is ethanol
  • the molar ratio of sodium hydroxide and the compound represented by formula (209) is 7:1-3:1
  • the concentration of sodium hydroxide aqueous solution is 2M-5M
  • the volume ratio of ethanol and sodium hydroxide aqueous solution is 7:1-3:1.
  • the pH value of the reaction solution can be adjusted to 5-6 by using an acidic solution, and then separated by column chromatography to obtain as follows: Compounds represented by formula (208).
  • the compound represented by formula (209) can be prepared by the following method: under the conditions of condensation reaction, make the compound represented by formula (210) After contacting with the compound represented by formula (211), the compound represented by formula (209) is separated:
  • the condensation reaction conditions are amidation reaction conditions, for example, in an organic solvent, there are 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl The conditions under which carbodiimide hydrochloride (EDC.HCl) and N,N-diisopropylethylamine (DIEA) exist.
  • the organic solvent is DMF.
  • the molar ratio of the compound represented by formula (210) and the compound represented by formula (211) is 1:2-1:5.
  • Those skilled in the art can isolate the compound represented by formula (209) through various methods.
  • the organic phase can be obtained by adding water and ethyl acetate to the reaction solution for extraction, and then washing the obtained organic phase with 10% citric acid, sodium bicarbonate, and saturated saline solution in sequence, and evaporating the solvent to dryness to obtain the formula: The compound shown in (209).
  • the compound represented by formula (210) is prepared according to the method of Scheme 3 disclosed in WO2014025805A1.
  • Those skilled in the art can obtain the compound represented by formula (211) through various methods.
  • the compound represented by formula (211) is commercially available.
  • the single-stranded oligonucleotide represented by formula (206) can obtain the single-stranded oligonucleotide represented by formula (206) through various methods, for example, by using phosphorus containing active groups at the corresponding positions during the synthesis of the single-stranded oligonucleotide.
  • the reactive group-containing phosphoramidite monomer is 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-ylidene Phosphamide monomer.
  • the phosphoramidite monomer is readily available to those skilled in the art, and in some embodiments, the phosphoramidite monomer is commercially available.
  • the polypeptide-single-stranded oligonucleotide conjugate contains multiple nucleotides connected to the polypeptide ligand, wherein a single core of the single-stranded oligonucleotide is connected to the polypeptide ligand.
  • the glycoside has the structure shown in formula (301A):
  • Nu represents a nucleotide in the single chain of the oligonucleotide
  • the Nu can be a nucleotide at any position in the single chain of the oligonucleotide, and it and the oligonucleotide
  • the remaining nucleotides in the single chain of nucleotides are connected through phosphate bonds or phosphorothioate bonds
  • PP represents the polypeptide ligand
  • p301 is 1 or
  • n 301 and m 301 are integers from 0 to 10.
  • the polypeptide ligand group replaces the hydrogen atom in the 2'-position hydroxyl group of a ribose ring adjacent to its left.
  • the p301 is 1 when the polypeptide ligand is attached to the 5' position of the ribose ring in the nucleotide.
  • the oligonucleotide single chain can be obtained by combining the click polypeptide containing the active group R The oligonucleotide single-stranded contact of x1 nucleotide is separated to obtain a polypeptide-single-stranded oligonucleotide conjugate.
  • the conditions for click chemistry are as described above:
  • Nu means one nucleotide in the single chain of the oligonucleotide.
  • the p301 when the polypeptide ligand is attached to the 2' position of the ribose ring in the nucleotide, the p301 is 0.
  • the oligonucleotide single chain can be made by combining the click polypeptide containing the active group R The oligonucleotide single-stranded contact of x1 nucleotide is separated to obtain a polypeptide-single-stranded oligonucleotide conjugate.
  • the conditions for click chemistry are as described above:
  • Nu represents a nucleotide in the single chain of the oligonucleotide.
  • the selection range of the polypeptide ligand is the same as the above description of the oligonucleotide conjugate of the present disclosure.
  • Those skilled in the art can obtain polypeptide ligands containing specific sequences in various ways.
  • the polypeptide ligands are commercially available.
  • oligonucleotide conjugates of the present disclosure contain a lipophilic group that is covalently bonded or linked to the functional double-stranded oligonucleotide via a linker group R II . of nucleotides connected.
  • the preparation method further includes the step of connecting the lipophilic group to the double-stranded oligonucleotide or single-stranded oligonucleotide in the above preparation method.
  • the conjugate contains h 0 lipophilic groups, h 0 is an integer taken from 1 to 5, and the oligonucleotide conjugate can be prepared by linking h 0 polypeptide complexes. Single-stranded oligonucleotides were prepared.
  • the oligonucleotide single chain connected with the lipophilic group can be prepared in the following manner: in a solvent, under coupling reaction conditions, an oligonucleotide with an active group R x1 The acid single chain contacts the lipophilic group with the active group R The molar ratio of the single-stranded oligonucleotides of group R x1 is 1:1-1:h 0 .
  • the polypeptide ligand and the lipophilic group are simultaneously attached to at least one nucleotide in the oligonucleotide conjugate of the present disclosure.
  • the nucleotide can be prepared by using nucleoside monomers containing more than one reactive group R x1 at different positions during the synthesis of a nucleotide single chain.
  • the nucleotide can be prepared by contacting a compound having a reactive group R x2 , a lipophilic group and a polypeptide ligand with a single-stranded oligonucleotide having a reactive group R x1 .
  • the nucleotide connected to the lipophilic group and the nucleotide connected to the polypeptide ligand are separated by at least 1 nucleotide.
  • the number of nucleotides spaced between the nucleotide and the nucleotide connected to the polypeptide ligand does not exceed 80% of the total number of nucleotides in the single strand of nucleotides, and the oligonucleotide conjugate can be provided in the sense strand 3
  • the first nucleotide at the 'end or 5' end is connected to 3 polypeptide ligands, and the distance between the first nucleotide at the 3' end or the 5' end of the sense strand does not exceed the nucleotides in
  • the lipophilic group is covalently linked to one nucleotide in the double-stranded oligonucleotide.
  • the lipophilic group is attached to the 2' position of the ribose ring of the nucleotide, and the oligonucleotide conjugate can be obtained by solid phase synthesis in the same phosphoramidite as described above.
  • the nucleoside monomers are connected at a time according to the order of the nucleotides in the single chain of the oligonucleotide, wherein at least one nucleoside monomer is a phosphorous acid with the lipophilic group connected to the 2' position of the sugar ring. Amide monomer.
  • the lipophilic group is a saturated or unsaturated, linear or branched hydrocarbon group with a length of 15-25 carbon atoms.
  • the lipophilic group is commercially available.
  • Those skilled in the art can activate the lipophilic group in various ways, in some embodiments, by using halogenated hydrocarbons as the lipophilic group containing the activating group.
  • the phosphoramidite monomer with a lipophilic group attached is easily available to those skilled in the art.
  • the phosphoramidite monomer is synthesized using a method as disclosed in Preparation Example 1 of WO2020257194Al.
  • the definitions and selection ranges of the sense strand and antisense strand of the present disclosure are the same as those described above for the oligonucleotide conjugates of the present disclosure.
  • the sense strand and antisense strand can be determined by routine methods in the art. Double-stranded oligonucleotide preparation methods, such as solid phase synthesis and liquid phase synthesis. Among them, solid-phase synthesis already has commercial customization services.
  • Modified nucleotide groups are introduced into double-stranded oligonucleotides according to the present disclosure, methods for preparing nucleoside monomers with corresponding modifications, and methods for introducing modified nucleotide groups into double-stranded oligonucleotides It is also well known to those skilled in the art.
  • the present disclosure provides a pharmaceutical composition containing an oligonucleotide conjugate as described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a carrier commonly used in the field of double-stranded oligonucleotide delivery, such as but not limited to magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ) , carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamide dendrimer (PAMAM) dendrimer ), poly(L-lysine), PLL, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly(D&L-lactic/glycolic acid)copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), PPEEA) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and one or more of their derivatives.
  • magnetic nanoparticles such as nanoparticles based on Fe 3 O 4 or Fe
  • the functional double-stranded oligonucleotide and pharmaceutically acceptable carrier in the pharmaceutical composition there are no special requirements on the content of the functional double-stranded oligonucleotide and pharmaceutically acceptable carrier in the pharmaceutical composition.
  • the functional double-stranded oligonucleotide The weight ratio of nucleotides to pharmaceutically acceptable carriers may be 1:(1-500). In some embodiments, the weight ratio is 1:(1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be, for example, sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the dosage of the preparation made from the pharmaceutical composition during administration will be adjusted due to different administration methods.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid The pharmaceutical composition can be, but is not limited to, administered by subcutaneous, intramuscular, intracerebroventricular or intrathecal injection, and can also be delivered by, but is not limited to, eye drops, nasal administration, oropharyngeal inhalation, spray administration, etc.
  • the pharmaceutical composition is delivered by intrathecal injection.
  • intrathecal injection of the pharmaceutical composition into the spinal fluid can be performed as a bolus injection or via micropumps that can be implanted under the skin to provide regular and constant delivery of siRNA to in spinal fluid.
  • intrathecal administration is via a surgically implanted osmotic pump.
  • an osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration. More details about this intrathecal delivery system can be found in PCT/US 2015/013253, filed on January 28, 2015, which is incorporated herein by reference in its entirety.
  • the present disclosure provides use of an oligonucleotide conjugate of the present disclosure, and/or a pharmaceutical composition, in the preparation of a medicament for inhibiting target mRNA expression of target gene expression in a cell.
  • the target gene is APP, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR or TTR, LRRK2, DUX4 , complement 3, complement 5, NMDA, complement factor B or RHO.
  • the target gene is SOD1 or RPTOR.
  • the disclosure also provides an oligonucleotide conjugate and/or pharmaceutical composition of the disclosure for use in the treatment and/or prevention of diseases related to target mRNA expression of target gene expression.
  • the target gene is APP, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR, TTR, LRRK2, DUX4 , complement 3, complement 5, NMDA, complement factor B or RHO.
  • the target gene is SOD1 or RPTOR.
  • the present disclosure also provides a method of inhibiting target gene expression in a cell, the method comprising contacting an effective amount of an oligonucleotide conjugate and/or pharmaceutical composition of the present disclosure with the cell.
  • the target gene is APP, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR, TTR, LRRK2, DUX4 , complement 3, complement 5, NMDA, complement factor B or RHO.
  • the target gene is SOD1 or RPTOR.
  • the oligonucleotide conjugates and/or pharmaceutical compositions provided by the present disclosure can be administered to a subject in need, prevention and/or treatment of diseases caused by the expression of cell-specific genes can be achieved through the mechanism of regulating gene expression.
  • the purpose of causing a pathological condition or disease Therefore, the oligonucleotide compositions and/or pharmaceutical compositions provided by the present disclosure can be used to prevent and/or treat the pathological conditions or diseases described herein, or for the preparation of preparations for preventing and/or treating the pathological conditions described herein, or Medicines for diseases.
  • the term "administration/administration” refers to the administration of an oligonucleotide by a method or pathway that at least partially localizes the oligonucleotide conjugate and/or pharmaceutical composition to the desired site to produce the desired effect.
  • the glycolic acid conjugate and/or pharmaceutical composition is placed in a subject.
  • Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. Generally speaking, administration results in greater oligonucleotide conjugates and/or pharmaceutical compositions being delivered to a specific site compared to the subject's entire body; whereas systemic administration results in The oligonucleotide conjugates and/or pharmaceutical compositions are delivered to substantially the entire body of the subject.
  • Administration to the subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, or transdermal.
  • Drugs such as intravenous, intramuscular, subcutaneous, or transdermal.
  • Drugs airway administration (aerosol), intracerebroventricular administration, intrathecal administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration), intravitreal injection Medication, eye drops.
  • the frequency of administration may be once or more every day, every week, every two weeks, every three weeks, every month, every 2 months, every 3 months, every six months, or once a year.
  • the dosage of the oligonucleotide conjugates and/or pharmaceutical compositions described in the present disclosure can be conventional dosages in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject.
  • Sure. Toxicity and efficacy can be measured in cell culture or experimental animals by standard pharmaceutical procedures, such as determining the LD50 (lethal dose that kills 50% of the population) and ED50 (the dose that causes 50% of the maximum response intensity in quantitative response, and qualitatively).
  • reaction refers to the dose that causes 50% of experimental subjects to have a positive reaction). Dosage ranges for humans can be derived based on data from cell culture assays and animal studies.
  • the methods provided by the present disclosure are used to inhibit the expression of specific genes in cells.
  • the dosage of the provided oligonucleotide conjugates and/or functional double-stranded oligonucleotides in the pharmaceutical composition is obtained by those skilled in the art as desired. The effect is easy to determine.
  • the amount of functional double-stranded oligonucleotide in provided oligonucleotide conjugates is an amount sufficient to reduce expression of the target gene and result in 1 pM at the surface of the target cell. To an extracellular concentration of 1 ⁇ M, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or about 5 nM.
  • the amount required to achieve this local concentration will vary depending on various factors, including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cells or tissue, whether the 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 cells or tissue.
  • the present disclosure provides a kit comprising an effective dose of an oligonucleotide conjugate and/or pharmaceutical composition provided by the present disclosure.
  • kits described herein can provide oligonucleotide conjugates and/or pharmaceutical compositions in one container.
  • a kit described herein may include a container providing a pharmaceutically acceptable excipient.
  • the kit may also include other ingredients, such as stabilizers or preservatives.
  • the kits described herein can include at least one additional therapeutic agent in a container other than the container in which the oligonucleotide conjugates and/or pharmaceutical compositions described herein are provided.
  • the kit may include a method for mixing the oligonucleotide conjugate and/or pharmaceutical composition with a pharmaceutically acceptable carrier and/or excipient or other ingredients, if any. instruction manual.
  • the oligonucleotide conjugate and pharmaceutically acceptable carrier and/or auxiliary material, and the pharmaceutical composition and/or pharmaceutically acceptable carrier and/or auxiliary material can be Available in any form, such as liquid form, dry form and/or lyophilized form.
  • the oligonucleotide conjugates and pharmaceutically acceptable carriers and/or excipients, and the pharmaceutical compositions and/or optional pharmaceutically acceptable excipients are substantially pure and/or or sterile.
  • this disclosure can Sterile water is provided in the opened kit.
  • the reagents and cultures used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory (1989)) .
  • the sequence of the siRNA sense strand in siRNA conjugate 1 in Table 2 was synthesized by a solid-phase synthesis method. The only difference is that during the solid-phase synthesis, the 3' Before the first nucleoside monomer at the 5' end and after the last nucleoside monomer at the 5' end, a phosphoramidite monomer containing a HO(CH 2 ) 6 -SS-(CH 2 ) 6 -group is connected.
  • Dissolve S2 (42.00 mg, 6.61 ⁇ mol) prepared in step (1-2) in a 15 ml centrifuge tube with 7.00 ml purified water. After complete dissolution, add Py-S-S-Py (0.1515 g) to the centrifuge tube. , 0.68mmol, purchased from Aladdin Company, batch number: F2015072). Vortex and mix, react at room temperature for 6 hours, and detect the reaction with an Agilent 1260 HPLC instrument.
  • HS-P7 (30.7 mg, 29.60 ⁇ mol, customized and synthesized from Tanzhen Biotech Company) was added to the reaction solution.
  • HS-P7 is in the N-terminal to C-terminal direction and contains the sequence shown in SEQ ID NO: 169 (HAIYPRH ) polypeptide ligand, mix well and react at room temperature for 6 hours. Dilute the reaction solution with an equal volume of purified water, filter, and use Agilent semi-preparative reverse-phase purification.
  • the antisense strand in oligonucleotide conjugate 1 was prepared according to the method described in Preparation Example 1 of WO2019105418 (A1), the only difference being that the nucleoside monomers were connected sequentially according to the sequence shown in SEQ ID NO: 129 in Table 2. body; use DEPC water to dissolve equal moles of the sense strand prepared in step (1-4) and the antisense strand prepared in step (1-5), followed by annealing to obtain conjugate 1 in Table 2. After the preparation is completed, use LC-MS to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand is: 8613.86, the measured value: 8612.76, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, the measured value and the theoretical value The values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 1 in Table 2.
  • the capital letters C, G, U, and A indicate the base composition of nucleotides;
  • the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • f indicates that the nucleotide adjacent to the left side of the letter f is a fluorine-modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group;
  • L3 represents a linking group R I.
  • P7 represents a polypeptide with the sequence shown in SEQ ID NO:169 in the direction from N-terminus to C-terminus;
  • RP7 represents a polypeptide with the sequence shown in SEQ ID NO:169 in the direction from C-terminus to N-terminus connected to the core.
  • P12 represents a polypeptide having the sequence shown in SEQ ID NO:170 in the direction from N-terminus to C-terminal;
  • RP12 represents a polypeptide having the sequence shown in SEQ ID NO:170 in the direction from C-terminus to N-terminus Connected to a nucleotide;
  • the capital letter L indicates that the nucleotide adjacent to the left of the capital letter L is a nucleotide formed by replacing the hydrogen in the hydroxyl group at the 2' position of the ribose ring with a hexadecyl group.
  • sequence shown in SEQ ID NO: 226 was prepared using the same method as in Preparation Example 13 of WO 2020/257194 Al. The only difference is that the sequence of the sense strand in siRNA is the sequence shown in SEQ ID NO: 226.
  • Conjugate 2 of the present disclosure was prepared according to the same method as in Preparation Example 1, the only difference being that for conjugate 2, instead of the sequence shown in SEQ ID NO: 127, the conjugate 2 was prepared according to the sequence shown in SEQ ID NO: 138 The sequence connects the nucleoside monomers of the sense strand in turn:
  • Conjugate 3 of the present disclosure was prepared according to the same method as in Preparation Example 1, the only difference being that polypeptide P12 as shown in SEQ ID NO:170 was used instead of polypeptide P7 as shown in SEQ ID NO:169.
  • the theoretical value of the sense strand 9211.59, the measured value: 9210.76, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, and the measured value is consistent with The theoretical values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 3 in Table 2.
  • Conjugate 4 of the present disclosure was prepared according to the same method as in Preparation Example 2, the only difference being that polypeptide P12 as shown in SEQ ID NO:170 was used instead of polypeptide P7 as shown in SEQ ID NO:169.
  • LC-MS is used to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand is: 9211.59, the measured value: 9211.89, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, the measured value and the theoretical value The values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 4 in Table 2.
  • the compound represented by formula (210) is synthesized according to the same method as disclosed in Scheme 3 (page 27) of WO2014025805A1.
  • the compound represented by formula (210) (9.00g, 15.97mmol) and HOBt (9.71g, 71.85mmol, McLean Company, product number: C10116218) were dissolved in DMF (90mL), and 2-((2-propargyl) was added baseoxy)ethoxy)ethylamine (8.57g, 59.88mmol) (compound represented by formula (211), purchased from Bide Pharmaceuticals, product number: ANL677) and EDC ⁇ HCl (11.48g, 59.88mmol, McLean Company, Catalog No.: C10087270), stir until completely dissolved and then add DIEA (14.43g, 111.77mmol, Anaiji Company, Catalog No.: EE300104).
  • phase A dichloromethane
  • DMF purchased from McLean Company, batch number: C10594019
  • nucleic acid precipitation solution 10% trichloroacetic acid aqueous solution, purchased from Biolab, product number: GL1252
  • nucleic acid precipitation solution 10% trichloroacetic acid aqueous solution, purchased from Biolab, product number: GL1252
  • the resulting precipitate is oligonucleotide single-stranded S5. (41.00mg, yield: 90.04%) (molecular weight: 7477.76, measured: 7475.85).
  • the antisense strand in oligonucleotide conjugate 5 was prepared according to the method described in Preparation Example 13 of WO2019105418 (A1). The only difference lies in that the nucleosides are connected sequentially according to the sequence shown in SEQ ID NO: 137 in Table 2. monomer to obtain the antisense strand sequence; use DEPC water to dissolve the sense strand prepared in step (5-6) and the antisense strand prepared above in equal moles, and anneal to obtain conjugate 5 in Table 2. After the preparation is completed, use LC-MS to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand is: 10741.00, the measured value: 10738.99, the theoretical value of the antisense chain: 7096.79.
  • the measured value: 7097.12, the measured value and the theoretical value The values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 5 in Table 2.
  • Conjugate 6 provided by the present disclosure was prepared according to the same method as in Preparation Example 1, the only difference being that HS-P7 in step (1-4) was replaced with HS-RP7 as shown in formula (601):
  • P7-N3 (26 mg, 24.8 umol, commercially ordered from Nanchang Probe Company) was dissolved in 1 mL DMF, and the oligonucleotide single-stranded S7A (26.2 mg, 4.13 umol) shown in formula (7-1-1) was , commercially ordered from Shanghai Zhaowei Company) was dissolved in 1 mL of purified water, mix the aforementioned solution evenly, and add copper sulfate solution (500ul, 24.8umol, purchased from McLean Company) and TBTA DMF solution (50mM, 500ul, Purchased from Shanghai Bid Company) and sodium ascorbate solution (1M aqueous solution, 124ul, purchased from McLean Company).
  • copper sulfate solution 500ul, 24.8umol, purchased from McLean Company
  • TBTA DMF solution 50mM, 500ul, Purchased from Shanghai Bid Company
  • sodium ascorbate solution (1M aqueous solution, 124ul, purchased from McLean Company).
  • the antisense strand in oligonucleotide conjugate 7 was prepared.
  • use LC-MS to detect the molecular weights of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand is: 8525.71, the measured value: 8525.88, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, the measured value and the theoretical value The values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 7 in Table 2.
  • Conjugate 8 provided by the present disclosure was prepared according to the same method as in Preparation Example 7, the only difference being that the oligonucleotide single-stranded S8A (27.1 mg, 4.13umol, commercially ordered from Shanghai Zhao Wei Company) to replace S7A, and RP12-N3 as shown in formula (802) to replace P7-N3 (42 mg, 24.8umol, commercially ordered from Nanchang Probe Company) to prepare the sense strand S8 (9 mg, yield: 22.0% , mass spectrometry theoretical value: 9914.30, measured value: 9912.90):
  • the antisense strand in oligonucleotide conjugate 8 was prepared.
  • use LC-MS to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand is: 9914.30, the measured value: 9912.90, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, the measured value and the theoretical value The values are consistent, indicating that the conjugate obtained has the sequence corresponding to conjugate 8 in Table 2.
  • Conjugate 9 provided by the present disclosure was prepared according to the same method as in Preparation Example 7, the only difference being that RP12-N3 as shown in formula (802) was used instead of P7-N3 (42.1 mg, 24.8umol, commercially ordered from Nanchang Tan needle company).
  • LC-MS was used to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the theoretical value of the sense strand was: 9721.18, the measured value: 9719.34, the theoretical value of the antisense strand: 7096.79.
  • the measured value: 7097.12, and the measured value was consistent with the theoretical value, indicating that the What was obtained was a conjugate with the sequence corresponding to conjugate 9 in Table 2.
  • Comparative conjugate 1 was prepared according to the same method as in Preparation Example 1. The only difference is that when preparing the sense strand, only after connecting the last nucleoside monomer at the 5' end, another conjugate containing HO(CH 2 ) 6 - was connected.
  • SS-(CH 2 ) 6 -group phosphoramidite monomer purchased from Hongene Biotech
  • HS-P7 replace HS-P7 in step (1-4) with HS- as shown in formula (601)
  • RP7 after the preparation is completed, use LC-MS to detect the molecular weight of the sense strand and the antisense strand respectively.
  • the measured values are consistent with the theoretical values, indicating that what is obtained is a conjugate with the sequence corresponding to the comparative conjugate 1 in Table 2. things.
  • Comparative conjugate 2 was prepared according to the same method as in Comparative Preparation Example 1. The only difference is that when preparing the sense strand, only before the first nucleoside monomer at the 3' end is connected, HO (CH 2 ) is additionally connected. 6 -SS-(CH 2 ) 6 -group phosphoramidite monomer (purchased from Hongene Biotech Company), after the preparation is completed, use LC-MS to detect the molecular weight of the sense strand and antisense strand respectively. The actual measured value and the theoretical value The values are consistent, indicating that what is obtained is a conjugate with the corresponding sequence in comparative conjugate 2 in Table 2.
  • Comparative conjugate 3 was prepared according to the same method as in Preparation Example 7, except that P7-N3 (13 mg, 12.4 umol, commercially ordered from Nanchang Probe Company) was dissolved in 1 mL DMF, and the formula was as follows: -1) The oligonucleotide single-stranded SS3A shown (26.2mg, 4.13umol, commercially ordered from Shanghai Zhaowei Company):
  • Comparative conjugate 4 was prepared according to the same method as in Comparative Preparation Example 3, except that the single-stranded oligonucleotide SS4A (26.3 mg, 4.13umol, commercially ordered from Shanghai) was used as shown in formula (12-1). Zhaowei Company) to replace SS3A in Comparative Preparation Example 3:
  • Comparative conjugate 5 was prepared according to the same method as in Comparative Preparation Example 3, the only difference being that RP12-N3 (20.1 mg, 12.4umol, commercially ordered from Nanchang Probe Co., Ltd.) as shown in formula (802) was used instead of the comparison.
  • Comparative conjugate 6 was prepared according to the same method as in Comparative Preparation Example 4, except that RP12-N3 (20.1 mg, 12.4umol, commercially ordered from Nanchang Probe Co., Ltd.) as shown in formula (802) was used instead of the comparative conjugate 6.
  • This experimental example examined the inhibitory activity of the prepared conjugate 1, conjugate 2 and conjugate 5 on the mRNA expression of SOD1 gene in mice, especially in the central nervous system.
  • mice in this experiment were purchased from Spefford Company.
  • the germ line is ICR, the grade is SPF, the gender is male, and the purchase weight is 25 ⁇ 1g.
  • the powdered conjugate was dissolved in PBS and diluted into an injection solution with a concentration of 20 ⁇ g/ ⁇ L for later use.
  • mice were randomly divided into 3 groups, with 6 mice in each group, numbered as blank group, test group 1, and test group 1.
  • Trial Group 2 and Test Group 3. No administration was given to the mice in the blank group.
  • conjugate 1, conjugate 2 and conjugate 5 were administered at a dose of 10 ⁇ L/mouse respectively.
  • the specific steps are: for each mouse The mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a dose of 400 mg/Kg body weight.
  • microinjection pump model: 78-8130, purchased from KDSCIENTIFIC Company
  • a 25 ⁇ L microinjection needle purchased from Hamilton Company
  • the injection time is 10 minutes, and the needle is retained after the injection. 5 minutes, then slowly remove the needle.
  • the pinholes were sealed with biological glue (purchased from Minnesota Mining Manufacturing Co., Ltd.), and the head skin was bonded. After the mice woke up, they were returned to the animal breeding room and continued to be raised. Record the day of injection as day one.
  • each mouse was intraperitoneally injected with 5% chloral hydrate at a dose of 400 mg/Kg body weight, and then the parietal cortex, hippocampus, cerebellum, oblongata, thalamus, and striatum of the mice were extracted and preserved.
  • RNA later No. MFCD03453003, purchased from SIGMA Company
  • RNAVzol purchased from Viglas Biotechnology (Beijing) Co., Ltd., Cat. No. N002
  • the conditions for reverse transcription are: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then incubate at 85°C for 5 minutes, and finally incubate at 4°C for 30 seconds. After the reaction is completed, proceed to the reverse transcription reaction. Add 80 ⁇ l of DEPC water to the system to obtain a solution containing cDNA.
  • each reverse transcription reaction system For each reverse transcription reaction system, take 5 ⁇ l of the above cDNA-containing solution as a template, and use the reagents provided by the SYBR TM Select Master Mix kit (purchased from Appliedbiosystems, Cat. No.: 50000332) to configure 20 ⁇ l of the qPCR reaction system, where:
  • the PCR primer sequences for amplifying the target gene SOD1 and the internal reference gene GAPDH are shown in Table 3.
  • the final concentration of each primer is 0.25 ⁇ M. Place each qPCR reaction system on the ABI StepOnePlus Real-Time PCR instrument and use a three-step method for amplification.
  • the amplification program is pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. After repeating the above-mentioned denaturation, annealing, and extension processes for a total of 40 times, a product W containing amplified target gene SOD1 and internal reference gene GAPDH was obtained. The product W was then incubated at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The real-time fluorescence quantitative PCR instrument collected the dissolution curves of the target gene SOD1 and the internal reference gene GAPDH in the product W, and obtained the Ct of the target gene SOD1 and the internal reference gene GAPDH. value.
  • the comparative Ct ( ⁇ Ct) method was used to calculate the relative quantification of the target gene APOC3 in each test group.
  • the calculation method is as follows:
  • ⁇ Ct (test group) Ct (test group target gene) – Ct (test group internal reference gene)
  • ⁇ Ct (control group) Ct (target gene in control group) – Ct (internal reference gene in control group)
  • ⁇ Ct control group average
  • ⁇ Ct control group average
  • the expression level of SOD1 mRNA in the test group was normalized, and the SOD1 mRNA expression level in the blank control group was defined as 100%.
  • SOD1 mRNA inhibition rate in the test group (1 - relative expression level of APOC3 mRNA in the test group) ⁇ 100%.
  • the inhibition rates of SOD1 mRNA in different regions of the brain by each siRNA conjugate are summarized in Table 4.
  • Conjugate 1 which is conjugated with the polypeptide ligand of the present disclosure at both ends of the sense strand, basically has an inhibition rate of more than 47.8% on SOD1 mRNA in different parts. Especially in the cortex area, the inhibition rate can be as high as 79.87%.
  • conjugate 2 conjugated with a lipophilic group showed higher inhibitory activity, with an inhibition rate of SOD 1 mRNA higher than 60% in all regions.
  • the inhibition rates were higher than 80%.
  • the inhibition rate in the cortex is as high as 88.33%.
  • Conjugate 5 which was conjugated with a lipophilic group at the 5' end of the sense chain and three polypeptide ligands at the 3' end, also showed up to 80.00% and 75.90% in the cortex and hippocampus areas, respectively.
  • Inhibition rate of SOD1 mRNA This shows that the conjugates of the present disclosure all display excellent SOD1 mRNA inhibitory activity and exhibit good pharmaceutical activity.
  • conjugate 3 of the present disclosure can inhibit SOD1 mRNA in the cortex area by up to 80.02%, while conjugate 4 further conjugated with a lipophilic group shows generally higher inhibitory activity against SOD in all areas.
  • the inhibition rates of 1 mRNA were all higher than 60%.
  • the inhibition rates were higher than 70%.
  • the inhibition rate in the cortex is as high as 86.53%. Displays excellent SOD1 mRNA inhibitory activity.
  • Conjugate 1 and Conjugate 2 of the present disclosure showed high levels of mRNA expression of SOD1 gene in different parts of the central nervous system in rats. inhibition rate.
  • Conjugate 1 of the present disclosure exhibits a pH higher than 55% in each region. Inhibitory activity, especially the inhibition rate of SOD1 mRNA in the cortex area can be as high as 81.55%.
  • Conjugate 2 which was further conjugated with a lipophilic group, showed generally higher inhibitory activity, with an inhibition rate of SOD 1 mRNA higher than 75% in all regions. Especially in the cortex, hippocampus, cerebellum and striatum, the inhibition rate of SOD1mRNA was higher than 85%.
  • the inhibition rate in the cerebellum is as high as 89.11%. Displays very high inhibitory activity on SOD1 mRNA.
  • the above results indicate that the conjugate of the present disclosure can effectively deliver siRNA to TfR-expressing tissues, such as various tissues within the central nervous system, and the delivered siRNA exhibits excellent target mRNA inhibition effect in these tissues.
  • conjugate 6 is an siRNA in which the first nucleotide at both ends of the sense strand is conjugated to one P7 polypeptide ligand.
  • comparative conjugate 1 and comparative conjugate 2 only have the P7 polypeptide ligand conjugated to the first nucleotide at the 5' end and the first nucleotide at the 3' end of the sense strand respectively. It can be seen from the results in Table 8 that Conjugate 6 of the present disclosure showed significantly higher inhibition rates than Comparative Conjugate 1 and Comparative Conjugate 2 in different regions of the central nervous system.
  • the inhibition rate of the conjugate provided by the present disclosure is as high as 48.18%, which is 18.68% and 10.88% higher than that of Comparative Conjugate 1 and Comparative Conjugate 2, respectively.
  • the inhibition rate of the conjugate provided by the present disclosure is as high as 65.11%, which is 29.36% and 16.72% higher than that of comparative conjugate 1 and comparative conjugate 2 respectively, showing significantly better Inhibitory effect.
  • Conjugate 7 is a siRNA in which the first nucleotide at both ends of the sense strand is conjugated to one RP7 polypeptide ligand, and Compare conjugates 3 and Comparative conjugate 4 only has the RP7 polypeptide ligand conjugated to the first nucleotide at the 5' end and the first nucleotide at the 3' end of the sense strand respectively. It can be seen from the results in Table 9 that in the cortex and hippocampus regions, Conjugate 7 of the present disclosure showed a significantly higher inhibition rate than Comparative Conjugate 3 and Comparative Conjugate 4, and the inhibition rates were both increased. About 10%.
  • the siRNA conjugate of the present disclosure is more effective in delivering siRNA to various tissues in the central nervous system expressing TfR, and the delivered siRNA exhibits excellent target mRNA inhibition effects in these tissues.
  • mice were randomly divided into 6 groups, with 6 mice in each group, numbered as blank group and test group 1-5. No administration was given to the mice in the blank group.
  • test groups 1-5 the conjugates used for injection corresponded to conjugate 8, conjugate 9 and comparative conjugate 5, comparative conjugate 6 respectively. The results are summarized in Table 10.
  • both Conjugate 8 and Conjugate 9 of the present disclosure showed significantly better inhibition rates.
  • the inhibition rates of the conjugates of the present disclosure were all higher than 60%, and the inhibition rate of conjugate 8 was as high as 74.45%, which was 2.5 times that of the comparative conjugate 5.
  • the inhibition rate of conjugate 8 of the present disclosure was also as high as 75.37%, which was twice the inhibition rate of comparative conjugate 5 and comparative conjugate 6.
  • the inhibition rate of conjugate 9 is as high as 59.91%, which is 24.01% and 23.78% higher than that of comparative conjugate 5 and comparative conjugate 6, respectively.
  • the conjugate of the present disclosure can more effectively deliver siRNA to various tissues within the central nervous system expressing TfR, and the delivered siRNA Exhibits excellent target mRNA inhibition effects in these tissues.
  • any combination of various embodiments of the present disclosure can also be carried out, and as long as they do not violate the idea of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

Abstract

本公开提供了一种新的寡核苷酸缀合物，以及含有该寡核苷酸缀合物的药物组合物。所述寡核苷酸缀合物包含功能性双链寡核苷酸和2-6个多肽配体，所述多肽配体与转铁蛋白受体具有亲和性；其中，所述功能性双链寡核苷酸和所述多肽配体通过共价键直接连接或者通过连接基团连接。本公开提供的寡核苷酸缀合物可以和细胞表面存在转铁蛋白受体的细胞结合，并经过转铁蛋白受体介导的内吞而进入细胞内，能够降低靶标mRNA水平，进而治疗和/或预防相关疾病。

Description

一种寡核苷酸缀合物、含有该寡核苷酸缀合物的组合物及制备方法和用途 技术领域
本公开涉及一种能够和转铁蛋白受体(TfR)结合的寡核苷酸缀合物，本公开还涉及含有这些寡核苷酸缀合物的组合物及其制备方法和用途。
背景技术
寡核苷酸包括但不限于小干扰RNA(siRNA)、小激活RNA(saRNA)，单链寡核苷酸等。近年来，寡核苷酸，特别是双链寡核苷酸作为药物活性成分已为公众所知，在双链寡核苷酸成药方面亦取得了相当程度的进展。然而，许多临床前药学研究中心显示出优异药学活性的双链寡核苷酸由于缺乏有效的递送载体而难以有效地到达特定的靶器官或靶组织，因此难以用于实际的药物研发，尤其是对于一些神经系统疾病，包括中枢神经系统疾病和外周神经系统疾病，肌肉疾病。因此，能够有效地递送到特定靶器官或靶组织，特别是如脑部，脊髓，视神经组织，嗅神经组织，耳神经以及其它外周神经组织，神经肌肉接头或肌肉组织，肿瘤组织的寡核苷酸药物在本领域中仍然存在重大的实际需求。
发明内容
本发明提供了一种具有高的递送效率的寡核苷酸缀合物，能够有效地将寡核苷酸递送至靶器官或靶组织，例如中枢神经系统，表现出很高的药学活性。
在一方面，本公开提供了一种寡核苷酸缀合物，所述寡核苷酸缀合物包含：
(a)功能性双链寡核苷酸，所述功能性双链寡核苷酸包含正义链和反义链，所述正义链和反义链分别包含15-25个核苷酸，每个所述核苷酸是修饰或未修饰的核苷酸；
(b)n0个多肽配体，每个所述多肽配体由5-12个修饰或未修饰的氨基酸组成，其中，n0为2-6的整数，所述多肽配体与转铁蛋白受体具有亲和性；
其中，每个所述多肽配体与所述功能性双链寡核苷酸通过共价键或者通过连接基团RI连接，每个所述多肽配体通过多肽配体的N端或C端连接至所述功能性双链寡核苷酸。
在另一方面，本公开还提供了一种药物组合物，所述药物组合物含有本公开的寡核苷酸缀合物和其药学上可接受的载体。
在又一方面，本公开还提供了本公开的寡核苷酸缀合物和/或药物组合物在制备用于抑制细胞中靶基因表达的靶mRNA表达的药物中的用途。
在又一方面，本公开还提供了一种抑制细胞中靶基因表达的方法，该方法包括将有效量的本公开的寡核苷酸缀合物和/或本公开的药物组合物与所述细胞接触。
另外，本公开还提供了一种试剂盒，所述试剂盒包含本公开的寡核苷酸缀 合物和/或药物组合物。
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
本公开提供的多肽缀合的寡核苷酸缀合物和/或药物组合物具有良好的稳定性，较高的目标基因表达调节活性，在中枢神经系统中具有很高的递送效率，表现出很高的药学活性。更进一步地，若在前述寡核苷酸缀合物中缀合亲脂基团，可以进一步提升前述寡核苷酸缀合物的递送效率，表现出普遍更高的药物活性。具体说明如下。
本公开的双链寡核苷酸和/或药物组合物在体内表现出优异的对中枢神经系统中靶基因表达的mRNA的抑制效果。例如，在小鼠体内，缀合有2个多肽配体的本公开的寡核苷酸缀合物对中枢神经不同部位的SOD1 mRNA的抑制率基本均达到50％，尤其是在皮层区域，抑制率可高达80.02％。更进一步地，缀合了亲脂基团的本公开的寡核苷酸缀合物对中枢神经不同部位的SOD1 mRNA的抑制率均高于60％，特别是在皮层、海马体和小脑，抑制率均高于70％。在皮层的抑制率更是高达88.33％。显示出优异的SOD1 mRNA的抑制活性。又例如，在大鼠体内，含有2个多肽配体的本公开的寡核苷酸缀合物在脑部各个区域均表现出高于55％的抑制活性，在皮层区域对SOD1 mRNA的抑制率可高达81.55％。更进一步地，缀合了亲脂基团的本公开的寡核苷酸缀合物对中枢神经不同部位的SOD1 mRNA的抑制率均高于75％。特别是在皮层、海马体、小脑和纹状体，对SOD1 mRNA的抑制率均高于85％。在小脑的抑制率更是高达89.11％。在正义链5’末端缀合有亲脂基团，并且在3’末端缀合有3个多肽配体的本公开缀合物在皮层区和海马区也分别显示出高达80.00％和75.90％的对SOD1 mRNA的抑制率。从而说明含有不同连接基团的、在不同位置含有不同数量的亲脂基团和/或多肽配体的本公开的缀合物均显示出优异的SOD1 mRNA的抑制活性，表现出良好的药学活性。
更进一步地，本公开的含有多个多肽配体的缀合物表现出比仅含有1个多肽配体的参比缀合物明显更高的抑制率，且含有不同多肽配体、多个配体连接在寡核苷酸中不同位置的本公开提供的缀合物均表现出类似效果。例如，在小鼠体内，与仅缀合有1个多肽配体的对比缀合物相比，本公开提供的缀合物在中枢神经系统内的不同区域均表现出明显更高的靶mRNA抑制率。其中，在延脑区域，本公开提供的缀合物的抑制率高达48.18％，相较于对比缀合物提高了18.68％。而在纹状体区域，本公开提供的缀合物的抑制率高达65.11％，相较于对比缀合物提高了29.36％，表现出明显更好的抑制效果。又例如，在海马体中，本公开的缀合物的抑制率均高于60％，最高高达74.45％，能达到对比缀合物的抑制率的2.5倍。在皮层中，本公开的缀合物的抑制率也高达75.37％， 比对比缀合物的抑制率的两倍更高。
上述结果表明本公开的缀合物和对比缀合物相比，更能够有效地将siRNA递送至表达TfR的中枢神经系统内的各种组织，并且所递送的siRNA在这些组织内表现出优异的靶mRNA抑制效果。
由此说明，本公开提供的寡核苷酸缀合物以及药物组合物能够在体内外有效调节目标基因的表达水平，具有优异的调控表达TfR的组织或细胞，如中枢神经系统内各种组织中靶基因表达的mRNA水平的功能，因此可以有效治疗和/或预防和靶基因表达的mRNA水平相关的疾病症状，具有良好的应用前景。
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，SOD1 mRNA是指Genbank注册号为NM_011434.2或NM_000454.5的mRNA；RPTOR mRNA是指Genbank注册号为NM_020761.3所示的序列。
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(G)始终与嘧啶碱基胞嘧啶(C)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。
在本文的上文及下文中，如无特别说明，在多肽序列中，每一个字母表示一个氨基酸，其中G表示甘氨酸，A表示丙氨酸，V表示缬氨酸，L表示亮氨酸，I表示异亮氨酸，P表示脯氨酸，F表示苯丙氨酸，Y表示酪氨酸，W表示色氨酸，S表示丝氨酸，T表示苏氨酸，C表示半胱氨酸，M表示蛋氨酸，N表示天冬酰胺，Q表示谷酰胺，D表示天冬氨酸，E表示谷氨酸，K表示赖氨酸，R表示精氨酸，H表示组氨酸。
在本文的上文及下文中，多肽序列是指由多个氨基酸单体通过羧基位置和氨基位置发生的脱水缩合反应形成的多肽序列，如无特别说明，多肽序列的N端指在缩合形成的多肽中含有未反应的氨基的一端，多肽序列的C端指在缩合形成的多肽中含有未反应的羧基的一端。
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，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
在上文及下文中，特别是在描述双链寡核苷酸、药物组合物或双链寡核苷酸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，根据欲制备的双链寡核苷酸或双链寡核苷酸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 RNAphosphoramidites，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，“寡核苷酸缀合物”或“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寡核苷酸或siRNA上而形成的化合物。寡核苷酸缀合物应根据上下文，理解为多个寡核苷酸缀合物或siRNA缀合物的总称或者某个化学式所示的、寡核苷酸缀合物或siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过 反应缀合至寡核苷酸，最终形成本公开的寡核苷酸缀合物或siRNA缀合物的特定化合物。
在上文或下文中，“经取代的”或“被取代的”基团包括但不限于经取代的烷基、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，“饱和烷基”是指所有碳原子之间通过单键相连，不含有碳碳双键和/或碳碳三键的烷基。
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：1,2,3-三氮唑亚基，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”是指指定数量的碳原子被一个或多个、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团对特定的反应条件不敏感，并且可以在分子中的该官能团上添加以及去除，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(DMTr)、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。
本公开所述寡核苷酸或寡核苷酸缀合物中，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。出于提高溶解性考虑，在一些实施方式中，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。因此，本公开所述寡核苷酸或寡核苷酸缀合物可以至少部分以盐的形式存在。在一些实施方式中，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，本公开所述寡核苷酸或寡核苷酸缀合物以钠盐或部分钠盐的形式存在。因此，在提及本公开所述的寡核苷酸或寡核苷酸缀合物，包括但不限于本公开所述的任何结构式表示的寡核苷酸缀合物时，均旨在涵盖该寡核苷酸或寡核苷酸缀合物的钠盐或部分钠盐形式。
本公开所述“转铁蛋白受体”是指转铁蛋白受体1(TfR1)和/或转铁蛋白受体2(TfR2)。转铁蛋白受体(TfR)介导含铁的铁蛋白从细胞外进入细胞内，其存在于许多细胞的表面，在许多靶组织或靶器官，诸如骨骼肌、平滑肌、脑部、眼部、肿瘤组织等中均有表达。
本公开所述“多肽配体”是指和寡核苷酸通过共价键或连接基团的由一定数量的氨基酸组成的结合肽。本公开所述“多肽配体与转铁蛋白受体具有亲和性”是指本公开的多肽配体可以和转铁蛋白受体特异性结合，并且本公开的多肽配体的序列是不含有转铁蛋白受体和转铁蛋白的结合域的序列。
本公开所述“功能性双链寡核苷酸”是指具有能够上调或者下调靶基因表达的靶mRNA水平功能的双链寡核苷酸，包括能够上调靶基因表达的靶mRNA水平的小激活RNA(saRNA)，和能够下调靶基因表达的靶mRNA水平的小干扰RNA(siRNA)。
本公开的寡核苷酸缀合物
在一方面，本公开提供了一种寡核苷酸缀合物，所述寡核苷酸缀合物包含：
(a)功能性双链寡核苷酸，所述功能性双链寡核苷酸包含正义链和反义链，所述正义链和反义链分别包含15-25个核苷酸，每个所述核苷酸是修饰或未修饰的核苷酸；
(b)n0个多肽配体，每个所述多肽配体由5-12个修饰或未修饰的氨基酸组成，其中，n0为2-6的整数，所述多肽配体与转铁蛋白受体具有亲和性；
其中，每个所述多肽配体与所述功能性双链寡核苷酸通过共价键或者通过连接基团RI连接，每个所述多肽配体通过多肽配体的N端或C端连接至所述功能性双链寡核苷酸。
在一些实施方式中，每个所述多肽配体连接至所述功能性双链寡核苷酸中的正义链或反义链。
本公开的寡核苷酸缀合物中，每一个多肽配体可以连接至任意至所述功能性双链寡核苷酸中的任意位置，即，有多个多肽配体存在的情况下，多个配体可以连接至双链寡核苷酸中的同一个核苷酸或分别连接至不同的核苷酸上。为了减小空间位阻对寡核苷酸缀合物活性的影响，在一些实施方式中，每个所述连接有多肽配体的共价键和/或连接基团RI分别和所述双链寡核苷酸中不同位置的核苷酸相连。在一些实施方式中，对于不同所述连接有多肽配体的共价键和/或连接基团RI，其所连接的核苷酸是相邻的核苷酸。在一些实施会中，其所连接的核苷酸之间间隔至少1个核苷酸。在一些实施方式中，其所连接的核苷酸之间间隔至少3个核苷酸。在一些实施方式中，其所连接的核苷酸之间间隔至少5个核苷酸。
在一些实施方式中，部分多肽配体连接至所述功能性双链寡核苷酸的正义链中，部分多肽配体连接至所述功能性双链寡核苷酸的反义链中。在一些实施方式中，全部多肽配体均连接至所述反义链。在一些实施方式中，全部多肽配体均连接至所述正义链。
在一些实施方式中，至少一个所述多肽配体连接至所述正义链或反义链的3'末端或5'末端的第一个核苷酸。在一些实施方式中，至少两个所述多肽配体分别连接至反义链的3'末端或5'末端的第一个核苷酸。为了获得更好的药学活性，在一些实施方式中，至少一个所述多肽配体连接至所述正义链的3'末端，至少一个多肽配体连接至所述正义链的5'末端。
在一些实施方式中，所述寡核苷酸缀合物中，多肽配体的数量n0为2-4的整数。
在一些实施方式中，n0＝2，一个所述多肽配体连接至所述正义链3'末端或5'末端的第一个核苷酸，另一个多肽配体连接至所述正义链中其他位置的核苷酸中的任意一个；或者，一个多肽配体连接至所述正义链3'末端的第一个核苷酸，另外一个多肽配体连接至所述正义链5'末端的第一个核苷酸。所述其他位置的核苷酸指的是正义链中除了3'末端或5'末端的第一个核苷酸以外的核苷酸。
在一些实施方式中，全部多肽配体均连接至所述正义链3'末端或5'末端的第一个核苷酸。在一些实施方式中，n0＝3。
在一些实施方式中，每个所述多肽配体连接至所述功能性双链寡核苷酸中核苷酸的核糖环，连接位置为核糖环的2'、3'或5'位置；或者，每个所述多肽配体连接至所述功能性双链寡核苷酸中核苷酸的碱基。为了简化合成工艺，在一些实施方式中，每个所述多肽配体连接至所述功能性双链寡核苷酸中核苷酸的核糖环，连接位置为核糖环的2'、3'或5'位置。
在一些实施方式中，每一个所述多肽配体分别通过一个共价键连接至所述 功能性双链寡核苷酸；或者，每一个所述多肽配体分别通过一个所述连接基团RI连接至所述功能性双链寡核苷酸。为了简化合成工艺，在一些实施方式中，所有的多肽配体均通过所述连接基团RI连接至所述功能性双链寡核苷酸，其中，任意2个、3个、4个、5个或6个所述多肽配体通过同一个所述连接基团RI连接至所述功能性双链寡核苷酸。
在一些实施方式中，本公开的寡核苷酸缀合物中，所述连接基团RI包含主链部分、1-6个侧链部分和1个缀合连接部，所述主链部分分别与所述缀合连接部和所述侧链部分连接，所述侧链部分分别与所述主链部分和所述多肽配体连接，所述缀合连接部分分别与所述主链部分和所述双链寡核苷酸连接。在一些实施方式中，所述连接基团RI具有式(101)所示的结构：
其中，LA为侧链部分，k为1-6的整数，LB为缀合连接部，LC为主链部分，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点；PP表示所述连接基团RI连接至所述多肽配体的位点；
所述主链部分LC为共价键或2-7价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
在一些实施方式中，LC为C5-C20饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-CONH2。在一些实施方式中，LC的长度为5-30个原子，其中所述LC的长度指LC中与LA直接连接的原子到与LB直接连接的原子形成的最长的原子链上的成链原子的个数。为了简化结构，在一些实施方式中，LC的长度为8-25个原子。
所述侧链部分LA为共价键，或者C1-C20亚烷基，或者，所述亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述亚烷基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH-SC1-C5烷基、-SC1-C5烷基苯基、-C1-C5烷基-SH、-OH、-SH、-NH2、-C1-C5烷基-NH2、-N(C1-C5烷基)(C1-C5烷基)、-NH(C1-C5烷基)、-N(C1-C5烷基)(C1-C5烷基苯基)、-NH(C1-C5烷基苯基)、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
所述缀合连接部LB为1-5个以下连接键中的一种或多种的连接组合：磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键。
在一些实施方式中，k为1-3的整数；LC含有如式(L1)-(L3)所示的基团中的任意一个，通过如式(L1)-(L3)所示的基团中的醚键与LA部分连接：
表示基团连接至分子其余部分的位点；
在一些实施方式中，k＝1,Lc含有如式(L1)所示的基团，基团(L1)中的O原子和LA直接相连。在一些实施方式中，k＝2，Lc含有如式(L2)所示的基团，基团(L1)中的2个O原子各自和1个LA直接相连。在一些实施方式中，k＝4，Lc含有如式(L3)所示的基团，基团(L3)中的3个O原子各自和1个LA直接相连。
LB为磷酸酯键或二硫键；
每个LA为共价键，或者每个LA选自于由基团(L4)-(L23)及其连接组合所组成的组：

式中，每个j1为1-10的整数；
每个R’为C1-C10烷基；
每个Ra为氢原子，C1-C10烷基，或者选自由基团(L24)-(L37)组成的组：

在一些实施方式中，LA的长度为3-35个原子，其中所述LA的长度指LA中与LC直接连接的原子到与LA中与PP直接连接的原子形成的最长的原子链上的成链原子的个数。在一些实施方式中，每个LA为基团(L4)-(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。在一些实施方式中，每个LA为基团(L4)、(L5)、(L7)、(L9)、(L13)、(L14)、(L18)的连接组合。
在一些实施方式中，所述连接基团RI具有如式(201)所示的结构：
其中，n201和m201为1-10的整数；
P201为1-3的整数；
LA具有如式(202)所示的结构，其中，LA中的亚氨基端和式(201)中的PP相连：
其中，n202、m202、p202、q202为1-5的整数，i202为0-5的整数。在一些实施方式中，n202、m202、p202和q202为2或3，i202为3或4。
在一些实施方式中，所述连接基团RI具有如式(103)所示的结构：
其中，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点，PP表示所述连接基团RI连接至所述多肽配体的位点；
n103和m103为1-10的整数。在一些实施方式中，n103为1-3的整数，m103为3-6的整数。为了合成简便，在一些实施方式中，所述连接基团RI具有如式(103)所示的结构，且n103为1，m103为4或6。在一些实施方式中，n103＝1,m103＝6。在一些实施方式中，在合成如式(103)所示的连接基团RI的过程中，为了原料易得，可以使用天然的半胱氨酸，此时，如式(103)所示的连接基团RI中的氨基变成羟基，即具有如式(104)所示的结构：
本领域的技术人员容易理解的是，所述位置是氨基或羟基均不会对如式(103)所示的结构作为连接基团的功能产生影响，所述氨基被羟基替换的如式(104)所示的连接基团也在本公开的保护范围中。
在一些实施方式中，所述连接基团RI具有式(301)所示的结构：
其中，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点，PP表示所述连接基团RI连接至所述多肽配体的位点；
p301为1或0，n301和m301为0-10的整数。
当所述多肽配体或亲脂基团连接至所述寡核苷酸中核糖环的5’位时，所述p301为1；所述多肽配体或亲脂基团连接至所述寡核苷酸中核糖环的2’位时，所述p301为0，所述多肽配体基团取代其左侧相邻的一个核糖环的2’位羟基中的氢原子。在一些实施方式中，为了合成简便，n301和m301为0-5的整 数。在一些实施方式中，其中n301和m301为0-3的整数
在一些实施方式中，所述连接基团RI还可以是如WO2019128611A1中公开的连接基团，以引用的方式将上述文献整体并入本文。
本公开的发明人出人意料地发现，对于上述本公开的寡核苷酸缀合物，如果进一步还含有亲脂基团，还可以进一步地提升前述寡核苷酸缀合物的递送效率从而取得普遍更高的药学活性。
在一些实施方式中，本公开的寡核苷酸缀合物还含有亲脂基团，所述亲脂基团通过共价键或连接基团RII和所述双链寡核苷酸中的核苷酸相连。在一些实施方式中，所述缀合物含有h0个亲脂基团，h0是取自1-5的整数。
在一些实施方式中，所述亲脂基团连接至所述功能性双链寡核苷酸中、至少含有一个多肽配体的正义链或反义链上。
在一些实施方式中，对于所述功能性双链寡核苷酸中的任意一个或多个核苷酸，同一个核苷酸上连接有所述多肽配体和所述亲脂基团。为了减小空间位阻对所述寡核苷酸缀合物活性的影响，在一些实施方式中，和所述亲脂基团连接的核苷酸，是和连接有多肽配体的核苷酸相邻的核苷酸。在一些实施方式中，和所述亲脂基团连接的核苷酸，和连接有多肽配体的核苷酸之间间隔1-21个核苷酸。在一些实施方式中，和所述亲脂基团连接的核苷酸，和连接有多肽配体的核苷酸之间间隔至少1个核苷酸。在一些实施方式中，和所述亲脂基团连接的核苷酸，和连接有多肽配体的核苷酸之间间隔至少5个核苷酸。
在一些实施方式中，n0＝3，h0＝1；其中全部多肽配体连接至正义链3'末端的第一个核苷酸，所述亲脂基团和正义链从3'末端-5'末端方向第2个到第15个核苷酸中的任意一个相连；或者，全部多肽配体连接至所述正义链5'末端的第一个核苷酸，连接有亲脂基团的核苷酸与连接有多肽配体核苷酸之间间隔的核苷酸数量不超过核苷酸单链中核苷酸总数的80％。在一些实施方式中，核苷酸单链的长度为19、21、23或25个核苷酸，相应地，核苷酸总数的80％分别为15、17、18、20个核苷酸。
在一些实施方式中，n0＝2，h0＝1；两个多肽配体和一个亲脂基团中的一个连接至所述正义链3'末端的第一个核苷酸，一个连接至所述正义链5'末端的第一个核苷酸，另外一个连接至所述正义链中其他位置的核苷酸中的任意一个；
或者，两个多肽配体分别连接至所述正义链3'末端的第一个核苷酸以及5'末端的第一个核苷酸，所述亲脂基团和正义链中一个的核苷酸相连，该核苷酸与所述正义链5'末端的第一个核苷酸距离及正义链3'末端的第一个核苷酸之间间隔的核苷酸数量不超过核苷酸单链中核苷酸总数的80％。
在一些实施方式中，所述亲脂基团通过连接基团RII连接至所述功能性双链寡核苷酸。在一些实施方式中，所述连接基团RII是前述连接基团RI，即，同一个连接基团上同时连接有所述多肽配体和所述亲脂基团。在一些实施方式中，同一个连接基团RI或RII上只连接有所述多肽配体或所述亲脂基团。
在一些实施方式中，为了简化工艺，降低成本，所述亲脂基团通过共价键连接至所述功能性双链寡核苷酸。
在一些实施方式中，连接位置为核苷酸中核糖环的2’、3’或5’位置。为了简化合成工艺，在一些实施方式中，所述连接位置为为核糖环的2’位置。
在一些实施方式中，每个所述多肽配体通过其N端连接至所述双链寡核苷酸，即，在合成所述寡核苷酸缀合物的过程中，通过多肽的N端的氨基和连接基团缀合，常见的缀合方法包括通过脱水缩合反应、点击化学反应等本领域技术人员所公知的化学反应缀合。
在一些实施方式中，其中每一个多肽配体是含有如SEQ ID NO:169–SEQ ID NO:183所示序列中的一种的多肽配体。在一些实施方式中，其中每一个多肽配体是含有如SEQ ID NO:169-SEQ ID NO:173所示的多肽配体中的一种。在一些实施方式中，每一个多肽配体是含有如SEQ ID NO：169或SEQ ID NO:170所示的序列的多肽配体。在一些实施方式中，每一个多肽配体是如SEQ ID NO:169或SEQ ID NO:170所示的多肽配体。在一些实施方式中，为了合成的方便，所述寡核苷酸缀合物中的每个所述多肽配体都是如SEQ ID NO:169所示的多肽配体；或者，每个所述多肽配体都是如SEQ ID NO:170所示的多肽配体。
本公开所述的多肽配体包括上述多肽的线性或环状形式，以及本领域技术人员所熟知的是，对于如表1中所示的多肽序列，当对其两端进行截短或者延长时，一些被截短或延长的多肽序列依旧能够和TfR受体特异性结合，因此，前述含有如SEQ ID NO:169–SEQ ID NO:183所示序列中的一种的多肽配体，也包含了此类被截短或延长的多肽序列。
表1.本公开的多肽配体序列

为了获得更高的寡核苷酸缀合物的药学活性，在一些实施方式中，所述多肽配体中至少一个氨基酸是修饰的氨基酸。在一些实施方式中，所述多肽配体中至少50％的氨基酸是修饰的氨基酸。在一些实施方式中，所述多肽配体中的所有氨基酸都是修饰的氨基酸。
在本公开的上文及下文中，所述“修饰的氨基酸”包括通过使用D-氨基酸替代天然的L-氨基酸构建本公开中的多肽配体。或者通过使用氨基酸模拟物对天然的L-氨基酸进行取代，所述氨基酸模拟物包括氨基酸的结构类似物，例如可以是天然氨基酸的盐或酯。此外，多肽的C末端可以是羧基或将羧基酰胺化得到的酰胺基，或由掺入一种上述氨基酸模拟物生成的其它物质。此外，上述肽中的一个或多个天然肽键可以由下述基团中的任意一个取代，所述基团包括但不限于：磺酰胺、逆酰胺、含氨氧基的键、酯、烷基酮、α,α-二氟酮、α-氟酮、类肽键(N-烷基化甘氨酰酰胺键)。此外，所述多肽配体中的天然氨基酸的侧链可以是被取代的氨基酸，例如可以被4-氟苯丙氨酸、4-疑赖氨酸、3-氨基脯氨酸、2-硝基酪氨酸或N-烷基组氨酸中的一种取代；或者在β-侧链碳原子处具有与天然手性相反的手性的β-支链氨基酸或β-支链氨基酸模拟物，例如别苏氨酸、别异亮氨酸及其衍生物。代表性的修饰的氨基酸公开于Baran等人，Biochemistry,2017,56(30):3863-3873.以及Mehta等人，Tetrahedron Letters,2017,58(14):1357-1372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，所述亲脂基团为长度为10-30个碳原子的饱和或不饱和、直链或支链的烃基；其中的一个或多个碳原子可以被羟基、氨基、羧基、磺酰基或磷酰基中的一种或多种替代。
在一些实施方式中，为了合成简便，所述亲脂基团为长度为15-25个碳原子的直链或支链的饱和烃基。在一些实施方式中，为了简化合成工艺，所述亲脂基团通过取代核苷酸中核糖环的2'羟基中的氢原子和所述功能性双链寡核苷酸连接。
在一些实施方式中，本公开的寡核苷酸组合物中的功能性双链寡核苷酸中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分反向互补地形成双链区，所述核苷酸序列II至少部分地与靶mRNA反向互补，所述靶mRNA为靶组织或靶器官中目标细胞中的靶基因表达的mRNA，所述目标细胞是细胞表面存在TfR的细胞。
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II均由19个核苷酸组成，所述核苷酸序列II与核苷酸序列I基本上反向互补、实质上反向互补、或完全反向互补；所述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；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；所述完全反向互补是指两个核苷酸序列之间没有碱基错配。
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II的至 少第2-19位的核苷酸与所述核苷酸序列I第1-18位的核苷酸完全反向互补。在一些实施方式中，所述核苷酸序列II与所述核苷酸序列I完全反向互补，或者按照5'末端到3'末端的方向，所述核苷酸序列II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I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功能性双链寡核苷酸的目标基因表达抑制活性。
本公开的寡核苷酸缀合物中的功能性双链寡核苷酸含有正义链和反义链，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。这样，本公开提供的功能性双链寡核苷酸正义链和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，所述功能性双链寡核苷酸中正义链和反义链的长度比为19/21、21/23或23/25。
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV的长度为1-4个核苷酸，所述核苷酸序列III连接在核苷酸序列I的所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端。在一些实施方式中，所述核苷酸序列III和所述核苷酸序列IV长度相等，实质上反向互补或完全反向互补。在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基组成，核苷酸序列IV的碱基组成也就确定了。
在一些实施方式中，所述正义链和所述反义链长度不同，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述所述反义链的3'末端，构成反义链的3'突出端。在一些实施方式中，所述正义链还含有核苷酸序列VI，核苷酸序列VI的长度为1至3个核苷酸，连接在所述正义链的3'末端，构成正义链的3'突出端。
在一些实施方式中，所述功能性双链寡核苷酸包括核苷酸序列V，但不包括核苷酸序列VI。由此，本公开提供的功能性双链寡核苷酸中正义链和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，本公开提供的功能性双链寡核苷酸包括核苷酸序列V和VI。在一些实施方式中，核苷酸序列V的长度与核苷酸序列VI的长度相同或不同。由此，本公开提供的功能性双链寡核苷酸中正义链和反义链的长度比可以是(19-26):(19-26)。在一些实施方式中，所述核苷酸序列V和/或VI的长度为2个核苷酸，由此，本公开提供的功能性双链寡核苷酸中正义链和反义链的长度比可以是19/21、21/21、21/23、23/23、23/25或25/25。
所述核苷酸序列V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，在一些实施方式中，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方式中，所述正义链和反义链长度之比为19/21或21/23，此时，所述功能性双链寡核苷酸具有更好的mRNA沉默活性。
所述核苷酸序列VI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合成成本，在一些实施方式中，所述核苷酸序列VI为连续的两个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的两个尿嘧啶核糖核苷酸(UU)；或者，为了提高功能性双链寡核苷酸中正义链与反义链的亲和力，核苷酸序列VI与靶mRNA的相应位置的核苷酸相同。因此，在一些实施方式中，所述功能性双链寡核苷酸包含核苷酸序列V和VI，正义链和反义链的长度之比为21/21或23/23，此时，所述功能性双链寡核苷酸具有更好的mRNA沉默活性。
靶mRNA的相应位置的核苷酸是指与靶mRNA的一段核苷酸序列在5'末端相邻的核苷酸或核苷酸序列，该段靶mRNA的核苷酸序列是与核苷酸序列II实质上反向互补或完全反向互补，或者与核苷酸序列II和核苷酸序列IV构成的核苷酸序列实质上反向互补或完全反向互补的那段核苷酸序列。
如前所述，所述功能性双链寡核苷酸中的核苷酸为修饰或未修饰的核苷酸。在一些实施方式中，所述功能性双链寡核苷酸中的核苷酸为未经修饰的核苷酸；在一些实施方式中，所述功能性双链寡核苷酸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所述功能性双链寡核苷酸对靶基因表达的B靶mRNA的调节功能明显削弱或丧失。
在一些实施方式中，所述正义链和所述反义链中的至少一个核苷酸为修饰的核苷酸。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，本公开提供的功能性双链寡核苷酸的正义链或反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；即，所述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和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。
在一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方式中，本公开提供的功能性寡核苷酸中的正义链和所述反义链中的每一个核苷酸为氟代修饰的核苷酸或非氟代修饰的核苷酸。
本公开的发明人惊奇地发现，本公开提供的寡核苷酸缀合物在动物实验中获得了血浆中稳定性和基因沉默效率的高度平衡。
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，所述核苷酸序列I中氟代修饰的核苷酸不多于5个，按照5'末端到3'末端的方向，所述核苷酸序列I的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列II中氟代修饰的核苷酸不多于7个，所述核苷酸序列II的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。
在一些实施方式中，按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5'末端到3'末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。
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'-经取代的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'-经取代的烷氧基修饰的核苷酸，例如可以是2'-O-甲氧基乙基修饰的核苷酸(2'-MOE)，如式(9)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH2)如式(10)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(11)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、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”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“具有2'-氟代核糖基的核苷酸”意义相同，均指核苷酸的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”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“具有2'-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基 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，所述功能性双链寡核苷酸是具有以下修饰的双链寡核苷酸：按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。
在一些实施方式中，所述功能性双链寡核苷酸是具有以下修饰的双链寡核苷酸：按照5'末端到3'末端的方向，所述功能性双链寡核苷酸的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，正义链的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，所述双链寡核苷酸的反义链中核苷酸序列II的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，所述反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5'末端到3'末端的方向，所述正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，所述正义链的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，所述反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，所述反义链其余位置的核苷酸为甲氧基修饰的核苷酸；
或者，按照5'末端到3'末端的方向，所述正义链中核苷酸序列I的第7、8和9位的核苷酸为氟代修饰的核苷酸，所述正义链的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，所述反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，所述反义链其余位置的核苷酸为甲氧基修饰的核苷酸。
具有上述修饰的双链寡核苷酸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酸酶水解的性能。同时，具有上述修饰的功能性双链寡核苷酸依旧具有较高的靶mRNA的调控功能。
在一些实施方式中，本公开提供的功能性双链寡核苷酸的正义链和反义链中至少一条单链的磷酸-糖骨架中的额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基：
这种修饰能稳定双链寡核苷酸的双链结构，保持碱基配对的高特异性和高亲和力。
在一些实施方式中，本公开提供的功能性双链寡核苷酸中，硫代磷酸酯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，硫代磷酸酯基连接存在于以下位置中的至少一处：
所述正义链的5'末端第1个核苷酸和第2个核苷酸之间；
所述正义链的5'末端第2个核苷酸和第3个核苷酸之间；
所述正义链的3'末端第1个核苷酸和第2个核苷酸之间；
所述正义链的3'末端第2个核苷酸和第3个核苷酸之间；
所述反义链的5'末端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'末端第2个核苷酸和第3个核苷酸之间；
所述反义链的3'末端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'末端第2个核苷酸和第3个核苷酸之间。
在一些实施方式中，所述功能性双链寡核苷酸中反义链的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，本公开的寡核苷酸缀合物中，所述功能性双链寡核苷酸是saRNA或siRNA。在一些实施方式中，所述功能性双链寡核苷酸是siRNA。
如前所述，本公开的寡核苷酸缀合物的目标细胞为细胞表面存在转铁蛋白受体的细胞。因此，本公开的寡核苷酸缀合物可以靶向受试者体内任意的有上述目标细胞存在的靶组织或靶器官。可以通过使用和不同的靶基因表达的靶mRNA的双链寡核苷酸，发挥调控在这些靶组织或靶器官中存在的靶基因的功能。
在一些实施方式中，所述靶组织或靶器官选自骨骼肌、平滑肌、心肌、眼部、脑部、脊髓、耳部、鼻部、心脏、视网膜、肌肉组织、肿瘤组织。
在一些实施方式中，所述靶基因选自，在一些实施方式中，所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR或TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。在一些实施方式中，所述靶基因是SOD1或RPTOR。
在一些实施方式中，所述功能性双链寡核苷酸是siRNA。
本公开的发明人意外发现，所述siRNA以及含有这些siRNA的寡核苷酸缀合物表现出具有显著提高的血浆中稳定性、低脱靶效应的同时，还表现出较高的靶mRNA沉默活性。因此，在一些实施方式中，所述siRNA可以为表11中示出的siRNA中的一种。
表11.本公开的寡核苷酸缀合物中的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s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；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，本公开的寡核苷酸缀合物中的功能性双链寡核苷酸是靶向小鼠SOD1 mRNA的siRNA。在一些实施方式中，本公开的寡核苷酸缀合物中的功能性双链寡核苷酸具有表11中列出的siRNA的序列。
本公开的寡核苷酸缀合物的合成
本领域技术人员可通过任何合适的方式制备获得本公开的寡核苷酸缀合物。
在一些实施方式中，本公开的寡核苷酸缀合物的合成方法包括提供双链寡核苷酸的正义链和反义链，将正义链和反义链退火得到本公开提供的双链寡核苷酸，其中，所述正义链和反义链分别包含15-25个核苷酸，每个所述核苷酸是修饰或未修饰的核苷酸，所述正义链或反义链中的至少一个为连接有所述多肽配体的正义链或反义链，连接在正义链和反义链上所述多肽配体的总数为n0个，n0为2-6的整数，所述多肽配体为与转铁蛋白受体具有亲和性的配体，每个所述多肽配体与正义链和/或反义链通过共价键或者通过连接基团RI连接，每个所述多肽配体由5-12个修饰或未修饰的氨基酸组成，每个所述多肽配体通过多肽配体的N端或C端连接至所述功能性双链寡核苷酸。在一些实施方式中，所述多肽配体的数量，连接至正义链和/或反义链的位置和方式如前所述。
在一些实施方式中，所述合成方法还包括双链寡核苷酸的分离提纯。
在一些实施方式中，连接有n0个所述多肽配体的核苷酸单链可以通过以下方式制备得到：在溶剂中，在偶联反应条件下，将具有活性基团Rx1的寡核苷酸单链和具有活性基团Rx2的多肽配体接触，反应获得连接有n0个多肽配体的寡核苷酸单链，具有活性基团Rx1的寡核苷酸单链和具有活性基团Rx2的多肽配体的摩尔比为1:1-1:n0。在一些实施方式中，所述多肽配体通过连接基团RI连接至所述寡核苷酸单链，所述活性基团Rx1和所述活性基团Rx2是能够通过偶联反应生成连接基团RI的基团。
本领域的技术人员可以通过各种方法获得具有活性基团Rx1的寡核苷酸单链。在一些实施方式中，所述具有活性基团Rx1的寡核苷酸单链可以通过本领域技术人员熟知的核酸合成方法，例如亚磷酰胺固相合成或者磷酸二酯法/磷 酸三酯法液相合成获得。在一些实施方式中，所述具有活性基团Rx1的寡核苷酸单链，采用亚磷酰胺固相合成法得到，该方法包括，在亚磷酰胺固相合成条件下，按照寡核苷酸单链中核苷酸的顺序，将核苷单体一次连接，其中，至少一个核苷单体为具有活性基团Rx1的核苷单体。亚磷酰胺固相合成法为本领域技术人员所公知，其过程和条件在Methods in Molecular Biology,vol.288:Oligonucleotide Synthesis:Methods and Applications，P17-P31中详细公开，以引用的方式将其全部内容整体并入本文。
在一些实施方式中，所述偶联反应条件是缩合反应条件或者巯基-二硫键交换反应的条件。
在一些实施方式中，所述偶联反应条件是缩合反应条件，所述缩合反应条件是酰基化缩合反应条件、脱水缩合反应条件或者点击化学反应的条件，活性基团Rx1与活性基团Rx2是能够发生前述缩合反应的基团。在一些实施方式中，所述缩合反应条件是酰基化缩合反应的条件，所述活性基团Rx1和Rx2是能够发生酰基化缩合反应形成RI的基团。在一些实施方式中，所述缩合反应条件是脱水缩合反应的条件，所述活性基团Rx1和Rx2中的一个是包含酰卤基团或羧基的基团，另一个是包含氨基或羟基的基团。在一些实施方式中，所述缩合反应条件是点击化学的条件，所述活性基团Rx1和Rx2中的一个是包含炔基的基团，另一个是包含叠氮基团的基团。
在一些实施方式中，所述具有活性基团Rx1的寡核苷酸单链是通过在偶联反应的条件下，将具有活性基团Rx0的寡核苷酸单链和交联剂接触制备得到的，所述交联剂含有点击化学活性基团和酰基化基团。所述活性基团和RX0与所述酰基化基团通过发生偶联反应而形成共价连接，使所述点击化学活性基团连接至所述寡核苷酸单链。
在一些实施方式中，活性基团Rx1是末端含有1-3个点击化学活性基团的活性基团，所述点击化学活性基团包含末端炔基。在一些实施方式中，所述酰基化基团是活性酯基团，例如可以是NHS酯基、亚氨酸酯基以及五氟苯酯基中的一种。本领域的技术人员可以通过各种方法获得所述交联剂，例如，当所述酰基化基团是五氟苯酯基，所述点击化学基团包含末端炔基时，所述交联剂可以按照如stergaard,Michael E.,et al."Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides."Bioconjugate chemistry 26.8(2015):1451-1455中Scheme 1a(A)中描述的方法制备获得。在一些实施方式中，所述活性基团Rx0是氨基。在一些实施方式中，所述偶联条件是碱性条件。在一些实施方式中，所述碱性条件是有弱碱水溶液存在的条件，例如有碳酸氢钠水溶液存在的条件。本领域的技术人员可以通过各种方式获得所述具有活性基团Rx0的寡核苷酸单链，在一些实施方式中，所述具有活性基团Rx0的寡核苷酸单链是通过在合成寡核苷酸单链的过程中在相应位置使用含有活性基团的亚磷酰胺单体制备得到的。本领域的技术人员可以通过各种方式获得含有活性基团的亚磷酰胺单体。在一些实施方式中，所述活性基团Rx0是氨基，含有Rx0的亚磷酰胺单体可以通过本领域技术人员熟知的方法商购获 得或制备获得，例如，含有Rx0的亚磷酰胺单体可以是容易商购获得的6-(三氟乙酰氨基)-己基-(2-氰乙基)-(N,N-二异丙基)-亚磷酰胺单体，其中，活性基团Rx0为氨基，该活性基团Rx0可以是通过亚磷酰胺固相合成法将所述亚磷酰胺单体连接至寡核苷酸单链后，经本领域技术人员容易实现的脱保护反应(如浓氨水氨解)脱除三氟乙酰基保护基而获得。
在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应中的一种，所述活性基团Rx1和Rx2中的一个是包含巯基的基团，另一个包含经二硫键连接的离去基团。为了避免副反应发生，在一些实施方式中，上述含有活性基团Rx1的亚磷酰胺单体中的Rx1以被保护的Rx1’形式存在，所述制备方法还包含在脱保护反应条件下，通过将制备得到的含有Rx1’的寡核苷酸单链和脱保护试剂接触，分离得到含有Rx1的寡核苷酸单链的步骤。在一些实施方式汇总，所述Rx1’含有二硫键离去基团，所述脱保护反应条件是巯基-二硫键交换反应条件，所述脱保护试剂是二硫键活化剂。在一些实施方式中，所述二硫键活化剂是二硫二吡啶。本领域的技术人员可以通过各种方法获得上述含有活性基团Rx1或Rx1’的亚磷酰胺单体，在一些实施方式中，所述含有活性基团Rx1或Rx1’的亚磷酰胺单体是商购得到的，例如可以通过商购得到如式(105)所示的亚磷酰胺单体。
其中n105是1-10的整数，m105＝m103；
本领域的技术人员可以通过各种方式获得所述具有活性基团Rx2的多肽配体。在一些实施方式中，所述偶联反应条件是巯基-二硫键交换反应条件，所述活性基团Rx2是包含巯基的活性基团，所述具有活性基团Rx2的多肽配体可通过商业订制获得。在一些实施方式中，所述偶联反应条件是点击化学反应条件，所述活性基团Rx2是包含叠氮基团的多肽配体，所述具有活性基团Rx2的多肽配体可通过商业订制获得。
在一些实施方式中，所述RI是如式(103)所示的连接基团，本公开的寡核苷酸缀合物可以通过以下方法制备得到：在溶剂中，在巯基-二硫键交换反应条件下，将式(106)所示的具有巯基交换基团，即Rx1的单链寡核苷酸和n0个如式(107)所示的含有Rx2的多肽配体接触，分离获得经连接基团RI连接的多肽配体-单链寡核苷酸缀合物；将该多肽配体-单链寡核苷酸缀合物与本公开的寡核苷酸缀合物的另一单链经退火形成双链寡核苷酸，分离获得本公开的寡核苷酸缀合物。
其中n107＝n103，pp表示所述多肽配体；
其中，Nu'代表寡核苷酸单链，m106＝m103；R106是巯基交换剂残基。在一些实施方式中，R106为C7-C12芳基或杂芳基。在一些实施方式中，R106为2-吡啶基。
所述溶剂和巯基-二硫键交换反应条件是本领域在进行巯基-二硫键交换反应中常用的溶剂和反应条件，例如在0.05-1M浓度的醋酸铵水溶液存在的条件下，在常温常压下反应2-10h，例如4-8h。所述溶剂与式(106)化合物的用量比例可以为100:1-2000:1L/mol；所述式(107)化合物与式(106)化合物的摩尔比可以为1:1-15:1，例如4:1-10:1。
可使用任何合适的分离方法从反应混合物中分离所述多肽-单链寡核苷酸缀合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离所述多肽-单链寡核苷酸缀合物，例如，可使用如下色谱条件进行分离：C18反向色谱柱作为固定相，100mM TEAA(PH＝7.0-7.3):乙腈的比例为5％-75％(V/V)的溶剂作为流动相进行梯度洗脱。在一些实施方式中，可以直接除去溶剂得到所述多肽-单链寡核苷酸缀合物粗产品，该粗产品可以直接用于后续反应。
式(107)所示的多肽配体可通过本领域技术人员熟知的方法制备获得。在一些实施方式中，式(107)所示的多肽配体可通过商业订制容易地获得。
本领域技术人员可通过各种方法获得式(106)所示的具有巯基交换基团的单链寡核苷酸。在一些实施方式中，式(106)所示化合物可通过如下方法制备获得：在溶剂中，在巯基-二硫键交换反应条件下，将式(108)所示的化合物与式(111)所示的巯基-二硫键交换剂接触，分离获得式(106)所示的化合物。

其中，m106、Nu'、R106的定义和选择范围与前述相同；所述溶剂和所述巯基-二硫键交换反应条件的选择与前述相同。式(111)化合物与式(108)化合物的摩尔比为大过量，例如可以是10:1-1000:1，在一些实施方式中为50:1–200:1。
可使用任何合适的分离方法从反应混合物中分离式(106)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(106)化合物，例如，可使用如下条件进行分离：以20％(V/V)的乙醇水溶液作为流动相，在纯化仪中进行凝胶脱盐纯化，在280nm波长下收集产品洗脱液。在一些实施方式中，可以过滤后直接除去溶剂得到式(106)化合物粗产品，该粗产品可以直接用于后续反应。
式(111)所示的巯基-二硫键交换试剂可通过本领域技术人员熟知的方法制备获得。在一些实施方式中，式(111)所示的化合物是可通过商购获得的2-2'-二硫二吡啶。
本领域技术人员可通过各种方法获得如式(108)所示的具有巯基的单链寡核苷酸。在一些实施方式中，如式(108)所示的单链寡核苷酸可以通过如下方法制备获得：在还原剂的水溶液中，在还原反应的条件下，将如式(109)所示的单链寡核苷酸和还原剂接触，分离获得式(108)所示的单链寡核苷酸，在一些实施方式中，所述还原剂是具有将二硫键还原成巯基的还原剂，在一些实施方式中，所述还原剂是TCEP，所述还原反应的条件是在常温常压的条件下，在TCEP的水溶液中反应。
其中，m106、Nu'的定义和选择范围与前述相同；所述还原剂和还原反应条件是本领域在进行还原反应中常用的还原剂和反应条件，例如，在一些实施方式中，所述还原剂是TCEP。所述反应条件是在常温常压下反应1-5个小时，在一些实施方式中，反应时长为2-3个小时，所述还原剂大量过量。在一些实施方式中，所述“大量过量”指所述还原剂和所述单链寡核苷酸的质量比可以是3:1-
1:1；在一些实施方式中，所述还原剂和所述单链寡核苷酸的质量比可以是
1.5:1。
可使用任何合适的分离方法从反应混合物中分离式(108)化合物。在一些实施方式中，可通过使用纯化水稀释反应液并过滤，再重复超滤和离心，直至测得超滤液体电导在100s以下，收集滤膜内的产品获得式(108)的化合物。
本领域技术人员可通过各种方法获得式(109)所示的单链寡核苷酸。在 一些实施方式中，如式(109)所示的单链核苷酸是通过在固相合成寡核苷酸单链的过程中，使用如式(105)所示的亚磷酰胺单体制备得到的，如式(105)所示的亚磷酰胺单体是本领域技术人员易得的，在一些实施方式中，如式(105)所示的亚磷酰胺单体是商购获得的。
其中n105是1-10的整数，m105＝m103；
同时，也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所述的功能性双链寡核苷酸中，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双链寡核苷酸的方法也是本领域技术人员所熟知的。
本领域技术人员可通过各种方法获得式(107)所示的含有巯基的多肽配体，在一些实施方式中，式(107)所示的含有巯基的多肽配体是通过如下方法制备得到的：在氨基酸脱水缩合的反应条件下，将所述多肽配体和天然或修饰的半胱氨酸或半胱氨酸类似物接触，所述半胱氨酸或半胱氨酸类似物具有如式(110)所示的结构，分离获得如式(107)所示的含有巯基的多肽配体。在一些实施方式中，所述如式(107)所示的多肽配体中，多肽配体的N端或C端连接有包含巯基的基团，从而，制备得到的连接有所述多肽配体的寡核苷酸单链和多肽配体的N端或C端相连。在一些实施方式中，所述制备方法包括将所述多肽配体的N端的氨基或C端的羧基保护从而制备得到只有N端或C端和巯基相连的反应。氨基或羧基的保护和脱保护方法为本领域技术人员所公知。
所述氨基酸脱水缩合的反应条件是本领域常用的氨基酸脱水缩合条件。其中，多肽配体本身的合成方法，即含有特定氨基酸序列的多肽的合成方法，包括含有修饰的氨基酸的多肽配体的合成方法已经是本领域技术人员所熟知的，目前已有成熟的公开商业化定制服务。在一些实施方式中，如本公开的表1中所示的多肽序列可以通过商业化订制获得。所述如式(110)所示的半胱氨酸或半胱氨酸类似物是本领域人员容易取得的，在一些实施方式中，是商购获得的。在一些实施方式中，如式(107)所示的含有巯基的多肽配体是商购获得的。在一些实施方式中，本公开的多肽序列中的氨基或羧基是被保护的氨基或羧基，例如，在一些实施方式中，羧基可以通过酰胺化转化为酰胺基进行保护。
其中，R107是-NH2或-OH，n107＝n103。
其中，R107是-NH2或-OH，n107＝n103。
在一些实施方式中，本公开的寡核苷酸缀合物中，每个多肽配体通过如式(201)所示的连接基团连接至所述双链寡核苷酸中的一条单链，此时，本公开的寡核苷酸缀合物可以通过如下方法制备得到：在溶剂中，在点击化学反应的条件下，将含有碳碳三键的寡核苷酸单链与含有-N3基团的点击多肽(Click Peptide)通过点击化学反应，将多肽配体引入含有活性基团的寡核苷酸单链，分离获得经连接基团RI连接的多肽配体-单链寡核苷酸缀合物，其中，点击多肽已经有商业化定制服务；将该多肽配体-单链寡核苷酸缀合物与本公开的寡核苷酸缀合物的另一单链经退火形成双链寡核苷酸，分离获得本公开的寡核苷酸缀合物。所述点击化学反应条件是本领域常用的点击化学反应条件，在一些实施方式中，所述点击化学的反应条件是在加热条件下，在催化剂存在的条件下，使点击多肽与寡核苷酸单链接触。在一些实施方式中，所述催化剂是含铜催化剂的水溶液。在一些实施方式中，所述催化剂是硫酸铜的水溶液，所述硫酸铜和点击多肽的摩尔比为1:6-1:1。在一些实施方式中，所述催化剂是硫酸铜、TBTA和抗坏血酸钠的混合溶液，其中，硫酸铜和TBTA的摩尔比是3:1-1:3，硫酸铜和抗坏血酸钠的摩尔比是1:5-1:10。优选地，所述硫酸铜和TBTA的摩尔比是1:1，硫酸铜和抗坏血酸钠的摩尔比是1:7.5。在一些实施方式中，所述加热条件是指在30-60摄氏度的水浴中反应。在一些实施方式中，所述加热条件是指在30-50摄氏度的水浴中反应。在一些实施方式中，所述点击多肽和所述寡核苷酸单链的摩尔比为10:1-3:1，在一些实施方式中，所述点击多肽和所述寡核苷酸单链的摩尔比为5:1-4:1。在一些实施方式中，通过HPLC监控反应混合物中的反应物和/或产物的含量确定反应终点。在一些实施方式中，所述点击化学反应条件是如Zengmin Li；Tae Seok Seo；Jingyue Ju(2004).1,3-Dipolar cycloaddition of azides with electron-deficient alkynes under mild condition in water.,45(15),3143–3146中所述的条件，以引用的方式将其全部内容整体并入本文。
可使用任何合适的分离方法从反应混合物中分离所述多肽-单链寡核苷酸缀合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离所述多肽- 单链寡核苷酸缀合物，例如，可使用如下色谱条件进行分离：C18反向色谱柱作为固定相，100mM TEAA(PH＝7.0-7.3)和乙腈/异丙醇混合溶剂(V/V＝1:1)的比例为5％-75％(V/V)的溶剂作为流动相进行梯度洗脱。在一些实施方式中，可以直接除去溶剂得到所述多肽-单链寡核苷酸缀合物粗产品，该粗产品可以直接用于后续反应。
在一些实施方式中，所述连接基团RI是如式(203)所示的连接基团，此时连接有多肽配体的寡核苷酸单链可以通过以下方式制备得到：
其中Nu'、PP的定义同与前述相同。
在点击化学反应的条件下，将如式(204)所示的含有活性基团Rx2点击多肽和如式(205)所示的含有活性基团Rx1的寡核苷酸单链接触，分离获得如式(203)所示的多肽-单链寡核苷酸缀合物，其中Rx1和Rx2是叠氮基团。所述点击化学反应的条件与前述相同。
本领域技术人员可以通过各种方法获得如式(204)所示的含有叠氮基团的多肽配体，在一些实施方式中，如式(204)所示的含有叠氮基团的多肽配体是商业订购获得的。
本领域技术人员可以通过各种方法获得如式(205)所示的单链寡核苷酸，例如，可以在常压常温下、在碱性条件下，将将如式(206)所示的单链寡核苷酸和如式(207)所示的活性酯接触，分离获得如式(205)所示的寡核苷酸。在一些实施方式中，所述碱性条件，是指有碳酸氢钠水溶液存在的条件。在一些实施方式中，如式(207)所示的活性酯相较于如式(205)所示的单链寡核苷酸是大过量的；在一些实施方式中，如式(207)所示的活性酯和如式(206)所示的单链寡核苷酸的摩尔比为200:1-50:1；在一些实施方式中，如式 (207)所示的活性酯和如式(206)所示的单链寡核苷酸的摩尔比为150:1-
80:1。
本领域技术人员可使用任何合适的分离方法从反应混合物中分离所述如式(205)所示的单链寡核苷酸。在一些实施方式中，可以通过向反应液中加入乙腈和PBS的混合溶剂，离心分离，向得到的上清液中加入核酸沉淀溶液(例如，可以是可容易商购获得的10％三氯乙酸水溶液)，分离出沉淀获得如式(205)所示的单链寡核苷酸。
本领域技术人员可以通过各种方法获得如式(207)所示的活性酯。在一些实施方式中，如式(207)所示的活性酯是通过使用如stergaard,Michael E.,et al."Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides."Bioconjugate chemistry 26.8(2015):1451-1455中Scheme 1a(A)中公开的制备方法得到，区别仅在于使用如式(208)所示的交联剂化合物取代了Scheme 1a(A)中的初始反应物RCO(CH2)3COOH，以引用的方式将其全部内容整体并入本文。
本领域人员可以通过各种方式获得如式(208)所示的化合物，在一些实施方式中，如式(208)所示的化合物是通过以下方式获得的：在溶剂中，在酯的水解条件下，使如式(209)所示的酯发生水解，分离获得如式(208)所示的化合物。在一些实施方式中，所述酯的水解条件是指在碱性水溶液中，有催化剂存在的条件下。在一些实施方式中，所述碱性水溶液是氢氧化钠的水溶液，所述催化剂是乙醇，其中氢氧化钠和如式(209)所示的化合物的摩尔比为7:1-3:1，氢氧化钠水溶液的浓度为2M-5M，乙醇和氢氧化钠水溶液的体积比为7:1-3:1。本领域技术人员可以使用各种方法分离如式(208)所示的化合物，在一些实施方式中，可以通过使用酸性溶液调节反应液的pH值到5-6，再用柱层析分离得到如式(208)所示的化合物。
本领域技术人员可以通过各种方法得到如式(209)所示的化合物，在一些实施方式中，可以通过如下方法制备得到：在缩合反应的条件下，使如式(210)所示的化合物和如式(211)所示的的化合物接触，分离得到如式(209)所示的化合物：
在一些实施方式中，所述缩合反应条件是酰胺化反应条件，例如可以是在有机溶剂中，有1-羟基苯并三唑(HOBt)、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC.HCl)和N,N-二异丙基乙胺(DIEA)存在的条件。在一些实施方式中，有机溶剂是DMF。在一些实施方式中，如式(210)所示的化合物和如式(211)所示的化合物的摩尔比是1:2-1:5。在一些实施方式中，如式(210)所示的化合物和1-羟基苯并三唑、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N,N-二异丙基乙胺的摩尔比为1:3-1:10，DMF的用量和所述如式(210)所示的化合物的用量比为7ml/g-20ml/g。本领域的技术人员可以通过各种方法分离如式(209)所示的化合物。在一些实施方式中，可以通过往反应液中加入水和乙酸乙酯萃取得到有机相，再依次用10％柠檬酸、碳酸氢钠、饱和食盐水溶液洗涤得到的有机相，蒸干溶剂获得如式(209)所示的化合物。本领域技术人员可以通过各种方法获得如式(210)所示的化合物，在一些实施方式中，如式(210)所示的化合物是按照如WO2014025805A1中公开的Scheme3的方法制备获得的。本领域技术人员可以通过各种方法获得如式(211)所示的化合物，在一些实施方式中，如式(211)所示的化合物是通过商购获得的。
本领域技术人员可以通过各种方法获得如式(206)所示的单链寡核苷酸，例如可以通过在合成寡核苷酸单链的过程中在相应位置使用含有活性基团的亚磷酰胺单体。在一些实施方式中，所述含有活性基团的亚磷酰胺单体是6-(三氟乙酰氨基)-己基-(2-氰乙基)-(N,N-二异丙基)-亚磷酰胺单体。所述亚磷酰胺单体是本领域技术人员容易获得的，在一些实施方式中，所述亚磷酰胺单体通过商购获得。
在一些实施方式中，所述多肽-单链寡核苷酸缀合物中含有多个连接有多肽配体的核苷酸，其中，寡核苷酸单链中单个连接有多肽配体的核苷酸具有式(301A)所示的结构：
其中，Nu”表示所述寡核苷酸单链中的一个核苷酸，所述Nu”可以是所述寡核苷酸单链中任意位置的1个核苷酸，其和所述寡核苷酸单链中其余核苷酸通过磷酸酯键或硫代磷酸酯键相连，PP表示所述多肽配体；p301为1或0，n301和m301为0-10的整数。当所述多肽配体连接至所述核苷酸核糖环的5’位时，所述p301为1；所述多肽配体连接至所述核苷酸核糖环的2’位时，所述p301为0，，所述多肽配体基团取代其左侧相邻的一个核糖环的2’位羟基中的氢原子。
在一些实施方式中，所述多肽配体连接至所述核苷酸中核糖环的5’位时，所述p301为1。所述寡核苷酸单链可以通过在点击化学的条件下，将如前述式(204)所示的含有活性基团Rx2点击多肽和含有如式(301A)所示的含有活性基团Rx1的核苷酸的寡核苷酸单链接触，分离获得多肽-单链寡核苷酸缀合物。所述点击化学的条件如前所述：
其中，Nu”表示表示所述寡核苷酸单链中的一个核苷酸。
在一些实施方式中，所述多肽配体连接至所述核苷酸中核糖环的2’位时，所述p301为0。所述寡核苷酸单链可以通过在点击化学的条件下，将如前述式(204)所示的含有活性基团Rx2点击多肽和含有如式(303)所示的含有活性基团Rx1的核苷酸的寡核苷酸单链接触，分离获得多肽-单链寡核苷酸缀合物。所述点击化学的条件如前所述：
其中，Nu”表示所述寡核苷酸单链中的一个核苷酸在一些实施方式中，多肽配体的选择范围和上文中对本公开的寡核苷酸缀合物的描述中相同，本领域的技术人员可以通过各种方式获得含有特定序列的多肽配体。在一些实施方式中，所述多肽配体通过商购获得。
在一些实施方式中，本公开的寡核苷酸缀合物含有亲脂基团，所述亲脂基团通过共价键或连接基团RII和所述功能性双链寡核苷酸中的核苷酸相连。所述制备方法还包含将所述亲脂基团连接至上述制备方法中的双链寡核苷酸或寡核苷酸单链上的步骤。在一些实施方式中，所述缀合物含有h0个亲脂基团，h0是取自1-5的整数，所述寡核苷酸缀合物可以通过制备连接有h0个多肽配体的寡核苷酸单链制备获得。在一些实施方式中，连接有所述亲脂基团的寡核苷酸单链可以通过以下方式制备得到：在溶剂中，在偶联反应条件下，将具有活性基团Rx1的寡核苷酸单链和具有活性基团Rx2的亲脂基团接触，反应获得连接有h0个多肽配体的寡核苷酸单链，具有活性基团Rx2的亲脂基团和具有活性基团Rx1的寡核苷酸单链的摩尔比为1:1-1:h0。
在一些实施方式中，本公开的寡核苷酸缀合物中至少一个核苷酸上同时连接有所述多肽配体和所述亲脂基团。在一些实施方式中，该核苷酸可以通过在合成核苷酸单链的过程中，使用在不同位置含有1个以上活性基团Rx1的核苷单体制备得到。在一些实施方式中，该核苷酸可以通过将具有活性基团Rx2、亲脂基团和多肽配体的化合物和具有活性基团Rx1的寡核苷酸单链接触制备得到。
在一些实施方式中，和所述亲脂基团连接的核苷酸，和连接有多肽配体的核苷酸之间间隔至少1个核苷酸。在一些实施方式中，n0＝3，h0＝1；其中全部多肽配体连接至所述正义链3'末端或5'末端的第一个核苷酸，连接有亲脂基团的核苷酸与连接有多肽配体核苷酸之间间隔的核苷酸数量不超过核苷酸单链中核苷酸总数的80％，所述寡核苷酸缀合物可以通过提供在正义链3'末端或5'末端的第一个核苷酸连接有3个多肽配体，在和正义链3'末端或5'末端的第一个核苷酸间隔不超过核苷酸单链中核苷酸总数的80％的正义链制备得到。
在一些实施方式中，所述亲脂基团通过共价键连接至所述双链寡核苷酸中的一个核苷酸上。在一些实施方式中，所述亲脂基团连接至核苷酸核糖环的2’位置，所述寡核苷酸缀合物可以通过以下方式获得：在和前述相同的亚磷酰胺固相合成条件下，按照寡核苷酸单链中核苷酸的顺序，将核苷单体一次连接，其中，至少一个核苷单体为在糖环2’位连接有所述亲脂基团的亚磷酰胺单体。
在一些实施方式中，所述亲脂基团为长度15-25个碳原子的饱和或不饱和、直链或支链的烃基。本领域的技术人员可以通过各种方式获得所述亲脂基团，在一些实施方式中，所述亲脂基团是商购得到的。本领域技术人员可以通过各种方式活化所述亲脂基团，在一些实施方式中，可以通过使用卤代烃作为含有活化基团的亲脂基团。所述连接有亲脂基团的亚磷酰胺单体是本领域技术人员容易获得的。在一些实施方式中，所述亚磷酰胺单体是通过使用如WO2020257194Al中制备例1中公开的方法合成获得的。
在一些实施方式中，本公开的正义链和反义链的定义和选择范围和上文中对本公开的寡核苷酸缀合物描述中相同，所述正义链和反义链可以通过本领域常规的双链寡核苷酸制备方法，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所述的双链寡核苷酸中，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双链寡核苷酸的方法也是本领域技术人员所熟知的。
药物组合物
在另一方面，本公开提供了一种药物组合物，所述药物组合物含有如上所述的寡核苷酸缀合物作为活性成分和药学上可接受的载体。
所述药学上可接受的载体可以是双链寡核苷酸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，对所述功能性双链寡核苷酸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，所述功能性双链寡核苷酸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，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pH缓冲液、保护剂和渗透压调节剂中的至少一种。
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％。
所述渗透压调节剂例如可以为氯化钠和/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、脑室内注射或鞘内注射给药，也可以但不限于通过眼药水、鼻腔给药、口咽吸入、喷雾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鞘内注射来递送。在一些实施方式中，将所述药物组合物鞘内注射至脊髓液中可以推注注射形式或经由微型泵来进行，这些微型泵可植入皮肤下方，从而提供将siRNA规律且恒定地递送至脊髓液中。在一些实施方式中，鞘内施用经由手术植入的渗透泵。在一些实施方式中，将渗透泵植入椎管的蛛网膜下腔以促进鞘内施用。关于此鞘内递送系统的更多细节可见于2015年1月28日提交的PCT/US 2015/013253，以引用的方式将该文献的全部内容并入本文。
本公开的寡核苷酸缀合物、药物组合物的应用
在另一方面，本公开提供了本公开的寡核苷酸缀合物，和/或药物组合物在制备用于抑制细胞中靶基因表达的靶mRNA表达的药物中的用途。在一些实施方式中，所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR或TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。在一些实施方式中，所述靶基因是SOD1或RPTOR。
在一些实施方式中，本公还提供了一种本公开的寡核苷酸缀合物和/或药物组合物在制备用于治疗和/或预防和靶基因表达的靶mRNA表达相关的疾病中的用途。在一些实施方式中，所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。在一些实施方式中，所述靶基因是SOD1或RPTOR。
在一些实施方式中，本公开还提供了一种抑制细胞中靶基因表达的方法，该方法包括将有效量的本公开的寡核苷酸缀合物和/或药物组合物与所述细胞接触。在一些实施方式中，所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。在一些实施方式中，所述靶基因是SOD1或RPTOR。
通过将本公开的提供的寡核苷酸缀合物和/或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，本公开所提供的寡核苷酸组合物和/或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寡核苷酸缀合物和/或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，将寡核苷酸缀合物和/或药物组合物放置与受试者体内。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，具备给药导致与受试者整个身体相比将更多的寡核苷酸缀合物和/或药物组合物递送至特定位点；而全身给药导致将 所述寡核苷酸缀合物和/或药物组合物递送至受试者的基本整个身体。
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)、脑室内给药、鞘内给药、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)，玻璃体注射给药，滴眼给药。给药频率可以是每天、每周、每两周、每三周、每个月、每2个月、每3个月、每半年或每年1次或多次。
本公开所述的寡核苷酸缀合物和/或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％的群体死亡的致死剂量)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。
采用本公开提供的方法抑制特定基因在细胞中表达，所提供的寡核苷酸缀合物和/或药物组合物中的功能性双链寡核苷酸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，所提供的寡核苷酸缀合物中的功能性双链寡核苷酸用量是这样的量：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，所述试剂盒包含有效剂量的本公开提供的寡核苷酸缀合物和/或药物组合物。
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寡核苷酸缀合物和/或药物组合物。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寡核苷酸缀合物和/或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寡核苷酸缀合物和/或药物组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，所述寡核苷酸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/或药学上可接受的载体和/或辅料可以以任何形式提供，例如液体形式、干燥形式和/或冻干形式。在一些实施方式中，所述寡核苷酸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/或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 Laboratory(1989))所记载的方法进行。
制备例1本公开提供的缀合物1的合成
(1-1)寡核苷酸单链S1的制备
按照WO2019105418(A1)制备例1记载的方法，通过固相合成方法合成表2中siRNA缀合物1中的siRNA正义链的序列，区别仅在于，在固相合成的过程中，在连接3’端的第一个核苷单体之前、以及连接完5’端最后一个核苷单体之后，另外连接含有HO(CH2)6-S-S-(CH2)6-基团的亚磷酰胺单体(购自Hongene Biotech公司)，从固相载体上切割下寡核苷酸单链，得到如式(1-1-1)所示的寡核苷酸单链S1(45.00mg，6.61μmol)(分子量：6807.15，实测：6807.91)：
式(1-1-1)；
式中，代表如SEQ ID NO:127所示的序列。
5’-UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm-3’(SEQ ID NO:127)。
(1-2)寡核苷酸单链S2的合成：
用5.00ml纯化水溶解(1-1)中制备得到的S1(45.00mg，6.61μmol)后，向所得溶液中按照TCEP和S1的质量比为1.5:1的比例加入TCEP水溶液(67.50mg，0.24mmol，购自毕得医药，批号：BD155793)。混匀，在室温下反应2个小时，将反应液用10mL纯化水稀释并过滤，得到反应液14mL，将反应液转移至15mL，3K规格的超滤管，在3900rpm的条件下离心30min。重复超滤和离心的步骤直至测得超滤液体电导在100S以下下，收集滤膜内产品，获得如SEQ ID NO:220所示的siRNA缀合物1中的正义链S2(42.00mg，6.42μmol，产率：97.10％)(分子量：6543.47，实测：6542.57)。
(1-3)寡核苷酸单链S3的合成：
将步骤(1-2)中制备得到的S2(42.00mg，6.61μmol)用7.00ml纯化水溶解在15ml的离心管中，待完全溶解后，向离心管中加入Py-S-S-Py(0.1515g，0.68mmol，购自阿拉丁公司，批号：F2015072)。旋涡混匀，在室温下反应6小时，用安捷伦1260HPLC仪器检测反应，待反应完全后，将反应液用20％浓度(V/V)的乙醇水溶液稀释，过滤，用纯化仪(购自利穗科技公司)凝胶脱盐纯化，使用的条件为流动相：20％浓度(V/V)的乙醇水溶液，收集波长：280nm，收集洗脱液，浓缩得到产物41.62mg(6.15μmol，产率：93.11％)(分子量：6761.76，实测：6761.06)。
(1-4)寡核苷酸缀合物1中正义链的合成
HS-P7：
将步骤(1-4)中制备得到的S3(40.00mg，5.92μmol)用8.00ml 0.1M浓度的醋酸铵水溶液溶解备用(醋酸铵购自阿拉丁公司，编号：A112061)。向反应液中加入HS-P7(30.7mg，29.60μmol，订制合成自Tanzhen Biotech公司)，其中，HS-P7是按照N端到C端方向，含有如SEQ ID NO:169所示序列(HAIYPRH)的多肽配体，混匀后再室温下反应6小时，将反应液用等体积纯化水稀释，过滤，使用安捷伦半制备反相纯化，反相纯化使用的柱子为Kromasil 100-10-C18,10um,21.2*250mm；流动相用100mM TEAA(PH＝7.0-7.3)：乙腈的比例为5％-75％(V/V)的溶剂进行梯度洗脱，收集产物峰洗脱液，浓缩得到产物20.50mg(2.53μmol，产率：41.19％)(分子量：8613.86，实测：8612.76)。
(1-5)缀合物1的合成
按照WO2019105418(A1)制备例1记载的方法，制备得到寡核苷酸缀合物1中的反义链，区别仅在于，按照表2中SEQ ID NO：129所示的序列依次连接核苷单体；使用DEPC水溶解等摩尔的步骤(1-4)中制备得到的正义链和(1-5)中制备得到的反义链，随后退火得到表2中的缀合物1。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：8613.86，实测值：8612.76，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物1对应的序列的缀合物。
表2缀合物的序列

表2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s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；大写字母组合-S-S-表示连接基团RI，所述连接基团RI具有式(103)所示的结构，其中n103＝1，m103＝6，大写字母H表示氢原子；N3表示连接基团RI，所述连接基团是如式(203)所示的连接基团：
-L5-表示连接基团RI，所述连接基团RI具有式(301)所示的结构，其中，p301＝1，n301＝2，m301＝2，所述连接基团RI和其右侧核苷酸中核糖环的5’位相连；
L3表示连接基团RI，所述连接基团RI具有式(301)所示的结构，其中，p301＝0，n301＝0，m301＝2，所述连接基团RI取代与其左侧相邻的第一个核苷酸中核糖环的2’位上羟基中的氢原子；
P7表示按照N端到C端方向，具有如SEQ ID NO:169所示序列的多肽；RP7表示按照将具有如SEQ ID NO:169所示序列的多肽，按照C端到N端的方向连接至核苷酸；P12表示按照N端到C到方向，具有如SEQ ID NO:170所示序列的多肽；RP12表示按照将具有如SEQ ID NO:170所示序列的多肽，按照C端到N端的方向连接至核苷酸；大写字母L表示该大写字母L左侧相邻的一个核苷酸为核糖环2’位置的羟基中的氢被十六烷基替代形成的核苷酸。
制备例2本公开提供的缀合物2的合成
用按照WO 2020/257194 Al的制备例13相同的方法制备如SEQ ID NO:226所示的序列，区别仅在于siRNA中正义链的序列是如SEQ ID NO:226所示的序列。
按照和制备例1中相同的方法，制备本公开的缀合物2，区别仅在于，对于缀合物2，代替如SEQ ID NO:127所示的序列，按照如SEQ ID NO:138所示的序列依次连接正义链的核苷单体：
5’-UmsUmsUmUmAmALUfCfCfUmCmAmCmUmCmUmAmAmAm-3’(SEQ ID NO:138)。
在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：8613.86，实测值：8789.46，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物2对应的序列的缀合物。
制备例3本公开提供的缀合物3的合成
按照和制备例1中相同的方法，制备本公开的缀合物3，区别仅在于，用如SEQ ID NO:170所示的多肽P12替代如SEQ ID NO:169所示的多肽P7。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，，其中正义链理论值：9211.59，实测值：9210.76，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物3对应的序列的缀合物。
制备例4本公开提供的缀合物4的合成
按照和制备例2中相同的方法，制备本公开的缀合物4，区别仅在于，用如SEQ ID NO:170所示的多肽P12替代如SEQ ID NO:169所示的多肽P7。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：9211.59，实测值：9211.89，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物4对应的序列的缀合物。
制备例5本公开提供的缀合物5的合成
(5-1)寡核苷酸单链S4的制备
按照WO2019105418(A1)制备例1记载的方法，通过固相合成方法合成表2中siRNA缀合物1中的siRNA正义链的序列，区别仅在于，对于连接有(N3-P7*3)的碱基，使用的亚磷酰胺单体另外连接含有6-(三氟乙酰氨基)-己基-(2-氰乙基)-(N,N-二异丙基)-亚磷酰胺单体(订购自Hongene Biotech公司)，其中，单体中的三氟乙酰保护基在前述记载的固相合成方法中氨水存在的条件下脱除，获得具有如式(5-1-1)所示结构的核苷酸单链S4：
其中，代表如SEQ ID NO:139所示的序列：5’-ULUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm-3’(SEQ ID NO:139)。
(5-2)式(209)所示的化合物的合成
按照如WO2014025805A1公开的Scheme3(27页)中相同的方法合成如式(210)所示的化合物。将如式(210)所示的化合物(9.00g,15.97mmol)和HOBt(9.71g,71.85mmol，麦克林公司，货号：C10116218)用DMF(90mL)溶解，加入2-((2-炔丙基氧)乙氧基)乙胺(8.57g,59.88mmol)(式(211)所示化合物，购自毕得医药，货号：ANL677)和EDC·HCl(11.48g，59.88mmol，麦克林公司，货号：C10087270)，搅拌至完全溶解后加入DIEA(14.43g，111.77mmol，安耐吉公司，货号：EE300104)。室温下反应20小时后，往反应液中加入100mL水和100mL乙酸乙酯，搅拌静置分层后分离有机相，依次用100mL10％柠檬酸水溶液、100mL碳酸氢钠水溶液和100mL食盐水溶液洗涤有机相，旋蒸去除溶剂，得到如式(209)所示的化合物(12.49g，13.29mmol)(分子量：939.15，实测：939.70)。
(5-3)如式(208)所示的化合物(AE1)的合成
将步骤(5-2)中制备得到的如式(209)所示的化合物(12.49g，13.29mmol)用100mL乙醇溶解，向溶液中加入氢氧化钠水溶液(3.2M，20mL)，室温下搅拌反应3小时，蒸发除去溶剂，加入50mL水溶解，用乙酸乙酯洗涤3次，每次50mL，分液，向水相中加入1M浓度的盐酸水溶液直至pH为5-6，加入100mL乙酸乙酯溶液萃取，有机相蒸发除去溶剂，柱层析分离提纯，使用的柱层析条件为A相：二氯甲烷，B相：二氯甲烷/甲醇＝10:1，(0～50(B％)，5min；50～100(B％)，10min；100(B％)，20min)梯度洗脱，在16-19min收集洗脱液，过滤并除去溶剂，得到产物AE1(6.37g，纯度：93.5％)(分子量：925.13，实测：924.80)。
(5-4)活性酯AE2的合成
将步骤(5-3)中制备得到的AE1(555.00mg，0.60mmol)用二氯甲烷(5.00ml，购自麦克林公司，批号：C10880269)溶解，加入DIEA(309.60mg，2.40mmol，购自安耐吉公司，批号：FA030154)，加入三氟乙酰五氟苯酯(336.00mg，购自西恩斯公司，批号：766000IKU)，室温下反应3小时，蒸发除去溶剂得到产物AE2(872.94mg，0.60mmol，产率：100％)。未经纯化直接用于下一步反应。
(5-5)寡核苷酸单链S5的合成
用1.00ml 100mM的碳酸氢钠水溶液溶解步骤(5-1)中制备得到的S4(40.00mg，6.09μmol)；用5.00ml DMF(购自麦克林公司，批号：C10594019)溶解步骤(5-4)中制备得到的AE2(872.94mg，0.60mmol)，在室温下反应4小时，向反应液中加入乙腈和1*PBS的混合溶剂(V：V＝14:1)，在16500rpm下离心10min，转移出上清液，用3ml核酸沉淀溶液(10％三氯乙酸水溶液，购自百奥莱博公司，货号：GL1252)洗涤3次，分离出沉淀，所得沉淀即为寡核苷酸单链S5(41.00mg，产率：90.04％)(分子量：7477.76，实测：7475.85)。
(5-6)寡核苷酸缀合物5中正义链的合成
将步骤(5-5)中制备得到的S5(31.25mg，4.18μmol)用2.5ml纯化水溶解，加入T7-N(27.20mg，25.08μmol，商业订购自Taizhen公司)，混合均匀。将167.00μl五水合硫酸铜(50mmol水溶液，8.36μmol，购自麦克林公司，批号：C10040033)和TBTA(100mmol-DMF溶液，8.36μmol，购自上海毕得公 司，批号：BLP365)以及抗坏血酸钠(1mol水溶液，62.63μmol，购自麦克林公司，批号：C10052338)混合均匀后加入前述反应体系。在四十摄氏度温度的水浴中反应6小时。用6ml DMF和纯化水的混合溶剂(V/V＝1:1)稀释反应液，过滤，通过反相柱纯化，柱材料为Kromasil 100-10-C18,10um,21.2*250mm，流动相为A-PA:100mM TEAA(PH＝7.0-7.3),PB:乙腈/异丙醇＝1/1，进行梯度洗脱，收集洗脱液，除去溶剂得到缀合物5中的正义链(2.00mg，产率：4.45％)(分子量：10741.00，实测：10738.99)。
(5-7)缀合物5的合成
按照WO2019105418(A1)制备例13记载的方法，制备得到寡核苷酸缀合物5中的反义链，区别仅在于，按照表2中SEQ ID NO：137所示的序列，依次连接核苷单体，获得反义链序列；使用DEPC水溶解等摩尔的步骤(5-6)中制备得到的正义链和上述制备得到的反义链，退火得到表2中的缀合物5。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：10741.00，实测值：10738.99，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物5对应的序列的缀合物。
制备例6本公开的缀合物6的合成
按照和制备例1中相同的方法，制备得到本公开提供的缀合物6，区别仅在于将步骤(1-4)中的HS-P7替换为如式(601)所示的HS-RP7：
在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：8613.86，实测值：8789.46，反义链理论值：7096.79.实测值：7097.12，实测值和理论值相符，表明所获得的的是具有表2中缀合物6对应的序列的缀合物。
制备例7本公开提供的缀合物7的合成
(7-1)缀合物7中正义链S7的制备
将P7-N3(26mg，24.8umol，商业订购自南昌探针公司)溶解于1mL DMF中，将如式(7-1-1)所示的寡核苷酸单链S7A(26.2mg，4.13umol，商业订购自上海兆维公司)溶解于1mL纯化水中，将前述溶液混合均匀，向其中依次加入硫酸铜溶液(500ul，24.8umol，购自麦克林公司)、TBTA的DMF溶液(50mM，500ul，购自上海毕得公司)和抗坏血酸钠溶液(1M水溶液，124ul，购自麦克林公司)，混合均匀后，加入前述混合均匀的寡核苷酸溶液，混合均匀后， 在40℃温度条件下，于恒温振动器中反应5h。将反应液用DMF稀释，安捷伦制备纯化仪反相纯化(反相柱：Kromasil 100-10-C18,10um，21.2*250mm；流动相：A-100mM TEAA，B-乙腈；梯度：5-65％B，0-40min；流速：10ml/min；柱温25℃，检测波长214/260nm双波长)，收集产物洗脱液，浓缩得到缀合物7的正义链(10mg，产率：38％，质谱理论值：8525.71，实测值：8505.27)。
其中，代表如SEQ ID NO:168所示的序列：5’-ULUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAm-3’(SEQ ID NO:168)。
(7-2)缀合物7的合成
按照WO2019105418(A1)制备例13记载的方法，制备得到寡核苷酸缀合物7中的反义链，区别仅在于，按照表2中SEQ ID NO：151所示的序列，依次连接核苷单体，获得反义链序列；使用DEPC水溶解等摩尔的步骤(7-1)中制备得到的正义链和上述制备得到的反义链，退火得到表2中的缀合物7。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：8525.71，实测值：8525.88，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物7对应的序列的缀合物。
制备例8本公开提供的缀合物8的合成
(8-1)缀合物8中正义链S8的制备
按照和制备例7中相同的方法制备本公开提供的缀合物8，区别仅在于用如式(801)所示的寡核苷酸单链S8A(27.1mg，4.13umol，商业订购自上海兆维公司)替代S7A，用如式(802)所示的RP12-N3替代P7-N3(42mg，24.8umol，商业订购自南昌探针公司)，制备得到正义链S8(9mg，产率：22.0％，质谱理论值：9914.30，实测值：9912.90)：
其中，代表如SEQ ID NO:168所示的序列：5’-ULUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAm-3’(SEQ ID NO:168)。
(8-2)缀合物8的合成
按照WO2019105418(A1)制备例13记载的方法，制备得到寡核苷酸缀合物8中的反义链，区别仅在于，按照表2中SEQ ID NO：153所示的序列，依次连接核苷单体，获得反义链序列；使用DEPC水溶解等摩尔的步骤(8-1)中制备得到的正义链和上述制备得到的反义链，退火得到表2中的缀合物8。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：9914.30，实测值：9912.90，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物8对应的序列的缀合物。
制备例9本公开提供的缀合物9的合成
按照制备例7中相同的方法制备得到本公开提供的缀合物9，区别仅在于用如式(802)所示的RP12-N3替代P7-N3(42.1mg，24.8umol，商业订购自南昌探针公司)。用LC-MS分别检测正义链和反义链的分子量，其中正义链理论值：9721.18，实测值：9719.34，反义链理论值：7096.79.实测值：7097.12，实测值与理论值相符，表明所获得的是具有表2中缀合物9对应的序列的缀合物。
对比制备例1对比缀合物1的合成
按照和制备例1中相同的方法制备对比缀合物1，区别仅在于制备正义链的时候，仅在连接完5’端最后一个核苷单体之后，另外连接含有HO(CH2)6-S-S-(CH2)6-基团的亚磷酰胺单体(购自Hongene Biotech公司)，以及用将步骤(1-4)中的HS-P7替换为如式(601)所示的HS-RP7,在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物1中对应的序列的缀合物。
对比制备例2对比缀合物2的合成
按照和对比制备例1中相同的方法制备对比缀合物2，区别仅在于制备正义链的时候，仅在连接完3’端第一个核苷单体之前，另外连接含有HO(CH2)6-S-S-(CH2)6-基团的亚磷酰胺单体(购自Hongene Biotech公司)，在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物2中对应的序列的缀合物。
对比制备例3对比缀合物3的制备
按照和制备例7中相同的方法制备得到对比缀合物3，区别仅在于用将P7-N3(13mg，12.4umol，商业订购自南昌探针公司)溶解于1mL DMF中，将如式(11-1)所示的寡核苷酸单链SS3A(26.2mg，4.13umol，商业订购自上海兆维公司)：
其中，代表如SEQ ID NO:168所示的序列：5’-ULUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAm-3’(SEQ ID NO:168)。
溶解于1mL纯化水中，向其中依次加入硫酸铜溶液(250ul，12.4umol，购自麦克林公司)、TBTA的DMF溶液(50mM，250ul，购自上海毕得公司)和抗坏血酸钠溶液(1M水溶液，65ul，购自麦克林公司)，在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物3中对应的序列的缀合物。
对比制备例4对比缀合物4的制备
按照和对比制备例3中相同的方法制备得到对比缀合物4，区别仅在于用如式(12-1)所示的寡核苷酸单链SS4A(26.3mg，4.13umol，商业订购自上海兆维公司)替代对比制备例3中的SS3A：
其中，代表如SEQ ID NO:168所示的序列：5’-ULUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAm-3’(SEQ ID NO:168)。在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物4中对应的序列的缀合物。
对比制备例5对比缀合物5的制备
按照和对比制备例3中相同的方法制备得到对比缀合物5，区别仅在于用如式(802)所示的RP12-N3(20.1mg，12.4umol，商业订购自南昌探针公司)替代对比制备例3中的P7-N3:
在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物5中对应的序列的缀合物。
对比制备例6对比缀合物6的制备
按照和对比制备例4中相同的方法制备得到对比缀合物6，区别仅在于用如式(802)所示的RP12-N3(20.1mg，12.4umol，商业订购自南昌探针公司)替代对比制备例4中的P7-N3:
在制备完成后，用LC-MS分别检测正义链和反义链的分子量，实测值与理论值相符，表明所获得的是具有表2中对比缀合物6中对应的序列的缀合物。
实验例1本公开的寡核苷酸缀合物在小鼠体内的活性
本实验例考察了制备得到的缀合物1、缀合物2和缀合物5在小鼠体内，尤其是中枢神经系统内，对SOD1基因表达的mRNA的抑制活性。
本实验的小鼠购自斯贝福公司，种系为ICR，等级为SPF，性别均为雄性，购入体重为25±1g。
详细步骤如下：
[1]小鼠侧脑室注射给药：
对于每一待测缀合物，用PBS将粉末状的缀合物溶解并稀释成浓度为20μg/μL的注射液备用。
将18只小鼠随机分为3组，每组6只小鼠，编号为空白组，测试组1，测 试组2和测试组3。对于空白组的小鼠，不给药。对于测试组1、测试组2的小鼠和测试组3的小鼠，按照10μL/只的剂量分别给药缀合物1、缀合物2和缀合物5，具体步骤为：对每只小鼠按照400mg/Kg体重的剂量腹腔注射5％水合氯醛(购自上海源叶生物科技有限公司)麻醉。待麻醉生效后，用微量给药泵(型号：78-8130，购自KDSCIENTIFIC公司)使用25μL的微量注射针(购自汉密尔顿公司)将药物注射到侧脑室，注射时间为10分钟，注射完留针5分钟，然后缓慢拔针。之后用生物胶水(购自明尼苏达矿业制造有限公司)封住针孔，将头部皮肤粘合好，待小鼠清醒后放回动物饲养室继续饲养。将注射日记录为第一天。
[2]样本采集
在注射后的第五天对每只小鼠按照400mg/Kg体重的剂量腹腔注射5％水合氯醛麻醉后提取小鼠的顶叶皮层、海马体、小脑、延脑、丘脑、纹状体保存于RNA later(编号：MFCD03453003，购自SIGMA公司)中，随后，使用RNAVzol(购自威格拉斯生物技术(北京)有限公司，货号N002)根据说明书记载的方法提取提取RNA后进行q-PCR检测靶基因SOD1的表达。
[3]检测
对于每只小鼠的脑部的每个区域，分别提取4件样本，对每件样本分别取1μg总RNA，使用反转录试剂盒GoldenstarTM RT6 cDNA Synthesis Kit(购自逸优科技，货号50000665)提供的试剂，其中选取GoldenstarTM Oligo(dT)17作为引物，按试剂盒说明书中反转录操作步骤配置反转录反应体系20μl，对各孔细胞的总RNA进行反转录。反转录的条件为：对于每一反转录反应体系，将反转录反应体系置于50℃孵育50min，然后85℃孵育5min，最后4℃孵育30s，反应结束后，向反转录反应体系中加入DEPC水80μl，得到含cDNA的溶液。
对于每一反转录反应体系，分别取上述含cDNA的溶液5μl做模板，使用SYBRTM Select Master Mix试剂盒(购自Appliedbiosystems，货号：50000332)提供的试剂配置qPCR反应体系20μl，其中，用于扩增目标基因SOD1和内参基因GAPDH的PCR引物序列如表3所示，每条引物的终浓度为0.25μM。将各qPCR反应体系置于ABI StepOnePlus Real-Time PCR仪上，使用三步法进行扩增，扩增程序为95℃预变性10min，然后95℃变性30s，60℃退火30s，72℃延伸30s，重复上述变性、退火、延伸的过程共40次后，得到含有扩增了目标基因SOD1和内参基因GAPDH的产物W。产物W随即依次经过95℃15s，60℃1min，95℃15s的孵育，实时荧光定量PCR仪分别收集产物W中目标基因SOD1和内参基因GAPDH的溶解曲线，得到目标基因SOD1和内参基因GAPDH的Ct值。
表3引物信息

采用比较Ct(ΔΔCt)法，对各测试组中目标基因APOC3进行相对定量计算，计算方法如下：
ΔCt(测试组)＝Ct(测试组目标基因)–Ct(测试组内参基因)
ΔCt(对照组)＝Ct(对照组目标基因)–Ct(对照组内参基因)
ΔΔCt(测试组)＝ΔCt(测试组)-ΔCt(对照组平均)
ΔΔCt(对照组)＝ΔCt(对照组)-ΔCt(对照组平均)
其中，ΔCt(对照组平均)是对4件样本各自的ΔCt(对照组)的算术平均值。。
以对照组为基准，对测试组SOD1 mRNA的表达水平进行归一化，定义空白对照组SOD1 mRNA表达水平为100％，
测试组SOD1 mRNA相对表达水平＝2-ΔΔCt(测试组)×100％
测试组SOD1 mRNA抑制率＝(1-测试组APOC3mRNA相对表达水平)×100％。各siRNA缀合物对SOD1 mRNA在脑部不同区域的抑制率总结于表4中。
表4缀合物在小鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
由表4结果可知，在200μg/每只的给药剂量条件下，本公开的缀合物1、缀合物2和缀合物5在小鼠体内对中枢神经不同部位的SOD1基因表达的mRNA均显示出很高的抑制率。其中在正义链两端均缀合有本公开的多肽配体的缀合物1对不同部位SOD1 mRNA的抑制率基本均达到47.8％以上，尤其是在皮层区域，抑制率可高达79.87％。更进一步地，缀合了亲脂基团的缀合物2显示出更高的抑制活性，在所有区域对SOD 1 mRNA的抑制率均高于60％。特别是在皮层、海马体和小脑，抑制率均高于80％。在皮层的抑制率更是高达88.33％。而在正义链5’末端缀合有亲脂基团，并且在3’末端缀合有3个多肽配体的缀合物5在皮层区和海马区也分别显示出高达80.00％和75.90％的对SOD1 mRNA的抑制率。从而说明本公开的缀合物均显示出优异的SOD1mRNA的抑制活性，表现出良好的药学活性。
实验例2本公开的寡核苷酸缀合物在小鼠体内的活性
按照和实验例1相同的方法，考察了制备得到的缀合物3和缀合物4在小鼠体内，尤其是中枢神经系统内，对SOD1基因表达的mRNA的抑制活性。区别仅在于，对于测试组1和测试组2，注射使用的缀合物为缀合物3和缀合物 4。结果总结于表5中。
表5.缀合物在小鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
由表5结果可知，在200μg/每只的给药剂量条件下，本公开的缀合物在小鼠体内对中枢神经不同部位的SOD1基因表达的mRNA均显示出很高的抑制率。其中本公开的缀合物3在皮层区域对SOD1 mRNA的抑制率可高达80.02％，而进一步缀合了亲脂基团的缀合物4显示出普遍更高的抑制活性，在所有区域对SOD 1 mRNA的抑制率均高于60％。特别是在皮层、海马体和小脑，抑制率均高于70％。在皮层的抑制率更是高达86.53％。显示出优异的SOD1 mRNA的抑制活性。
实验例3本公开的寡核苷酸缀合物在大鼠体内的活性
按照和实验例1相同的方法，考察了制备得到的缀合物1和缀合物2在大鼠体内，尤其是中枢神经系统内，对SOD1基因表达的mRNA的抑制活性。使用的大鼠购自斯贝福公司，种系为SD，等级为SPF，性别为雄性。在注射后的第8天取样；在检测步骤中，使用的大鼠β-ACTIN基因替代小鼠GAPDH基因，作为内参基因，具体使用的引物序列如表6所示：
表6引物信息
结果总结于表7中。
表7.缀合物在大鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
由表7结果可知，在200μg/每只的给药剂量条件下，本公开的缀合物1和缀合物2在大鼠体内对中枢神经不同部位的SOD1基因表达的mRNA均显示出很高的抑制率。其中本公开的缀合物物1在各个区域均表现出高于55％的抑 制活性，尤其是皮层区域对SOD1 mRNA的抑制率可高达81.55％。而进一步缀合了亲脂基团的缀合物2显示出普遍更高的抑制活性，在所有区域对SOD 1mRNA的抑制率均高于75％。特别是在皮层、海马体、小脑和纹状体，对SOD1mRNA的抑制率均高于85％。在小脑的抑制率更是高达89.11％。显示出非常高的SOD1 mRNA的抑制活性。上述结果表明，本公开的缀合物能够有效地将siRNA递送至表达TfR的组织，例如中枢神经系统内的各种组织，并且所递送的siRNA在这些组织内表现出优异的靶mRNA抑制效果。
实验例4本公开的寡核苷酸缀合物在小鼠体内的活性
按照和实验例1相同的方法，考察了制备得到的缀合物6、缀合物7和对比缀合物1、对比缀合物2、对比缀合物3以及对比缀合物4在小鼠体内，尤其是中枢神经系统内，对SOD1基因表达的mRNA的抑制活性。区别仅在于，将42只小鼠随机分为7组，每组6只小鼠，编号为空白组，测试组1-6。对于空白组的小鼠，不给药。对于测试组1-8，注射使用的缀合物分别对应为缀合物6、缀合物7和对比缀合物1、对比缀合物2、对比缀合物3以及对比缀合物4。结果总结于表8和表9中。
表8.缀合物在小鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
表9.缀合物在小鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
其中，对比缀合物1和对比缀合物2与缀合物6的不同点仅为，缀合物6是正义链两端第一个核苷酸各缀合1个P7多肽配体的siRNA，而对比缀合物1和对比缀合物2分别仅在正义链5’端第一个核苷酸和3’端第一个核苷酸缀合有P7多肽配体。由表8的结果可以看出，本公开的缀合物6在中枢神经系统内的不同区域均表现出明显高于对比缀合物1和对比缀合物2的抑制率，在延脑区域，本公开提供的缀合物的抑制率高达48.18％，相较于对比缀合物1和对比缀合物2分别提高了18.68％和10.88％。而在纹状体区域，本公开提供的缀合物的抑制率高达65.11％，相较于对比缀合物1和对比缀合物2分别提高了29.36％和16.72％，表现出明显更好的抑制效果。
对比缀合物3和对比缀合物4与缀合物7的不同点仅为，缀合物7是正义链两端第一个核苷酸各缀合1个RP7多肽配体的siRNA，而对比缀合物3和 对比缀合物4分别仅在正义链5’端第一个核苷酸和3’端第一个核苷酸缀合有RP7多肽配体。由表9的结果可以看出，在皮层和海马体区域中，本公开的缀合物7表现出比对比缀合物3和对比缀合物4明显更高的抑制率，抑制率均增加了约10％。
上述结果表明，与仅含有1个多肽配体的缀合物相比，本公开的siRNA缀合物更能够有效地将siRNA递送至表达TfR的中枢神经系统内的各种组织，并且所递送的siRNA在这些组织内表现出优异的靶mRNA抑制效果。
实验例5本公开的寡核苷酸缀合物在小鼠体内的活性
按照和实验例1相同的方法，考察了制备得到的缀合物8、缀合物9和对比缀合物5、对比缀合物6在小鼠体内，尤其是中枢神经系统内，对SOD1基因表达的mRNA的抑制活性。区别仅在于，将30只小鼠随机分为6组，每组6只小鼠，编号为空白组，测试组1-5。对于空白组的小鼠，不给药。对于测试组1-5，注射使用的缀合物分别对应为缀合物8、缀合物9和对比缀合物5、对比缀合物6，结果总结于表10中。
表10.缀合物在小鼠中枢神经系统内对SOD 1基因表达的mRNA的抑制率
其中，缀合物8、缀合物9、对比缀合物5和对比缀合物6中siRNA的序列相同，区别仅在于缀合物8中正义链的5’端的第一个核苷酸通过点击化学连接了2个RP12多肽配体，缀合物9中正义链的5’端和3’端的第一个核苷酸分别各自通过点击化学连接了1个RP12多肽，对比缀合物5和对比缀合物6分别仅在5’端第一个核苷酸和3’端第一个核苷酸缀合有1个RP12多肽配体的缀合物。
由表10中结果可知，与对比缀合物相比，本公开的缀合物8和缀合物9均表现出了明显更为优异的抑制率。其中，在海马体中，本公开的缀合物的抑制率均高于60％，缀合物8的抑制率更是高达74.45％，是对比缀合物5的抑制率的2.5倍。在皮层中，本公开的缀合物8的抑制率也高达75.37％，比对比缀合物5和对比缀合物6的抑制率的两倍更高。缀合物9的抑制率高达59.91,％，相较于对比缀合物5和对比缀合物6分别提高了24.01％和23.78％。
上述结果表明，与仅含有1个多肽配体的缀合物相比，本公开的缀合物能够更有效地将siRNA递送至表达TfR的中枢神经系统内的各种组织，并且所递送的siRNA在这些组织内表现出优异的靶mRNA抑制效果。
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 (60)

  1. 一种寡核苷酸缀合物，所述寡核苷酸缀合物包含：
    (a)功能性双链寡核苷酸，所述功能性双链寡核苷酸包含正义链和反义链，所述正义链和反义链分别包含15-25个核苷酸，每个所述核苷酸是修饰或未修饰的核苷酸；
    (b)n0个多肽配体，每个所述多肽配体由5-12个修饰或未修饰的氨基酸组成，其中，n0为2-6的整数，所述多肽配体与转铁蛋白受体具有亲和性；
    其中，每个所述多肽配体与所述功能性双链寡核苷酸通过共价键或者通过连接基团RI连接，每个所述多肽配体通过多肽配体的N端或C端连接至所述功能性双链寡核苷酸。
  2. 如权利要求1所述的寡核苷酸缀合物，其中，每个所述连接有多肽配体的共价键和/或连接基团RI分别和所述双链寡核苷酸中不同位置的核苷酸相连。
  3. 如权利要求2所述的寡核苷酸缀合物，其中，与共价键或连接基团RI连接的核苷酸之间间隔至少1个核苷酸。
  4. 如权利要求1-3中任意一项所述的寡核苷酸缀合物，全部多肽配体均连接至所述正义链。
  5. 如权利要求4所述的寡核苷酸缀合物，其中，至少一个所述多肽配体连接至所述正义链的3'末端的第一个核苷酸或5'末端的第一个核苷酸。
  6. 如权利要求5所述的寡核苷酸缀合物，其中，至少一个所述多肽配体连接至所述正义链的3'末端第一个核苷酸，且至少一个所述多肽配体连接至所述正义链的5'末端第一个核苷酸。
  7. 如权利要求1-6中任意一项所述的寡核苷酸缀合物，其中n0为2-4的整数。
  8. 如权利要求7所述的寡核苷酸缀合物，其中，n0＝2，一个所述多肽配体连接至所述正义链3'末端或5'末端的第一个核苷酸，另一个所述多肽配体连接至所述正义链中其他位置的核苷酸中的任意一个；
    或者，一个所述多肽配体连接至所述正义链3'末端的第一个核苷酸，另一个所述多肽配体连接至所述正义链5'末端的第一个核苷酸。
  9. 如权利要求7所述的寡核苷酸缀合物，其中，全部所述多肽配体均连接至所述正义链3'末端或5'末端的第一个核苷酸。
  10. 如权利要求1-9中任意一项所述的寡核苷酸缀合物，其中，全部多肽配体均通过连接基团RI连接至所述功能性双链寡核苷酸。
  11. 如权利要求1-10中任意一项所述的寡核苷酸缀合物，其中，所述连接有多肽配体的连接基团RI或共价键与所述功能性双链寡核苷酸中核苷酸核糖环的2'、3'或5'位置相连。
  12. 如权利要求1-11中任意一项所述的寡核苷酸缀合物，其中，所述连接基团RI具有式(101)所示的结构：
    其中，LA为侧链部分，k为1-6的整数，LB为缀合连接部，LC为主链部分，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点；PP表示所述连接基团RI连接至所述多肽配体的位点；
    所述主链部分LC为共价键或2-7价、直链或支链的C1-C25饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、-C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
    所述侧链部分LA为共价键，或者C1-C20亚烷基，或者，所述亚烷基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述亚烷基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH-SC1-C5烷基、-SC1-C5烷基苯基、-C1-C5烷基-SH、-OH、-SH、-NH2、-C1-C5烷基-NH2、-N(C1-C5烷基)(C1-C5烷基)、-NH(C1-C5烷基)、-N(C1-C5烷基)(C1-C5烷基苯基)、-NH(C1-C5烷基苯基)、硝基、-C(O)O(C1-C5烷基)、-CON(C1-C5烷基)(C1-C5烷基)、-CONH(C1-C5烷基)、-CONH2，-NHC(O)(C1-C5烷基)、-NHC(O)(苯基)、-N(C1-C5烷基)C(O)(C1-C5烷基)、-N(C1-C5烷基)C(O)(苯基)、 -C(O)C1-C5烷基、-C(O)C1-C5烷基苯基、-OC(O)C1-C5烷基、-SO2(C1-C5烷基)、-SO2(苯基)、-SO2NH2、-SO2NH(C1-C5烷基)、-SO2NH(苯基)、-NHSO2(C1-C5烷基)和-NHSO2(苯基)；
    所述缀合连接部LB为1-5个以下连接键中的一种或多种的连接组合：磷酸酯键、硫代磷酸酯键、酰胺键、酯键、醚键、二硫键。
  13. 如权利要求12所述的寡核苷酸缀合物，其中LC为C5-C20饱和烃基，或者，所述饱和烃基中的一个或多个碳原子被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、OP(O)2、C5-C8亚糖苷基、C2-C5亚烯基、C2-C5亚炔基、C6-C10亚芳基、C3-C8亚杂环基和C5-C10亚杂芳基；其中，所述饱和烃基可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-C5烷基、C6-C10芳基、C5-C10杂芳基、-O-C1-C5烷基、-OC1-C5烷基苯基、-C1-C5烷基-OH、-SC1-C5烷基、硝基、-CONH2。
  14. 如权利要求12或13所述的寡核苷酸缀合物，其中，k为1-3的整数；
    LC含有如式(L1)-(L3)所示的基团中的任意一个，通过如式(L1)-(L3)所示的基团中的醚键与LA部分连接：
    表示基团连接至分子其余部分的位点；
    LB为磷酸酯键或二硫键；
    每个LA为共价键，或者每个LA选自于由基团(L4)-(L23)及其连接组合所组成的组：

    式中，每个j1为1-10的整数；
    每个R’为C1-C10烷基；
    每个Ra为氢原子，C1-C10烷基，或者选自由基团(L24)-(L37)组成的组：

  15. 如权利要求14所述的寡核苷酸缀合物，其中，LA的长度为3-35个原子，其中所述LA的长度指LA中与LC直接连接的原子到与LA中与PP直接连接的原子形成的最长的原子链上的成链原子的个数。
  16. 如权利要求15所述的寡核苷酸缀合物，其中，每个LA为基团(L4)-(L9)、(L13)、(L14)、(L18)中至少2个的连接组合。
  17. 如权利要求16所述的寡核苷酸缀合物，其中，所述连接基团RI具有如式(201)所示的结构：
    其中，n201和m201为1-10的整数；
    P201为1-3的整数；
    LA具有如式(202)所示的结构：
    其中，n202、m202、p202、q202为1-5的整数，i202为0-5的整数。
  18. 如权利要求17所述的寡核苷酸缀合物，其中，n202、m202、p202和q202为2或3，i202为3或4。
  19. 如权利要求13所述的寡核苷酸缀合物，其中所述连接基团RI具有如 式(103)所示的结构：
    其中，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点，PP表示所述连接基团RI连接至所述多肽配体的位点；
    n103和m103为1-10的整数。
  20. 如权利要求19所述的寡核苷酸缀合物，其中，n103为1-3的整数，m103为3-6的整数。
  21. 如权利要求19所述的寡核苷酸缀合物，其中n103＝1,m103＝6。
  22. 如权利要求11所述的寡核苷酸缀合物，其中，所述连接基团RI具有式(301)所示的结构：
    其中，Nu表示所述连接基团RI连接至所述双链寡核苷酸的位点，PP表示所述连接基团RI连接至所述多肽配体的位点；
    p301为1或0，n301和m301为0-10的整数。
  23. 如权利要求22所述的寡核苷酸缀合物，其中n301和301为0-3的整数。
  24. 如权利要求1-23中任意一项所述的寡核苷酸缀合物，其中，所述寡核苷酸缀合物还含有亲脂基团。
  25. 如权利要求24所述的寡核苷酸缀合物，所述缀合物含有h0个亲脂基团，h0是取自1-5的整数。
  26. 如权利要求24或25所述的寡核苷酸缀合物，其中所述亲脂基团连接至所述功能性双链寡核苷酸的正义链或反义链上，该正义链或反义链至少含有一个多肽配体。
  27. 如权利要求24-26中任意一项所述的寡核苷酸缀合物，其中，所述功能性双链寡核苷酸中包括至少一个核苷酸，该核苷酸上同时连接有所述多肽配 体和所述亲脂基团。
  28. 如权利要求24-27中任意一项所述的寡核苷酸缀合物，其中和所述亲脂基团连接的核苷酸，和连接有多肽配体的核苷酸之间间隔至少1个核苷酸。
  29. 如权利要求24-28中任意一项所述的寡核苷酸缀合物，其中，n0＝3，h0＝1；其中全部多肽配体连接至所述正义链3'末端或5'末端的第一个核苷酸，连接有亲脂基团的核苷酸与连接有多肽配体核苷酸之间间隔的核苷酸数量不超过核苷酸单链中核苷酸总数的80％。
  30. 如权利要求24-28中任意一项所述的寡核苷酸缀合物，其中，n0＝2，h0＝1；
    两个多肽配体和一个亲脂基团中的一个连接至所述正义链3'末端的第一个核苷酸，一个连接至所述正义链5'末端的第一个核苷酸，另外一个连接至所述正义链中其他位置的核苷酸中的任意一个；
    或者，两个多肽配体分别连接至所述正义链3'末端的第一个核苷酸以及5'末端的第一个核苷酸，所述亲脂基团和正义链中一个的核苷酸相连，该核苷酸与所述正义链5'末端的第一个核苷酸距离及正义链3'末端的第一个核苷酸之间间隔的核苷酸数量不超过核苷酸单链中核苷酸总数的80％。
  31. 如权利要求24-30中任意一项所述的寡核苷酸缀合物，其中所述亲脂基团通过共价键连接至所述功能性双链寡核苷酸，所述多肽配体通过连接基团RI连接至所述功能性双链寡核苷酸。
  32. 如权利要求31所述的寡核苷酸缀合物，其中所述亲脂基团连接至核苷酸核糖环的2'位置。
  33. 如权利要求1-32中任意一项所述的寡核苷酸缀合物，其中每个所述多肽配体通过其N端连接至所述双链寡核苷酸。
  34. 如权利要求1-33中任意一项所述的寡核苷酸缀合物，其中每一个多肽配体是含有如表1中的SEQ ID NO:169-SEQ ID NO:183中任意一条所示序列的多肽配体。
  35. 如权利要求34所述的寡核苷酸缀合物，其中，按照从N端到C端的方向，每个多肽配体是含有如SEQ ID NO:169或SEQ ID NO:170所示序列的多肽配体：
    HAIYPRH(SEQ ID NO:169)；
    THRPPMWSPVWP(SEQ ID NO:170)。
  36. 如权利要求35所述的寡核苷酸缀合物，其中每一个多肽配体是如SEQ ID NO:169或SEQ ID NO:170所示序列的多肽配体。
  37. 如权利要求36所述的寡核苷酸缀合物，其中每个多肽配体都是如SEQ ID NO:169所示序列的多肽配体；或者，其中每个多肽配体都是如SEQ ID NO:170所示序列的多肽配体。
  38. 如权利要求33-37中任意一项所述的寡核苷酸缀合物，其中每一个多肽配体中所有的氨基酸都是修饰的氨基酸。
  39. 如权利要求24-38中任意一项所述的寡核苷酸缀合物，其中所述亲脂基团为长度为10-30个碳原子的饱和或不饱和、直链或支链的烃基；其中的一个或多个碳原子可以被羟基、氨基、羧基、磺酰基或磷酰基中的一种或多种替代。
  40. 如权利要求39所述的寡核苷酸缀合物，其中所述亲脂基团为长度为15-25个碳原子的饱和或不饱和、直链或支链的烃基。
  41. 如权利要求1-40中任意一项所述的寡核苷酸缀合物，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分反向互补形成双链区，所述核苷酸序列II至少部分地与靶mRNA反向互补，所述靶mRNA为靶组织或靶器官中目标细胞中的靶基因表达的mRNA，所述目标细胞是细胞表面存在转铁蛋白受体的细胞。
  42. 如权利要求41所述的寡核苷酸缀合物，其中，所述核苷酸序列I和所述核苷酸序列II均由19个核苷酸组成，所述核苷酸序列II与核苷酸序列I基本上反向互补、实质上反向互补、或完全反向互补；所述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；所述实质上反向互补是指两个核苷酸序列之间存在不多于1个的碱基错配；所述完全反向互补是指两个核苷酸序列之间没有碱基错配。
  43. 如权利要求41或42所述的寡核苷酸缀合物，其中所述正义链和所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基。
  44. 如权利要求43所述的寡核苷酸缀合物，其中，所述正义链和所述反义链中的每一个核苷酸为氟代修饰的核苷酸或非氟代修饰的核苷酸。
  45. 如权利要求44所述的寡核苷酸缀合物，其中，按照5'末端到3'末端的方向，所述核苷酸序列II的至少第2、6、14、16个核苷酸为2'-氟代修饰的 核苷酸，或者，所述核苷酸序列II的至少第2、14、16个核苷酸为2'-氟代修饰的核苷酸。
  46. 如权利要求1-45中任意一项所述的寡核苷酸缀合物，其中所述功能性双链寡核苷酸是saRNA或siRNA。
  47. 如权利要求46所述的寡核苷酸缀合物，其中所述双链寡核苷酸是siRNA。
  48. 如权利要求41-47中任意一项所述的寡核苷酸缀合物，其中所述靶组织或靶器官可以是骨骼肌、平滑肌、心肌、眼部、脑部、脊髓、耳部、鼻部、心脏、视网膜、肌肉组织、脾脏或肿瘤组织。
  49. 如权利要求41-48中任意一项所述的寡核苷酸缀合物，其中所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。
  50. 如权利要求49所述的寡核苷酸缀合物，其中所述靶基因是SOD1或RPTOR。
  51. 如权利要求50所述的寡核苷酸缀合物，其中所述双链寡核苷酸是siRPTORa1、siRPTORa2、siRPTORa3、siRPTORa1-M1、siRPTORa1-M2、siRPTORa1-M3、siRPTORa1-M1S、siRPTORa1-M1X、siRPTORa1-M2S、siRPTORa1-M2X、siRPTORa1-M3S、siRPTORa1-M3X、siRPTORa1-M1P1、siRPTORa1-M2P1、siRPTORa1-M3P1、siRPTORa1-M1SP1、siRPTORa1-M1SP1X、siRPTORa1-M2SP1、siRPTORa1-M2SP1X、siRPTORa1-M3SP1、siRPTORa1-M3SP1X、siRPTORb1、siRPTORb2、siRPTORb3、siRPTORb1-M1、siRPTORb1-M2、siRPTORb1-M3、siRPTORb1-M1S、siRPTORb1-M1X、siRPTORb1-M2S、siRPTORb1-M2X、siRPTORb1-M3S、siRPTORb1-M3X、siRPTORb1-M1P1、siRPTORb1-M2P1、siRPTORb1-M3P1、siRPTORb1-M1SP1、siRPTORb1-M1SP1X、siRPTORb1-M2SP1、siRPTORb1-M2SP1X、siRPTORb1-M3SP1、siRPTORb1-M3SP1X、siRPTORc1、siRPTORc2、siRPTORc3、siRPTORc1-M1、siRPTORc1-M2、siRPTORc1-M3、siRPTORc1-M1S、siRPTORc1-M1X、siRPTORc1-M2S、siRPTORc1-M2X、siRPTORc1-M3S、siRPTORc1-M3X、siRPTORc1-M1P1、siRPTORc1-M2P1、siRPTORc1-M3P1、siRPTORc1-M1SP1、siRPTORc1-M1SP1X、siRPTORc1-M2SP1、siRPTORc1-M2SP1X、siRPTORc1-M3SP1或siRPTORc1-M3SP1X中的一种。
  52. 一种药物组合物，该药物组合物含有如权利要求1-50中任意一项所 述的缀合物和其药学上可接受的载体。
  53. 如权利要求52所述的药物组合物，其中，所述双链寡核苷酸与药学上可接受的载体的重量比为1:(1-50)。
  54. 如权利要求1-53中任意一项所述的寡核苷酸缀合物和/或权利要求50-51中任意一项所述的药物组合物在制备用于抑制细胞中靶基因表达的靶mRNA表达的药物中的用途。
  55. 如权利要求54所述的用途，其中所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。
  56. 如权利要求1-51中任意一项所述的寡核苷酸缀合物和/或权利要求50-51中任意一项所述的药物组合物在制备用于治疗和/或预防和靶基因表达的靶mRNA表达相关的疾病中的用途。
  57. 如权利要求56所述的用途，其中所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。
  58. 一种抑制细胞中靶基因表达的方法，该方法包括将有效量的权利要求1-51中任意一项所述的寡核苷酸缀合物和/或权利要求52-53中任意一项所述的药物组合物与所述细胞接触。
  59. 如权利要求58所述的方法，其中所述靶基因是APP、ATXN2、C9orf72、TARDBP、MAPT、HTT、SNCA、FUS、ATXN3、ATXN1、SCA7、SCA8、MeCP2、PRNP、SOD1、DMPK、RPTOR、TTR、LRRK2、DUX4、补体3、补体5、NMDA、补体因子B或RHO。
  60. 一种试剂盒，该试剂盒包含权利要求1-51中任意一项所述的寡核苷酸缀合物和/或权利要求52-53中任意一项所述的药物组合物。
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YU MIN-ZHI; PANG WEN-HAO; YANG TING; WANG JIAN-CHENG; WEI LIN; QIU CHONG; WU YI-FAN; LIU WEI-ZHONG; WEI WEI; GUO XI-YING; ZHANG QI: "Systemic delivery of siRNA by T7 peptide modified core-shell nanoparticles for targeted therapy of breast cancer", EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES, ELSEVIER AMSTERDAM, NL, vol. 92, 26 June 2016 (2016-06-26), NL , pages 39 - 48, XP029666078, ISSN: 0928-0987, DOI: 10.1016/j.ejps.2016.06.020 *

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