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WO2019027015A1 - Complexe d'acide nucléique - Google Patents

Complexe d'acide nucléique Download PDF

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Publication number
WO2019027015A1
WO2019027015A1 PCT/JP2018/029113 JP2018029113W WO2019027015A1 WO 2019027015 A1 WO2019027015 A1 WO 2019027015A1 JP 2018029113 W JP2018029113 W JP 2018029113W WO 2019027015 A1 WO2019027015 A1 WO 2019027015A1
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compound
nucleic acid
formula
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mmol
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Japanese (ja)
Inventor
陽史 山田
麻奈 牧野
長谷川　尚
宏徒 岩井
上原　啓嗣
康裕 鈴木
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Kyowa Kirin Co Ltd
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Kyowa Hakko Kirin Co Ltd
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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
    • A61K47/54—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 an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 invention relates to a nucleic acid complex, a pharmaceutical composition containing the nucleic acid complex, and the like.
  • nucleic acid drugs As nucleic acid drugs, aptamers, antisenses, decoy nucleic acids, ribozymes, siRNAs, miRNAs and antimiRNAs are known. Nucleic acid drugs are expected to be clinically applicable to various diseases that have been considered difficult to treat up to now, because of their high versatility in which all genes in cells can be controlled. In addition, nucleic acid drugs are expected as next-generation drugs next to antibodies and small molecule drugs because of high target selectivity and activity in cells. However, the problem with nucleic acid medicines is that delivery to target tissues is difficult.
  • nucleic acid complex conjugate of a targeting compound and a nucleic acid
  • Targeting compounds include ligands capable of binding to extracellularly expressed receptors.
  • GalNAc N-acetyl-D-galactosamine
  • ASGPR asialoglycoprotein receptor
  • Patent Documents 1 and 2 disclose, for example, nucleic acid complexes shown below as complexes of a targeting compound and an oligonucleotide.
  • Patent Document 3 discloses a nucleic acid complex having the following structure having the same sugar ligand-tether unit as Patent Documents 1 and 2.
  • Patent Document 4 discloses a nucleic acid complex having a structure shown below as a sugar ligand-tether unit.
  • Complement is a group of blood proteins that mediate the immune response, and the action of complement is to promote phagocytosis of pathogenic bacteria by phagocytes and damage the virus with an outer membrane to lose infectivity etc. Can be mentioned.
  • C3 is most abundant in serum, and in the alternative pathway, its action is controlled by activation by complement factor B (CFB) etc. (non-patented) Literature 2).
  • Atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria when C3 continues to be activated due to abnormalities in regulatory factors involved in the alternative pathway of complement and stabilization with autoantibodies against C3 converting enzyme Disease (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute nephropathy (AKI), It is known to be associated with the onset of diseases such as ANCA-related vasculitis, lupus nephritis, asthma, autoimmune diseases (eg, systemic lupus erythematosus (SLE), psoriasis, neuromyelitis optica, myasthenia gravis, etc.) Patent documents 3, 4).
  • ANCA-related vasculitis lupus nep
  • complement factor B is present in blood at a relatively high concentration of 300 ⁇ g / mL ( Non-patent document 5), it is not easy to continue to inhibit all these complement factor B by, for example, general antibody drugs.
  • An object of the present invention is to provide a nucleic acid complex capable of suppressing the expression of complement factor B (CFB).
  • Formula 1 (In the formula 1, X is a double stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a duplex region of at least 11 base pairs, The double-stranded nucleic acid is any one of the target CFB mRNA sequences described in Tables 1-1 to 1-3 in a 17 to 30 nucleotides long oligonucleotide chain in the antisense strand.
  • Complementary and The 3 'or 5' end of the sense strand is linked to S3, L1 and L2 are each independently a sugar ligand, S1, S2 and S3 are each independently a linker.
  • nucleic acid complex according to [1], which has a structure represented by the following formula 2.
  • Formula 2 (In the formula 2, X, L1, L2 and S3 are as defined above, P1, P2, P3, P4, P5 and P6, and T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH 2 CH 2 O) n -CH 2 CH 2- , N is an integer of 0 to 99, B1 and B2 are each independently a bond or a structure represented by the following formula 2-1, and the terminal black dot in each structure is P2 or P3 or P5, respectively.
  • Formula 5 (In the equation 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 are as defined above. ) [7] The nucleic acid complex according to [6], wherein P1 is -CO-NH-, -NH-CO- or -O-. [8] The nucleic acid complex according to [6] or [7], which has any one of structures represented by the following formulas 6-1 to 6-9.
  • Formula 6-1 Formula 6-2: Equation 6-3: Equation 6-4: Formula 6-5: Formula 6-6: Formula 6-7: Formula 6-8: Formula 6-9: (In the formulas 6-1 to 6-9, X, S3, P3, Q2, T1, L1 and q2 are as defined above.
  • nucleic acid complex according to any one of [1] to [11], wherein the 3 'end of the sense strand and the 5' end of the antisense strand form a blunt end.
  • nucleic acid complex according to [11] wherein the double stranded nucleic acid comprises a sugar moiety modified nucleotide.
  • Formula 7-8-1 (In formula 7-8-1, X is as defined above.) [15] Any of [1] to [14], wherein X is a pair of sense strand / antisense strand selected from the group consisting of sense strand / antisense strand described in Tables 1-1 to 1-3. The nucleic acid complex according to item 1. [16] Any of [1] to [14], wherein X is a pair of sense strand / antisense strand selected from the group consisting of sense strand / antisense strand described in Table M1-1 to Table M1-3 The nucleic acid complex according to item 1.
  • [22] Administering the nucleic acid complex of any one of [1] to [18] or the pharmaceutical composition of any one of [19] to [21] to a patient in need thereof And methods of treating or preventing the disease.
  • [23] Introducing a double stranded nucleic acid into cells using the nucleic acid complex according to any one of [1] to [18] or the pharmaceutical composition according to any one of [19] to [21]
  • a method of suppressing the expression of the CFB gene including [24] A CFB related disease, which comprises administering to a mammal the nucleic acid complex according to any one of [1] to [18] or the pharmaceutical composition according to any one of [19] to [21]. Treatment method.
  • a medicament for use in the treatment of a CFB related disease comprising the nucleic acid complex according to any one of [1] to [18] or the pharmaceutical composition according to any one of [19] to [21].
  • a therapeutic agent for a CFB related disease comprising the nucleic acid complex according to any one of [1] to [18] or the pharmaceutical composition according to any one of [19] to [21].
  • CFB related diseases include atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN).
  • RPGN rapidly progressive glomerulonephritis
  • the treatment method according to [24] which is acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, neuromyelitis optica or myasthenia gravis.
  • AKI acute kidney injury
  • SLE systemic lupus erythematosus
  • psoriasis neuromyelitis optica or myasthenia gravis.
  • CFB related diseases include atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN).
  • the drug according to [25] which is acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, neuromyelitis optica or myasthenia gravis.
  • AKI acute kidney injury
  • SLE systemic lupus erythematosus
  • psoriasis neuromyelitis optica or myasthenia gravis.
  • CFB related diseases include atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN).
  • the therapeutic agent according to [26] which is acute kidney injury (AKI), ANCA-related vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, neuromyelitis optica or myasthenia gravis.
  • AKI acute kidney injury
  • SLE systemic lupus erythematosus
  • psoriasis neuromyelitis optica or myasthenia gravis.
  • a pharmaceutical composition containing the nucleic acid complex of the present invention can be administered to a mammal to treat various related diseases in vivo.
  • the nucleic acid complex of the present invention is a nucleic acid complex represented by the following formula 1.
  • Formula 1 :
  • X is a double stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a duplex region of at least 11 base pairs
  • the double-stranded nucleic acid is any one of the target CFB mRNA sequences described in Tables 1-1 to 1-3 in a 17 to 30 nucleotides long oligonucleotide chain in the antisense strand.
  • Complementary and The 3 'or 5' end of the sense strand is linked to S3, L1 and L2 are each independently a sugar ligand, S1, S2 and S3 are each independently a linker.
  • S1 and S2 can bind to the benzene ring in the ortho position, meta position and para position respectively with respect to the substitution position on the benzene ring of S3, but the nucleic acid complex represented by the following formula 1-1 It is preferred that it is a body. It is meant that the bond of S1 and S2 to the benzene ring in Formula 1 may be at any position other than the substitution position of S3 on the benzene ring.
  • Formula 1-1 :
  • Formula 1-1 X, L1, L2, S1, S2 and S3 are as defined above. In the present specification, the same meaning as described above will be described by exemplifying Formula 1-1: X, L1, L2, S1, and S2 in Formula 1-1, X, L1, It means that it may be the same group as the definition for each of L2, S1 and S2.
  • X is a double stranded nucleic acid consisting of a sense strand and an antisense strand and comprising a double stranded region of at least 11 base pairs.
  • the double-stranded nucleic acid is a target CFB mRNA described in Tables 1-1 to 1-3 described later in the oligonucleotide chain of 17 to 30 nucleotides in chain length in the antisense strand. Complementary to any of the sequences.
  • the 3 'or 5' end of the sense strand is linked to S3.
  • L1 and L2 are each independently a sugar ligand.
  • a sugar ligand means a group derived from a saccharide (such as monosaccharide, disaccharide, trisaccharide and polysaccharide) capable of binding to a receptor expressed in a target cell.
  • the moiety excluding the hydroxyl group involved in the binding of the saccharide constituting the sugar ligand is a group derived from the saccharide.
  • sugar ligand is meant.
  • a sugar ligand to be a target cell of an oligonucleotide may be selected.
  • monosaccharides include allose, altose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fusitol, L-fusitol, fucosamine, fucose, fucose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, Galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycero-D-manno-heptose, glycerol, glycerone, gulose, idoses, liquisose, mannosamine , Mannose, mannose-6-phosphate, psicose, quinobose, quinovosamine, rhamnitol, rhamnosamine,
  • disaccharides, trisaccharides and polysaccharides include avequase, aclavose, amysetose, amylopectin, amylose, apiose, alkanose, alkanose, alkanose, alkanose, ascorbic acid, boibinose, cellobiose, cellotriose, cellulose, cacotriose, chalcose, chitin, colitos, Cyclodextrin, Dextrose, Dextrin, 2-Deoxyribose, 2-Deoxyglucose, Diginose, Digitulose, Digitose, Evalose, Evemitrose, Fructooligosaccharide, Galto-oligosaccharide (galto-oligosaccharide), Gentianose, Gentiobiose, Glucan, Glucogen, Glycogen , Hammellose, heparin, inulin, isolevoglucosenone, isoma Tose
  • Each monosaccharide in the saccharide may be in D-form or L-form, or may be a mixture of D-form and L-form in any proportion.
  • an amino sugar in saccharides galactosamine, glucosamine, glucosamine, mannosamine, fucosamine, quinosamine, neuraminic acid, muramic acid, lactose acid, lactose diamine, acosamine, dacrosamine, daunosamine, desosamine, forosamine, garosamine, canosamine, kansosamine (kansamine) Mikaminose, mycosamine, perosamine, pneuimosamine, purpurosamine, rhodosamine etc. are mentioned.
  • the amino group of the amino sugar may be substituted with an acetyl group or the like.
  • sialic acid-containing sugar chains include sugar chains containing NeuAc at the non-reducing end of the sugar chain, such as NeuAc-Gal-GlcNAc-containing sugar chains, Neu5Ac ⁇ (2-6) Gal ⁇ (1-3) GlcNAc, etc. Can be mentioned.
  • Each monosaccharide in the saccharide may be substituted by a substituent, as long as it can bind to the receptor expressed in the target cell, for example, a hydroxyl group may be substituted, and each monosaccharide
  • the hydrogen atom in may be substituted one or more with an azide and / or an aryl group which may be substituted.
  • sugar ligand it is preferable to select a sugar ligand that binds to a receptor expressed on the surface of a target cell corresponding to each target organ, for example, when the target cell is a hepatocyte, on the surface of a hepatocyte Sugar ligands to the expressed receptor are preferred, and sugar ligands to the asialoglycoprotein receptor (ASGPR) are more preferred.
  • ASGPR asialoglycoprotein receptor
  • sugar ligand for ASGPR mannose or N-acetylgalactosamine is preferable, and N-acetylgalactosamine is more preferable.
  • sugar ligands having higher affinity to ASGPR for example, sugar derivatives described in Bioorganic Medicinal Chemistry, 17, 7254 (2009), and Journal of American Chemical Society, 134, 1978 (2012), etc. are known. These may be used.
  • S1, S2 and S3 are linkers.
  • S1 and S2 are not particularly limited as long as they are structures that link the sugar ligands L1 and L2 with the benzene ring, and a known structure used in a nucleic acid complex may be adopted.
  • S1 and S2 may be the same or different.
  • the sugar ligands L1 and L2 are preferably linked to S1 and S2 by glycosidic bonds, and S1 and S2 may be combined with a benzene ring, for example, -CO-, -NH-, -O-, -S- And -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S-, or -CO-NH- bond.
  • S3 is not particularly limited as long as it is a structure linking X, which is a double-stranded nucleic acid, and a benzene ring, and a known structure used in a nucleic acid complex may be adopted.
  • the oligonucleotide X is preferably linked to S3 by a phosphodiester bond, and S3 is linked to a benzene ring, for example, -CO-, -NH-, -O-, -S-, -O-CO. -, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- bond may be linked.
  • linkers of S1, S2 and S3 for example, WO 2009/073809, WO 2013/075035, WO 2015/105083, WO 2014/179620, WO 2015/006740
  • the structure disclosed in the above may be adopted.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 2.
  • Formula 2 :
  • X, L1, L2 and S3 are as defined above, P1, P2, P3, P4, P5 and P6, and T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH 2 CH 2 O) n -CH 2 CH 2- , N is an integer of 0 to 99.
  • P1 and P4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but preferably -O-, -O-CO-, -NH-CO- or -CO-NH-, -O -, -NH-CO- or -CO-NH- is more preferable, and -NH-CO- is more preferable.
  • P1 or P4 is, for example, -NH-CO-, it has a partial structure of -NH-CO-benzene ring.
  • Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH 2 CH 2 O) n -CH 2 CH 2- And n is an integer of 0 to 99, preferably substituted or unsubstituted alkylene having 1 to 12 carbon atoms, more preferably unsubstituted alkylene having 1 to 12 carbon atoms, and unsubstituted More preferably, it is an alkylene having 1 to 6 carbon atoms, and still more preferably an unsubstituted alkylene having 1 to 4 carbon atoms.
  • P2 and P5 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but not present, preferably -CO-O- or -CO-NH-, preferably absent, -CO-NH It is more preferable that it is-.
  • P2 and P5 are, for example, -CO-NH-, they have partial structures of B1-CO-NH-Q1 and B2-CO-NH-Q3.
  • m5 and m6 are each independently an integer of 0 to 10, and the terminal black dot in the structure of Formula 3-1 to Formula 3-3 is a bonding point to B1 or B2 or P1 or P4, respectively is there.
  • B1 and B2 are each independently a bond or a structure represented by the following formula, and the terminal black dot in each structure is P2 or P3 or P5 or P6, respectively.
  • m1, m2, m3 and m4 are each independently an integer of 0 to 10.
  • B1 and B2 are preferably groups derived from amino acids including non-natural amino acids such as glutamic acid, aspartic acid, lysine and iminodiacetic acid, or amino alcohols such as 2-amino-1,3-propanediol, And when B2 is a group derived from glutamic acid and aspartic acid, it is preferable that the amino groups of glutamic acid and aspartic acid are respectively bonded to form -NH-CO- as P2 and P5, and B1 and B2 are lysine When it is a group derived from, it is preferable that the carboxyl group of lysine is respectively bonded to form -CO-NH- bond as P2 and P5, and when B1 and B2 are groups derived from iminodiacetic acid, The amino group of iminodiacetic acid is bonded to form -CO- bonded as P2 and P5, respectively. It is preferable to be. Specifically, B1 and B2 preferably have the following structures.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formulas 4-1 to 4-9.
  • Formula 4-1 :
  • X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2 and q4 are as defined above.
  • P3 and P6 each independently do not exist, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but preferably -O-CO- or -NH-CO-, and more preferably -NH-CO-.
  • P3 and P6 are, for example, -NH-CO-, they have partial structures of B1-NH-CO-Q2 and B2-NH-CO-Q4, respectively.
  • T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- is preferably -O- or -S-, more preferably -O-.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 5.
  • P1 and P4, P2 and P5, P3 and P6, Q1 and Q3, Q2 and Q4, B1 and B2, T1 and T2, L1 and L2, p1 and p2, q1 and q3, and q2 and q4 in Equation 2. are the same.
  • X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 are as defined above. Further, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 in Formula 5 may be the above-mentioned suitable groups, but P1 is —CO— It is preferable that it is NH-, -NH-CO- or -O-. It is preferable that — (P 2 ⁇ Q 1) q 1 ⁇ in the formula 5 is absent or has any structure represented by the above formulas 3-1 to 3-3.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formulas 6-1 to 6-9.
  • Formula 6-1 :
  • Equation 6-3
  • Equation 6-4
  • X, S3, P3, Q2, T1 and L1 are as defined above.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by any one of the following formulas 7-1 to 7-9.
  • Formula 7-1 :
  • X, L1, L2 and S3 are as defined above.
  • L1 and L2 may be identical or different, and are preferably identical.
  • each alkylene group moiety is introduced with an alkylene chain having a different chain length, or an amide bond or the like is substituted with another bond to form formulas 7-1 to 7
  • Nucleic acid derivatives other than the nucleic acid complex having the structure represented by -9 can also be produced.
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 11.
  • Formula 11 :
  • L1, L2, S1 and S2 are each as defined above, P7 and P8 each independently do not exist, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q5, Q6 and Q7 are each independently absent or alkylene substituted or unsubstituted C 1-12 or - (CH 2 CH 2 O) n8 -CH 2 CH 2 - and is, n8 Is an integer from 0 to 99, B3 is herein referred to as a blanker unit and is any structure represented by the following formula 11-1 and means a bond with Q5 and Q6, respectively, in a broken line: Formula 11-1:
  • substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
  • q5 and q6 are each independently an integer of 0 to 10.
  • P7 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO-S- or -CO-NH-, preferably -O-, -NH-CO- or -CO-NH-, more preferably -O- or -NH-CO- .
  • P7 is, for example, -O-, it has a partial structure of a benzene ring -O-.
  • P8 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO—S— or —CO—NH—, when present, is preferably —CO—O— or —CO—NH—, more preferably —CO—NH—.
  • P8 is, for example, -CO-NH-, it has a partial structure of Q6-CO-NH-.
  • Q5, Q6 and Q7 are each independently absent or alkylene substituted or unsubstituted C 1-12 or - (CH 2 CH 2 O) n8 -CH 2 CH 2 - and is, n8 Is an integer of 0 to 99, preferably substituted or unsubstituted alkylene having 1 to 12 carbon atoms, more preferably unsubstituted alkylene having 1 to 12 carbon atoms, and unsubstituted unsubstituted carbon atoms An alkylene of 1 to 6 is more preferable, and an unsubstituted alkylene of 1 to 4 carbon atoms is still more preferable.
  • q5 - is, -O- (CH 2) m15 -NH- and -NH-CO- (CH 2) a m16 -NH, m15 and m16 are each independently 1 to 10 It is preferable that it is an integer of
  • the nucleic acid complex is preferably a nucleic acid complex having a structure represented by any one of the following formulas 12-1 to 12-12.
  • Formula 12-1 :
  • X, L1, L2, S1 and S2 are as defined above, and n1 'to n12' are each independently an integer of 1 to 10.
  • the nucleic acid complex of the present invention is a nucleic acid complex obtained by combining the structure described in formula 2 corresponding to S1 and S2 with the structure described in formula 11 corresponding to S3 in the nucleic acid complex represented by formula 1 Is preferred.
  • Formula 2 may be Formula 4-1 to Formula 4-9, Formula 6-1 to Formula 6-9, Formula 7-1 to Formula 7-9, and Formula When 2 is Expression 4-1 to Expression 4-9, Expression 6-1 to Expression 6-9, or Expression 7-1 to Expression 7-9, Expression 11 has Expression 12-1 to Expression 12-. It may be twelve.
  • the nucleic acid complex of the present invention is a nucleic acid complex represented by formula 1, which has a structure according to any one of formulas 4-1 to 4-9 corresponding to S1 and S2, and a formula 12 corresponding to S3.
  • a nucleic acid complex combining any one of the structures described in any one of the structures 1 to 12-12, any one of the structures described in the formulas 6-1 to 6-9 corresponding to S1 and S2, and A nucleic acid complex combining any one of the structures described in the corresponding formula 12-1 to the formula 12-12, any one of the structures described in the formula 7-1 to the formula 7-9 corresponding to S1 and S2 More preferably, it is a nucleic acid complex obtained by combining any one of the structures described in formulas 12-1 to 12-12 corresponding to S3.
  • X in Formula 1 is a double-stranded nucleic acid consisting of a sense strand and an antisense strand and containing a double-stranded region of at least 11 base pairs, said double-stranded nucleic acid being in the antisense strand 17 It is complementary to any of the target CFB mRNA sequences described in Table 1-1 to Table 1-3 in oligonucleotide chains of 30 to 30 nucleotides long.
  • X binding to S3 is a sense strand constituting a double-stranded nucleic acid, which will be described later in Table 1-1 to Table 1- 3 or the sense strand represented by the sense strand sequence in Table M1-1 to Table M1-3.
  • a nucleic acid containing a base sequence complementary to CFB mRNA is referred to as an antisense strand nucleic acid
  • a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is also referred to as a sense strand nucleic acid.
  • the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention is a double-stranded nucleic acid having the ability to reduce or stop the expression of the CFB gene when introduced into mammalian cells, and comprises a sense strand and an anti It is a double stranded nucleic acid having a sense strand.
  • the sense strand and the antisense strand have at least 11 base pairs, and at least 17 nucleotides and at most 30 nucleotides, ie, 17 to 30 nucleotides in the antisense strand.
  • An oligonucleotide strand of the following length is complementary to a target CFB mRNA sequence selected from the group described in Tables 1-1 to 1-3.
  • the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention may be any molecule obtained by polymerizing a nucleotide or a molecule having the same function as the nucleotide, for example, a polymer of ribonucleotide And RNA which is a polymer of deoxyribonucleotide, a chimeric nucleic acid consisting of RNA and DNA, and a nucleotide polymer in which at least one nucleotide of these nucleic acids is substituted with a molecule having the same function as the nucleotide.
  • uracil (U) can be read unambiguously as thymine (T).
  • nucleotide derivatives examples include nucleotide derivatives and the like.
  • the nucleotide derivative may be any molecule as long as the nucleotide is modified.
  • the affinity to the complementary strand nucleic acid is selected.
  • a ribonucleotide or a deoxyribonucleotide modified molecule is preferably used.
  • nucleotides examples include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and nucleotides in which at least one of a sugar moiety, a phosphodiester bond and a base has been modified.
  • the sugar moiety-modified nucleotide may be any one in which part or all of the chemical structure of the nucleotide sugar is modified or substituted with an arbitrary substituent or substituted with an arbitrary atom, but '-Modified nucleotides are preferably used.
  • the 2'-modified nucleotides such as 2'-OH group of the ribose is H, OR, R, R'OR, SH, SR, NH 2, NHR, NR 2, N 3, CN, F, Cl, Br and Substituted with a substituent selected from the group consisting of I, wherein R is alkyl or aryl, preferably alkyl of 1 to 6 carbons, and R ′ is alkylene, preferably alkylene of 1 to 6 carbons It is a nucleotide, more preferably a nucleotide in which the 2'-OH group is substituted with H, F or a methoxy group, still more preferably a nucleotide in which the 2'-OH group is substituted with F or a methoxy group.
  • 2′-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) etoxy group and 2-cyanoetoxy group Also included are substituted nucleotides and the like.
  • the content of 2'-modified nucleotides is preferably 50 to 100%, more preferably 70 to 100%, still more preferably 90 to 100% with respect to nucleotides in the double stranded nucleic acid region. .
  • the content of 2′-modified nucleotides is preferably 20 to 100%, more preferably 40 to 100%, still more preferably 60 to 100%, based on the nucleotides of the sense strand.
  • the 2'-modified nucleotide-modified nucleotide is preferably contained in an amount of 20 to 100%, more preferably 40 to 100%, still more preferably 60% to 100%, based on the nucleotides of the antisense strand. More preferable.
  • a phosphodiester bond modified nucleotide any one of those obtained by modifying or substituting a part or all of the chemical structure of phosphodiester bond of the nucleotide with an arbitrary substituent, or substituting with an arbitrary atom
  • a nucleotide in which a phosphodiester bond is substituted with a phosphorothioate bond a nucleotide in which a phosphodiester bond is substituted with a phosphorodithioate bond
  • a nucleotide in which a phosphodiester bond is substituted with an alkyl phosphonate bond phosphoric acid
  • the nucleotide etc. by which the diester bond was substituted by the phosphoroamidate bond etc. are mentioned.
  • any part or all of the chemical structure of the base of the nucleotide may be modified or substituted with any substituent, or any one substituted with any atom, for example, a base
  • the oxygen atom is substituted by a sulfur atom
  • the hydrogen atom is substituted by an alkyl group having 1 to 6 carbon atoms, halogen or the like
  • the methyl group is hydrogen, hydroxymethyl, an alkyl group having 2 to 6 carbon atoms, etc.
  • the amino group is substituted by an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group or the like.
  • nucleotide derivative a nucleotide or a nucleotide derivative in which at least one of sugar moiety, phosphodiester bond or base is modified, peptide, protein, sugar, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, Fluorescein, rhodamine, coumarin, dyes, etc., which are added with another chemical substance directly or through a linker, can also be mentioned, and specifically, 5'-polyamine adduct nucleotide derivative, cholesterol adduct nucleotide derivative, steroid adduct nucleotide derivative And bile acid-added nucleotide derivatives, vitamin-added nucleotide derivatives, Cy5-added nucleotide derivatives, Cy3-added nucleotide derivatives, 6-FAM-added nucleotide derivatives, biotin-added nucleo
  • the nucleotide derivative may form a cross-linked structure, such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.
  • complementary means a relationship capable of base pairing between two bases, for example, a loose hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It refers to one that takes on a double helical structure as a whole double stranded region via a bond.
  • an antisense strand complementary to CFB mRNA means that one or more bases may be substituted in the base sequence completely complementary to the partial base sequence of the mRNA.
  • the antisense strand is 1 to 8, preferably 1 to 6, preferably 1 to 4, 1 to 3, particularly 2 or 1 mismatched bases relative to the target sequence of the target gene. You may have.
  • the antisense strand when the antisense strand is 21 bases long, it may have 6, 5, 4, 3, 2, 2 or 1 mismatched bases with respect to the target sequence of the target gene, The position of the mismatch may be at the 5 'end or 3' end of each sequence.
  • “complementary” includes a case where one nucleotide sequence is a sequence in which one or more bases are added and / or deleted in a base sequence completely complementary to the other nucleotide sequence.
  • CFB mRNA and the antisense strand nucleic acid of the present invention have one or two bulge bases in the antisense strand and / or target CFB mRNA region by addition and / or deletion of bases in the antisense strand. You may
  • the double-stranded nucleic acid as a drug used in the present invention is a nucleic acid containing a base sequence complementary to a part of the base sequence of CFB mRNA and / or a base sequence complementary to the base sequence of the nucleic acid. As long as it contains the nucleic acid, it may be composed of any nucleotide or its derivative.
  • the double-stranded nucleic acid of the present invention comprises at least 11 bases of a nucleic acid containing a base sequence complementary to a target CFB mRNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid.
  • the length of the sequence capable of forming duplexes is usually 11 to 27 bases, preferably 15 to 25 bases, and 15 to 23 bases. Is more preferable, and 17 to 21 bases are more preferable.
  • a nucleic acid containing a base sequence complementary to the target CFB mRNA sequence is used, among which 1 to 3 bases, preferably 1 to 2 bases, Preferably, one in which one base is deleted, substituted or added may be used.
  • the nucleic acid that suppresses the expression of CFB is a nucleic acid that has a base sequence complementary to the target CFB mRNA sequence, and is a single-stranded nucleic acid that suppresses the expression of CFB, or is complementary to the target CFB mRNA sequence
  • a double-stranded nucleic acid consisting of a nucleic acid containing a unique base sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid, and suppressing the expression of CFB is preferably used.
  • the single-stranded antisense strand nucleic acid and the single-stranded antisense strand nucleic acid constituting the double-stranded nucleic acid are the same or different, usually 11 to 30 bases, preferably the same or different 17 to 27 bases And 17 to 25 bases are more preferable, 19 to 25 bases are more preferable, and 21 to 23 bases are even more preferable.
  • the double-stranded region if having an additional nucleotide or nucleotide derivative which does not form a double strand at the 3 'or 5' side following the double-stranded region, the overhang Call it (overhang).
  • the nucleotides constituting the overhang may be ribonucleotides, deoxyribonucleotides or derivatives thereof.
  • a double-stranded nucleic acid having an overhang one having an overhang consisting of 1 to 6 bases, usually 1 to 3 bases, at the 3 'end or 5' end of at least one strand is used, but from 2 bases Those having a protruding portion are preferably used, for example, those having a protruding portion consisting of dTdT (dT represents deoxythymidine) or UU (U represents uridine).
  • the overhang can be present only in the antisense strand, only in the sense strand, and in both the antisense and sense strands, but in the present invention, a double-stranded nucleic acid having an overhang in the antisense strand is preferably used.
  • the antisense strand is a target CFB mRNA described in Tables 1-1 to 1-3 in an oligonucleotide strand consisting of 17 to 30 nucleotides, which includes a double-stranded region followed by an overhang. Fully complementary to any of the sequences.
  • double-stranded nucleic acid of the present invention for example, a nucleic acid molecule (WO 2005/089287) that produces a double-stranded nucleic acid by the action of ribonuclease such as Dicer or the like, or a protrusion at the 3 'end or the 5' end
  • ribonuclease such as Dicer or the like
  • a protrusion at the 3 'end or the 5' end A double stranded nucleic acid which forms a blunt end, a double stranded nucleic acid (US2012 / 0040459) etc. which only the sense strand protruded can also be used.
  • a nucleic acid consisting of the same sequence as the base sequence of the target gene or the base sequence of its complementary strand may be used.
  • a double-stranded nucleic acid consisting of a nucleic acid in which the 5 ′ end or 3 ′ end of the strand has been deleted by 1 to 4 bases and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.
  • the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention is a double-stranded RNA (dsRNA) in which the RNAs form a duplex, and a double-stranded DNA (dsDNA) in which the DNAs form a duplex.
  • dsRNA double-stranded RNA
  • dsDNA double-stranded DNA
  • a hybrid nucleic acid in which RNA and DNA form a duplex a hybrid nucleic acid in which RNA and DNA form a duplex.
  • one or both strands of the double strand may be a chimeric nucleic acid of DNA and RNA.
  • it is a double stranded RNA (dsRNA).
  • the second nucleotide from the 5 'end of the antisense strand of the nucleic acid complex of the present invention is preferably complementary to the second deoxyribonucleotide from the 3' end of the target CFB mRNA sequence, and the 5 'end of the antisense strand More preferably, the 2nd to 7th nucleotides are completely complementary to the 2nd to 7th deoxyribonucleotides from the 3 'end of the target CFB mRNA sequence, and the 2nd to 11th nucleotides from the 5' end of the antisense strand It is further preferred that is completely complementary to the 2nd to 11th deoxyribonucleotides from the 3 'end of the target CFB mRNA sequence.
  • the 11th nucleotide from the 5 'end of the antisense strand in the nucleic acid of the present invention is preferably complementary to the 11th deoxyribonucleotide from the 3' end of the target CFB mRNA sequence, and the 5 'end of the antisense strand More preferably, the 9th to 13th nucleotides are completely complementary to the 9th to 13th deoxyribonucleotides from the 3 'end of the target CFB mRNA sequence, and the 7th to 15th nucleotides from the 5' end of the antisense strand More preferably, it is completely complementary to the 7th to 15th deoxyribonucleotides from the 3 'end of the target CFB mRNA sequence.
  • the antisense strand and the sense strand of the nucleic acid complex of the present invention are based on, for example, the base sequence (SEQ ID NO: 3541) of the cDNA (sense strand) of full length mRNA of human CFB registered as Genbank Accession No. NM — 000042 It can be designed.
  • Double stranded nucleic acids can be designed to interact with target sequences within the CFB gene sequence.
  • sequence of one strand of double stranded nucleic acid is complementary to the target site sequence described above.
  • Double stranded nucleic acids can be chemically synthesized using the methods described herein.
  • RNA may be produced enzymatically or by partial / total organic synthesis, and modified ribonucleotides can be introduced in vitro by enzymatic or organic synthesis.
  • each strand is chemically prepared. Methods for chemically synthesizing RNA molecules are known in the art [see Nucleic Acids Research, 1998, Volume 32, p. 936-948]. In general, double stranded nucleic acids can be synthesized by using solid phase oligonucleotide synthesis methods (e.g., Usman et al., U.S. Patent 5,804,683; U.S. Patent 5,831,071; U.S. Patent No. 5,998,203; U.S. Patent No.
  • Single-stranded nucleic acids are synthesized, deprotected, and prepared using the solid phase phosphoramidite method (see Nucleic Acids Research, 1993, Vol. 30, p. 2435-2443). Desalted on a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ). Oligomers are Amersham Source 15Q columns using a 15 minute linear gradient-1.0 cm. Purified using ion exchange high performance liquid chromatography (IE-HPLC) at .25 cm height (Amersham Pharmacia Biotech, Piscataway, NJ).
  • IE-HPLC ion exchange high performance liquid chromatography
  • Samples are monitored at 260 nm and peaks corresponding to full-length oligonucleotide species are collected, pooled, desalted on a NAP-5 column, and lyophilized.
  • each single stranded nucleic acid is determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).
  • the CE capillary has an internal diameter of 100 ⁇ m and contains ssDNA 100R Gel (Beckman-Coulter).
  • ssDNA 100R Gel (Beckman-Coulter).
  • about 0.6 nmole of oligonucleotide is injected into the capillary and run at an electric field of 444 V / cm, detected by UV absorbance at 260 nm.
  • Denatured Tris-Borate-7 mol / L-Urea running buffer is purchased from Beckman-Coulter.
  • Single stranded nucleic acids that are at least 90% pure as assessed by CE are obtained for use in the experiments described below.
  • Compound identity is determined using the matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass on Voyager DE.TM. Biospectrometry workstation (Applied Biosystems, Foster City, Calif.) According to the manufacturer's recommended protocol. Verified by spectroscopy.
  • the relative molecular mass of single stranded nucleic acid can be obtained within 0.2% of the expected molecular mass.
  • Single stranded nucleic acid is resuspended at a concentration of 100 ⁇ mol / L in a buffer consisting of 100 mmol / L potassium acetate, 30 mmol / L HEPES, pH 7.5.
  • the complementary sense and antisense strands are mixed in equal molar amounts to obtain a final solution of 50 ⁇ mol / L double stranded nucleic acid.
  • the sample is heated to 95 ° C. for 5 minutes and allowed to cool to room temperature before use.
  • Double stranded nucleic acids are stored at -20 ° C.
  • Single stranded nucleic acids are lyophilized or stored at -80 ° C. in nuclease free water.
  • the sense strand and the antisense strand according to the present invention which comprises a duplex region of at least 11 base pairs, in the antisense strand, an oligonucleotide of 11 to 30 nucleotides in length, A group consisting of the antisense strand described in Tables 1-1 to 1-3 as a double-stranded nucleic acid complementary to a target CFB mRNA sequence selected from the group described in 1-1 to Table 1-3 Or a sequence selected from the group consisting of a sense strand described in Table M1-1 to Table M1-3, and Tables 1-1 to 1-3 described later.
  • a pair of senses selected from the group consisting of a double stranded nucleic acid, or a sense strand / antisense strand described in Tables M1-1 to M1-3 and Tables 1-1 to 1-3 described later.
  • Double stranded nucleic acids comprising the strand / antisense strand sequence may be used. That is, specific examples of the double stranded nucleic acid constituting the nucleic acid complex used in the present invention are the sense strand and the anti in the Tables M1-1 to M1-3 and the Tables 1-1 to 1-3 described later. It is a double stranded nucleic acid consisting of a sense strand.
  • N (M) represents 2'-O-methyl-modified RNA
  • N (F) represents 2'-fluorine-modified RNA
  • ⁇ represents phosphorothioate.
  • the 5 'terminal nucleotide of the antisense strand sequence described in Table 1-1 to Table 1-3 and Table M1-1 to Table M1-3 may be phosphorylated at the 5' end, It does not have to be, but is preferably phosphorylated.
  • the double-stranded nucleic acid comprising the sense / antisense strand sequences described in Tables 1-1 to 1-3 described later has a relative expression amount of CFB of 0.40 in the measurement of knockdown activity when 0.1 nM is added. The following is preferred.
  • the method for producing the nucleic acid complex of the present invention will be described.
  • introduction and removal of protecting groups commonly used in synthetic organic chemistry Methods eg, Protective Groups in Organic Synthesis, third edition, by T. Greene, John Wiley & Sons Inc.
  • the target compound can be produced by using the method described in (1999), etc.].
  • the order of reaction processes, such as substituent introduction can also be changed as needed.
  • the nucleic acid polymer represented by Formula 1 can also be synthesized by solid phase synthesis.
  • the nucleic acid polymer represented by the formula 1 can be synthesized with reference to a synthesis method of a linker structure known as a nucleic acid complex.
  • the synthesis of the L1-benzene ring unit using S1 as a linker and the L2-benzene ring unit using S2 as a linker in the nucleic acid complex represented by formula 1 can be performed, for example, using the nucleic acid complex represented by formula 2 as an example. explain.
  • the L1-benzene ring unit and the L2-benzene ring unit in the nucleic acid complex represented by the formula 2 are linked by P1, P2, P3, P4, P5, and P6, and T1 and T2.
  • the partial structure of the L1-benzene ring unit can be produced by combining a compound having Q1 as a partial structure and a compound having B1 as a partial structure sequentially from the benzene ring. Separately synthesizing a compound having L1 and Q2 as a partial structure, and binding a compound having L1 and Q2 as a partial structure to a compound having a partial structure of an L1-benzene ring unit having a benzene ring and Q1 and B1 as a partial structure
  • L 1 -benzene ring unit structure can be produced.
  • the partial structure of L2-benzene ring unit can be produced by sequentially binding a compound having Q3 as a partial structure and a compound having B2 as a partial structure sequentially from the benzene ring . Separately synthesizing a compound having L2 and Q4 as a partial structure, and combining a compound having L2 and Q4 as a partial structure with a compound having a partial structure of L2-benzene ring unit having a benzene ring and Q3 and B2 as partial structures Thus, an L2-benzene ring unit structure can be produced.
  • an alkylene having 1 to 10 carbon atoms or a — (CH 2 CH 2 O) n —CH 2 CH 2 — terminal group is a hydroxyl group or a carboxyl group at both ends
  • compounds having an amino group and a thiol group are represented by the compound having B1 as a partial structure and the compound having B2 as a partial structure.
  • the compound having B1 as a partial structure and the compound having B2 as a partial structure have one of the structures represented by the following formula 2-1, and a hydroxyl group and a carboxyl group are represented by the black dots at the end of each structure, respectively.
  • compounds having an amino group or a thiol group are represented by the black dots at the end of each structure, respectively.
  • the compound having B1 as a partial structure and the compound having B2 as a partial structure include glycol, glutamic acid, aspartic acid, lysine, Tris, iminodiacetic acid, 2-amino-1,3-propanediol and the like. Glutamic acid, aspartic acid, lysine and iminodiacetic acid are preferred.
  • B1 and B2 preferably have the following structures.
  • a compound having L1, Q2 and B1 as a partial structure may be synthesized and then coupled to a compound having Q1 and a benzene ring to produce an L1-benzene ring unit structure.
  • a compound having L2, Q4 and B2 as a partial structure may be synthesized and then combined with Q3 and a compound having a benzene ring to produce an L2-benzene ring unit structure.
  • [L1-T1- (Q2- P3) q2 -] p1 -B1- and substructure is (P2-Q1) q1 a -P1-, [L2-T2- (Q3 -P6) q4 -]
  • the partial structure which is p2- B2- (P5-Q3) q3- P2- may be identical or different, but is preferably identical.
  • Examples of the unit corresponding to L1-T1-Q2 of the sugar ligand include L3-T1-Q2-COOH, L3-T1- (Q2-P3) q2-1 -Q2-NH 2 and the like.
  • L3-O-alkylene-COOH having 1 to 12 carbon atoms L-alkylene-CO-NH having 1 to 12 carbons, alkylene-NH 2 having 2 to 12 carbons, etc. may be mentioned.
  • L3 is not particularly limited as long as it is a sugar ligand derivative that becomes L1 by deprotecting.
  • the substituent of the sugar ligand is not particularly limited as long as it is a substituent generally used in the field of carbohydrate chemistry, but an Ac group is preferable.
  • the number of carbon atoms of the alkylene chain is appropriately increased or decreased, with reference to the method described in the Examples, and synthesis of L1-benzene ring unit using S1 as a linker and L2 benzene ring unit using S2 as a linker
  • the terminal amino group and the terminal carboxyl group can be selected from -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-
  • the compound can be synthesized by using a compound converted into a group capable of forming a -CO-S- or -CO-NH- bond.
  • mannose or N-acetylgalactosamine is exemplified in the examples also for L1 sugar ligands, but other sugar ligands can be used instead.
  • the synthesis of the X-benzene ring unit using the S3 as a linker in the nucleic acid complex represented by the formula 1 will be described, for example, by taking the nucleic acid complex represented by the formula 12 as an example.
  • the X-benzene ring unit in the nucleic acid complex represented by Formula 12 has a bond represented by P7 and P8 in addition to the bond of the oligonucleotide.
  • the compound can be appropriately synthesized by selecting an appropriate raw material for forming the structure represented by Formula 12 with reference to the method of the binding reaction described in 1.).
  • the partial structure of the X-benzene ring unit can be produced by combining a compound having Q5 as a partial structure and a compound having B3 as a partial structure sequentially from the benzene ring.
  • a compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure is separately synthesized, and a compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure is a benzene ring and Q5
  • An X-benzene ring unit structure can be produced by combining with a compound having a partial structure of the X-benzene ring unit having a partial structure to construct a B3 portion.
  • an X-benzene ring unit structure can be produced by reacting a formed oligonucleotide to cause cycloaddition to form a triazole ring to construct a B3 moiety.
  • a compound having Q5 as a partial structure a compound having Q6 as a partial structure, and a compound having Q7 as a partial structure, an alkylene having 1 to 10 carbon atoms or- (CH 2 CH 2 O) n 8 -CH 2 CH 2-
  • the compound which has a hydroxyl group, a carboxyl group, an amino group, and a thiol group is mentioned at both ends.
  • the L1-benzene ring unit structure, the L2-benzene ring unit structure, and the X-benzene ring unit structure can be sequentially produced, but the L1-benzene ring unit structure and the L2-benzene ring unit structure are synthesized. Then, it is preferable to combine the X-benzene ring unit structure.
  • X having an oligonucleotide moiety is preferably introduced into the compound near the final step of sugar ligand complex synthesis.
  • R1 and R2 are each independently a hydrogen atom, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group), 9-fluorenylmethyloxycarbonyl group (Fmoc group), -CO-R4 Or -CO-B4-[(P9-Q8) q7- T3-L3] p3
  • P9 and T3 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-
  • Q8 is absent or substituted or unsubstituted alkylene having 1 to 12 carbon atoms, or-(CH 2 CH 2 O) n 1 -CH 2 CH 2- , and n 1 is an integer of 0 to 99
  • B4 is each independently a bond or a structure represented by Formula 8-1 below, and
  • p3 is an integer of 1, 2 or 3 and q7 is an integer of 0 to 10
  • L3 is a sugar ligand
  • Y is —O— (CH 2 ) m 11 —NH— and —NH—CO— (CH 2 ) m 12 —NH
  • m 11 and m 12 are each independently an integer of 1 to 10
  • R3 is a hydrogen atom, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, -CO-R4, -CO- (CH 2 CH 2 O) n2 -CH 2 CH 2 -N 3 or -CO-Q9- B5- (Q10-P10) q8- X1, and n2 is an integer of 0 to 99
  • P10 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO
  • substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
  • q8 is an integer of 0 to 10
  • X 1 is a hydrogen atom or a solid support
  • R 4 is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group It is an alkyl group having 2 to 10 carbon atoms which is substituted by 1 or 2 substituents. )
  • R5 and R6 each independently represent a hydrogen atom, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, -CO-R4 ', or -CO-Q11- (P11-Q11) ') Q9 -T4-L4,
  • P11 and T4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-
  • Q11 and Q11 ′ are absent or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or- (CH 2 CH 2 O) n 4 -CH 2 CH 2- , and n 4 is an integer of 0 to 99
  • q9 is an integer of 0 to 10
  • L4 is a sugar ligand
  • substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
  • q8 ' is an integer of 0 to 10
  • X1 ' is a hydrogen atom or a solid support
  • R 4 ′ is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group An alkyl group of 2 to 10 carbon atoms substituted with one or two substituents.
  • R7 and R8 are each independently a hydroxy group, a t-butoxy group, a benzyloxy group, -NH-R10 or -NH-Q12- (P12-Q12 ') q10- T4-L4, P12 and T4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q12 and Q12 'is absent or alkylene substituted or unsubstituted C 1-12 or - (CH 2 CH 2 O) n2-CH 2 CH 2 - and is, n2 is an integer 0-99
  • L4 is a sugar ligand
  • Y 2 is —O— (CH 2 ) m 9 —NH— and —NH—CO— (CH 2 ) m 10 —NH
  • m 9 and m 10 are
  • substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
  • q11 is an integer of 0 to 10
  • X2 is a hydrogen atom or a solid support
  • R 10 is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group It is an alkyl group having 2 to 10 carbon atoms which is substituted by 1 or 2 substituents. )
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (I ′).
  • P 1 represents a protecting group which can be deprotected by a base such as Fmoc
  • DMTr represents p, p′-dimethoxytrityl group
  • R represents a sugar ligand-tether unit
  • R ′ represents R in Each hydroxyl group of the sugar ligand is a group protected by a protecting group which can be deprotected by a base such as an acetyl group
  • Polymer is a solid phase carrier
  • Q ' is -CO-.
  • Step 1 Compound (I-B) is a compound (IA) and p, p'-dimethoxytrityl chloride in a solvent such as pyridine, optionally in the presence of a cosolvent, at a temperature between 0 ° C. and 100 ° C. Can be produced by reacting for 5 minutes to 100 hours.
  • a solvent such as pyridine
  • co-solvent for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide (DMF) And N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, and these may be used alone or in combination.
  • DMF N-dimethylformamide
  • N-dimethylacetamide N-methylpyrrolidone
  • pyridine water and the like
  • Step 2 Compound (IC) is reacted with Compound (IB) without solvent or in the presence of 1 to 1000 equivalents of a secondary amine in a solvent at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
  • a solvent for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide (DMF), Examples thereof include N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, which may be used alone or in combination.
  • secondary amines include diethylamine, piperidine and the like.
  • Step 3 Compound (1-E) is a compound (IC) and compound (ID), 1 to 30 equivalents of a base, a condensing agent, and optionally 0.01 to 30 equivalents without solvent or in a solvent C. for 5 minutes to 100 hours at a temperature between room temperature and 200.degree. C. in the presence of an additive.
  • a base for example, cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo [5.4.
  • DBU 1,3-dicyclohexanecarbodiimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide ⁇ hydrochloride
  • carbonyldiimidazole benzotriazol-1-yloxytris (Dimethylamino) phosphonium hexaflurophosphate, (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate, O- (7-azabenzotriazol-1-yl) -N, N, N ′, N '-Tetramethyluronium hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -N, N, N', N'-tetramethyl
  • additive examples include 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP) and the like.
  • HOBt 1-hydroxybenzotriazole
  • DMAP 4-dimethylaminopyridine
  • ID can be obtained by a known method (see, for example, Journal of American Chemical Society, 136, 16958, (2014)) or a method analogous thereto.
  • Step 4 Compound (IF) is prepared by reacting Compound (IE) and succinic anhydride in a solvent in the presence of 1 to 30 equivalents of a base at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
  • IE Compound (IE)
  • succinic anhydride succinic anhydride
  • step 2 As the solvent, those exemplified in step 2 can be mentioned.
  • Step 5 The compound (IG) is obtained by using a compound (IF) and a terminally aminated solid phase carrier, 1 to 30 equivalents of a base, a condensing agent, and optionally 0. After reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of 01 to 30 equivalents of additive, for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. It can be produced by reaction.
  • the solvent those exemplified in step 2 can be mentioned.
  • the base, the condensing agent and the additive include those exemplified in Step 3.
  • the aminated solid phase carrier include long-chain alkylamine pore glass (LCAA-CPG) and the like, which can be obtained as commercial products.
  • the nucleic acid complex having a sugar ligand-tether-brancher unit represented by the formula (I ′) is a compound (IG) after extending the corresponding nucleotide chain by a known oligonucleotide chemical synthesis method. It can be produced by removal from the solid phase, deprotection of the protective group and purification.
  • oligonucleotide chemical synthesis methods include the phosphoroamidite method, the phosphorothioate method, the phosphotriester method, the CEM method (see Nucleic Acids Research, 35, 3287 (2007)), and the like, for example, ABI 3900 high. It can be synthesized by a throughput nucleic acid synthesizer (manufactured by Applied Biosystems).
  • Removal from solid phase, deprotection may be prepared by treatment with a base for 10 seconds to 72 hours after chemical synthesis of oligonucleotide in a solvent or in the absence of solvent, at a temperature between -80 ° C and 200 ° C. it can.
  • ammonia for example, ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), carbonate Potassium and the like can be mentioned.
  • DBU 1,8-diazabicyclo [5.4.0] -7-undecene
  • Examples of the solvent include water, methanol, ethanol, THF and the like.
  • oligonucleotide Purification of the oligonucleotide is possible by combining a C18 reverse phase column or an anion exchange column, preferably the two methods described above.
  • the nucleic acid complex purity after purification is desirably 90% or more, preferably 95% or more.
  • the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (IC).
  • the compound in step 3 Ethyl ester of the compound obtained by condensing (IC) and CH 3 CH 2 -O-CO-Q 4 '-CO-OH (Q 4' is as defined above) in the same manner as in step 3.
  • the compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH 2 (R ′ is as defined above) to give the desired compound.
  • a base such as lithium hydroxide
  • R′ is as defined above
  • CH 3 CH 2 -O-CO -Q4'-CO-OH Q4 ' is as defined above
  • R'-NH 2 R' is as defined above
  • Q 4 ′ the substituted or unsubstituted C 1 to C 12 alkylene substituent and alkylene moiety are as defined above.
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (II ′).
  • TBDMS represents a t-butyldimethylsilyl group
  • Fmoc represents a 9-fluorenylmethyloxycarbonyl group
  • Step 7 Compound (II-A) can be prepared by reacting compound (IA), t-butyldimethylsilyl chloride and dimethylaminopyridine in a solvent such as N, N-dimethylformamide (DMF), preferably in the presence of 2 equivalents of a base The reaction can be carried out at a temperature between 0 ° C. and 100 ° C. for 5 minutes to 100 hours.
  • a solvent such as N, N-dimethylformamide (DMF)
  • a base those exemplified in Step 3 of Production Method 1 can be mentioned.
  • Step 8 Compound (II-B) can be produced using compound (II-A) under the same conditions as in step 1 of production method 1.
  • Step 9 Compound (II-C) is produced by reacting compound (II-B) with n-tetrabutylammonium fluoride (TBAF) in a solvent at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours. can do.
  • TBAF n-tetrabutylammonium fluoride
  • Step 10 Compound (II-D) can be produced using compound (II-C) under the same conditions as in step 2 of production method 1.
  • Step 11 Compound (II-E) can be produced using compound (II-D) and compound (ID) under the same conditions as in step 3 of production method 1.
  • Steps 12 to 14 Compound (II ′) can be produced using compound (II-E) under the same conditions as steps 4 to 6 of production method 1.
  • the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (II-C).
  • R-Q ' is -NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms)
  • compound (II- C) and CH 3 CH 2 -O-CO-Q 4 '-CO-OH (Q 4' is as defined above) are condensed in the same manner as in step 11, and the ethyl ester of the obtained compound is ethanol or After hydrolysis with a base such as lithium hydroxide in a solvent such as water, the compound is further condensed with R′—NH 2 (R ′ is as defined above) to give the desired compound.
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (III ′).
  • Compound (III ′) can be produced using compound (III-A) under the same conditions as steps 1 to 6 of production method 1.
  • Compound (III-A) can be obtained as a commercial product.
  • Step 15 Compound (III-B) can be produced using compound (III-A) under the same conditions as in step 1 of production method 1.
  • Compound (III-A) can be purchased as a commercial product.
  • Step 16 Compound (III-C) can be produced using compound (III-B) under the same conditions as in step 2 of production method 1.
  • Step 17 Compound (III-E) can be produced using compound (III-C) under the same conditions as in step 3 of production method 1.
  • Steps 18 to 20 Compound (III ′) can be produced using compound (III-E) under the same conditions as in Steps 4 to 6 of Production Method 1.
  • the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (III-C).
  • the compound (III-) is C) and CH 3 CH 2 -O-CO-Q 4 '-CO-OH (wherein Q 4' is as defined above) are condensed in the same manner as in step 17 to obtain ethyl ester of the obtained compound as ethanol or After hydrolysis with a base such as lithium hydroxide in a solvent such as water, the compound is further condensed with R′—NH 2 (R ′ is as defined above) to give the desired compound.
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (IV ′).
  • Compound (IV ′) can be produced using compound (IV-A) under the same conditions as steps 1 to 6 of production method 1.
  • Compound (IV-A) can be obtained as a commercial product.
  • compound (ID) can be divided into two units, divided into two steps, and condensed with compound (IV-C).
  • compound (IV-C) when R'-Q 'is -NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms), the compound in step 23 (IV-C) and CH 3 CH 2 —O—CO—Q 4 ′ —CO—OH (Q 4 ′ is as defined above) are condensed in the same manner as in step 23 to obtain ethyl ester of the compound obtained
  • the compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH 2 (R ′ is as defined above) to give the desired compound.
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (V ′).
  • Compound (V ′) can be produced using compound (IV-A) under the same conditions as steps 1 to 7 of production method 2.
  • Compound (IV-A) can be obtained as a commercial product.
  • the compound (ID) can also be divided into two units if necessary, divided into two steps, and condensed with the compound (VD).
  • the compound in step 31 (VD) and CH 3 CH 2 —O—CO—Q 4 ′ —CO—OH (Q 4 ′ is as defined above) are condensed in the same manner as in step 31 to obtain ethyl ester of the compound obtained
  • the compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH 2 (R ′ is as defined above) to give the desired compound.
  • Manufacturing method 6 The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.
  • Step 35 Compound (I-H) comprises compound (II-E) and 2-cyanoethyl-N, N, N ′, N′-tetraisopropylphosphodiamidite in the presence of a base and a reaction accelerator either in the absence or in the presence of a solvent.
  • a reaction accelerator either in the absence or in the presence of a solvent.
  • C. at a temperature between room temperature and 200.degree. C., for 5 minutes to 100 hours.
  • the solvent those exemplified in step 2 of production method 1 can be mentioned.
  • As the base those exemplified in step 3 of production method 1 can be mentioned.
  • reaction accelerators include 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthiotetrazole, 5-benzylthiotetrazole and the like, and they can be purchased as commercial products.
  • Step 36 The oligonucleotide chain is extended, and finally the compound (IH) is used to modify the 5 'end of the oligonucleotide with a sugar ligand-tether-brancher unit, followed by removal from the solid phase, deprotection of the protecting group Compound (I ′ ′) can be produced by performing and purification.
  • the removal from the solid phase, the deprotection of the protective group and the purification can be carried out in the same manner as in step 7 of production method 1, respectively.
  • Manufacturing method 7 The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.
  • Manufacturing method 8 The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.
  • Manufacturing method 9 The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.
  • Manufacturing method 10 The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.
  • Manufacturing method 11 Water or a suitable buffer of the sense strand having a sugar ligand-tether-brancher unit at the 3 'end or 5' end of the sense strand constituting the double stranded nucleic acid, and the antisense strand constituting the double stranded nucleic acid
  • a nucleic acid complex having double-stranded nucleic acid can be obtained by dissolving each in a liquid and mixing.
  • the buffer include acetate buffer, Tris buffer, citrate buffer, phosphate buffer, water and the like, and these may be used alone or in combination.
  • the mixing ratio of the sense strand to the antisense strand is preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, and 0.95 equivalents to 1 equivalent of the antisense strand per equivalent of the sense strand. More preferred is .05 equivalents.
  • an annealing treatment may be appropriately performed.
  • the annealing treatment is carried out by heating the mixture of sense strand and antisense strand to preferably 50 to 100 ° C., more preferably 60 to 100 ° C., still more preferably 80 to 100 ° C., and then gradually cooling to room temperature.
  • the annealing treatment is carried out by heating the mixture of sense strand and antisense strand to preferably 50 to 100 ° C., more preferably 60 to 100 ° C., still more preferably 80 to 100 ° C., and then gradually cooling to room temperature.
  • the antisense strand can be obtained according to the known oligonucleotide synthesis method described above.
  • the nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (VI ′).
  • TBS represents a t-butyldimethylsilyl group
  • R 0 and R x are the same or different, and are hydrogen atoms
  • W is a C1-C10 alkylene, a C3-C8 cycloalkylene, or together with R0, a C4-C8 nitrogen-containing heterocyclic ring You may form.
  • Step 45 Compound (VI-B) can be produced using compound (VI-A) under the same conditions as in step 1 of production method 1.
  • Compound (VI-A) can be obtained as a commercially available product, or by a known method (eg, Bioorganic & Medical Chemistry Letters, 11: 383-386) or a method analogous thereto.
  • Step 46 Compound (VI-C) can be produced using compound (VI-B) under the same conditions as in step 2 of production method 1.
  • Step 47 Compound (VI-D) can be produced using compound (VI-C) under the same conditions as in step 3 of production method 1.
  • Step 48 Compound (VI-E) can be produced using compound (VI-D) under the same conditions as in step 2 of production method 1.
  • Step 49 Compound (VI-G) can be produced under the same conditions as in step 3 of production method 1 using compound (VI-E) and compound (VI-F).
  • Step 50 Compound (VI-H) can be produced using compound (VI-G) under the same conditions as in step 9 of production method 2.
  • Steps 51-53 Compound (VI ′) can be produced using compound (VI-H), compound (VI-I) and compound (VI-J) under the same conditions as in steps 4 to 6 of production method 1.
  • Steps 45 to 53 can be carried out by a known method (for example, the method described in WO 2015/105083) or a method analogous thereto.
  • Compound (VI-F) can be obtained by a known method (for example, a method described in Journal of American Chemical Society, 136, 16958, Trio, 2014), or a method according thereto be able to.
  • the sugar ligand-tether unit in formula 2 wherein P1 and P4 are -NH-CO-, -O-CO- or -S-CO- can be produced by the following method.
  • q 2 ′ is represents an integer smaller than 1 by q2
  • q4 ' represents an integer smaller by 1 than q4
  • Z represents H, OH, NH 2 , SH, a chlorine atom, a bromine atom, an iodine atom, methanesulfonyloxy, p-toluenesulfonyloxy or a carboxylic acid
  • B1 'and B2' are any of the structures of the following formulas And PG1, PG2, PG3, PG4, PG5, PG6 and PG7 each represent a suitable protecting group.
  • n1, m2, m3 or m4 each independently represent an integer of 0 to 10.
  • Step 54 Compound (VII-C) is obtained by adding Compound (VII-A) and Compound (VII-B) in a solvent such as tetrohydrofuran etc, triphenylphosphine polymer support, and under ice-cooling, diisopropylazodicarboxylate toluene It can be produced by reacting a solution.
  • a solvent such as tetrohydrofuran etc, triphenylphosphine polymer support, and under ice-cooling, diisopropylazodicarboxylate toluene It can be produced by reacting a solution.
  • a solvent such as tetrohydrofuran etc, triphenylphosphine polymer support, and under ice-cooling, diisopropylazodicarboxylate toluene It can be produced by reacting a solution.
  • the solvent those exemplified in step 2 of production process 1 can be mentioned.
  • Compound (VII-A) can
  • Step 55 Compound (VII-D) can be produced by reacting Compound (VII-C) in a solvent such as methanol under ice-cooling in the presence of a base.
  • a solvent such as methanol
  • a base those exemplified in Step 3 of Production Process 1 can be mentioned.
  • Step 56 Compound (VII-F) can be produced using compound (VII-D) and compound (VII-E) under the same conditions as in step 3 of production process 1.
  • Step 57 Compound (VII-H) can be produced using compound (VII-F) and compound (VII-G) under the same conditions as in step 3 of production process 1.
  • Step 58 Compound (VII-J) can be produced using compound (VII-H) and compound (VII-I) under the same conditions as in step 3 of production process 1. Also, by repeatedly performing the DP step and the step 58, a compound (VII-J) having a desired value of q1 can be produced.
  • Step 59 Compound (VII-L) can be produced using compound (VII-J) and compound (VII-K) under the same conditions as in step 3 of production process 1.
  • Step 60 Compound (VII-N) can be produced using compound (VII-L) and compound (VII-M) under the same conditions as in step 3 of production process 1.
  • Steps 61 to 63 Compound (VII ′) can be produced using compound (VII-O), compound (VII-P) and compound (VII-Q) under the same conditions as in step 3 of production process 1. Also, by repeatedly performing the DP step and the step 61, a compound (VII ′) having a desired q3 value can be produced.
  • Manufacturing method 14 A unit in which P7 is -O- in Formula 4 can be manufactured by the following method.
  • q5 '' represents an integer smaller than 2 by q5
  • Z2 represents H, OH, NH 2 or SH
  • PG8 and PG9 each represent a suitable protecting group
  • LC represents a sugar ligand-tether unit
  • E represents a carboxylic acid or maleimide.
  • Step 64 Compound (VIII-C) can be produced using compound (VIII-A) and compound (VIII-B) under the same conditions as in step 3 of production process 1. Also, by repeatedly performing the DP step and the step 64, a compound (VIII-C) having a desired q5 ′ ′ value can be produced.
  • Compound (VIII-B) is commercially available, or “Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)”, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 th It can obtain by combining the method as described in Edition, or the method according to it.
  • Step 65 Compound (VIII ′) can be produced using compound (VIII-C) and compound (VIII-D) under the same conditions as in step 3 of production process 1.
  • Compound (VIII-D) is commercially available, or “Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)”, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 th It can obtain by combining the method as described in Edition, or the method according to it.
  • a sugar ligand-tether unit in which P1 and P4 in the formula 2 are -O- can be produced by the following method.
  • n1, m2, m3 and m4 are as defined above.
  • Step 66 Compound (IX-C) is prepared by dissolving Compound (IX-A) and Compound (IX-B) in a solvent such as N, N′-dimethylformamide, adding a base such as potassium hydrogen carbonate, and the like to room temperature to 200 ° C. The reaction can be carried out by reacting for 5 minutes to 100 hours.
  • a solvent such as N, N′-dimethylformamide
  • a base such as potassium hydrogen carbonate, and the like
  • Step 67 Compound (IX-E) is prepared by dissolving Compound (IX-C) and Compound (IX-D) in a solvent such as N, N′-dimethylformamide, adding a base such as potassium hydrogen carbonate, and the like to room temperature to 200 ° C. The reaction can be carried out by reacting for 5 minutes to 100 hours.
  • a solvent such as N, N′-dimethylformamide
  • a base such as potassium hydrogen carbonate, and the like
  • the reaction can be carried out by reacting for 5 minutes to 100 hours.
  • As the solvent those exemplified in step 2 of production process 1 can be mentioned.
  • As the base those exemplified in Step 3 of Production Process 1 can be mentioned.
  • Compound (IX-A) can be obtained as a commercial product.
  • Step 68 Compound (IX-G) can be produced using compound (IX-E) and compound (IX-F) under the same conditions as in step 3 of production process 1.
  • Step 69 Compound (IX-I) can be produced using compound (IX-G) and compound (IX-H) under the same conditions as in step 3 of production process 1. Also, by repeatedly performing the DP step and the step 69, it is possible to produce the compound (VII-J) having a desired value of q1.
  • Step 70 Compound (IX-K) can be produced using compound (IX-I) and compound (IX-J) under the same conditions as in step 3 of production process 1.
  • Step 71 Compound (IX-M) can be produced using compound (IX-K) and compound (IX-L) under the same conditions as in step 3 of production process 1.
  • Steps 72-74 The compound (IX ′) can be produced using compound (IX-M), compound (IX-N), compound (IX-O) and compound (IX-P) under the same conditions as in step 3 of production process 1. Can. Also, by repeatedly performing the DP step and the step 72, the compound (IX ′) having a desired value of q3 can be produced.
  • Compound (IX'-B), Compound (IX'-D), Compound (IX'-F), Compound (IX'-H), Compound (IX'-J), Compound (IX'-L), Compound (IX) IX'-N), compound (IX'-O) and compound (IX'-P) are commercially available products, or "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)", "March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 th Edition "combining the methods described, or can be obtained by a method analogous thereto.
  • Manufacturing method 16 The following method can also be used as a method for producing the nucleic acid complex of Formula 1 to Formula 8.
  • Step 75 Compound (XB) can be produced by reacting compound (XIII ′ ′) with compound (XA) in a solvent at 0 ° C. to 100 ° C. for 10 seconds to 100 hours.
  • the solvent include water, phosphate buffer, sodium acetate buffer, dimethyl sulfoxide and the like, and these can be used alone or in combination.
  • Compound (VIII ′) can be obtained by using production method 14.
  • the compound (X-A) can be prepared by a known method (for example, Bioconjugate Chemistry, Vol. 21, pp. 187-202, 2010, or Current Protocols in Nucleic Acid Chemistry (Current Protocols) in Nucleic Acid Chemistry), September, 2010; CHAPTER: Unit 4.41) or an equivalent method.
  • Step 76 The compound (X ′) is produced by reacting the compound (X-B) at a temperature of room temperature and 200 ° C. for 5 minutes to 100 hours under conditions of pH 8 or more such as aqueous sodium carbonate solution or ammonia water Can.
  • Manufacturing method 17 The following method can also be used as a method for producing the nucleic acid complex of Formula 1 to Formula 8.
  • Step 77 Compound (XI-A) can be produced by a known method (for example, the method described in Bioconjugate Chemistry, Vol. 26, pp. 1451-1455, 2015) or the compound using Compound (VIII ′ ′) It can be obtained by a similar method. Compound (VIII ′ ′ ′) can be obtained by using production method 14.
  • Step 78 The compound (XI ′) can be produced using a compound (XI-A) and a compound (XI-B) according to a known method (eg, Bioconjugate Chemistry, vol. 26, p. 1451-1455, 2015) The methods described) or can be obtained by methods analogous thereto.
  • Compound (XI-B) can be obtained by the method described in Bioconjugate Chemistry, vol. 26, p. 1451-1455 (2015) or a method analogous thereto.
  • Step 79 As another method, compound (XI ′) can be obtained by a known method (see, for example, Bioconjugate Chemistry, 22: 1723-1728, 2011) or a method according thereto. It can be obtained directly from XI-A).
  • the nucleic acid complex in the present specification can also be obtained as a salt such as an acid addition salt, a metal salt, an ammonium salt, an organic amine addition salt, an amino acid addition salt and the like.
  • acid addition salts include inorganic acid salts such as hydrochlorides, sulfates and phosphates, and organic acid salts such as acetates, maleates, fumarates, citrates and methanesulfonates.
  • metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts and the like, and examples of ammonium salts include ammonium and tetramethyl Salts such as ammonium may be mentioned, organic amine addition salts may include addition salts such as morpholine and piperidine, and amino acid addition salts may include addition salts such as lysine, glycine and phenylalanine.
  • the complex When it is desired to prepare a salt of the nucleic acid complex of the present invention, the complex may be purified as it is when it is obtained in the form of the desired salt, or when it is obtained in free form, the complex Is dissolved or suspended in an appropriate solvent, the corresponding acid or base is added, and isolation and purification may be performed.
  • the complex salt when converting the counter ion that forms the complex salt to a different counter ion, the complex salt is dissolved or suspended in an appropriate solvent, and then an acid, a base and / or a salt (sodium chloride, chloride It may be isolated and purified by adding several equivalent to a large excess amount of an inorganic salt such as ammonium).
  • nucleic acid complexes of the present invention may have geometric isomers, stereoisomers such as optical isomers, tautomers etc., all possible isomers and their mixtures are also possible. Included in the present invention.
  • nucleic acid complex of the present invention may exist in the form of an adduct with water or various solvents, and these adducts are also included in the present invention.
  • nucleic acid complex of the present invention also includes those in which a part or all of the atoms in the molecule are substituted by atoms (isotopes) having different mass numbers (for example, deuterium atoms etc.).
  • the pharmaceutical composition of the present invention comprises the nucleic acid complex represented by Formula 1.
  • the nucleic acid complex of the present invention is recognized by the target cell by having the L1 and L2 sugar ligands, and is introduced into the cell.
  • the nucleic acid complex of the present invention can be administered to a mammal to suppress or reduce the expression of a target gene in vivo, and can be used to treat a disease associated with the target gene.
  • the nucleic acid complex of the present invention is used as a therapeutic agent or a prophylactic agent, it is desirable to use the most effective administration route for treatment as a therapeutic agent or a prophylactic agent, and it is not particularly limited.
  • Administration, subcutaneous administration, intramuscular administration and the like can be mentioned, preferably subcutaneous administration.
  • the dose varies depending on the condition, age, administration route and the like of the administration subject, it may be administered, for example, such that the daily dose converted to double-stranded oligonucleotide is 0.1 ⁇ g to 1000 mg, daily administration More preferably, the amount is 1 to 100 mg.
  • preparations suitable for intravenous administration or intramuscular administration include injections, and it is possible to use the prepared solution as it is, for example, in the form of injections, etc.
  • the solvent is removed and used by lyophilization, the solution is used by lyophilization, and / or the solution with an excipient such as mannitol, lactose, trehalose, maltose or glycine is used by lyophilization. It can also be done.
  • an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA, or an isotonic agent such as glycerin, glucose or sodium chloride.
  • an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA
  • an isotonic agent such as glycerin, glucose or sodium chloride.
  • a cryopreservative such as glycerin can be added and cryopreserved.
  • the double stranded nucleic acid in the composition of the present invention can be introduced into cells by administering the composition of the present invention to mammalian cells.
  • the introduction of the nucleic acid complex of the present invention into mammalian cells in vivo may be carried out according to known transfection procedures which can be carried out in vivo.
  • the composition of the present invention can be delivered to the liver by intravenous administration to mammals including humans, and the double stranded nucleic acid in the composition of the present invention can be introduced into the liver or hepatocytes.
  • the expression of the CFB gene in the hepatocytes is reduced to treat a disorder mediated by a complement pathway abnormality. Or it can be prevented.
  • CFB related diseases include atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 nephritis, Membrane nephropathy, asthma, autoimmune diseases (eg, systemic lupus erythematosus (SLE), psoriasis, neuromyelitis optica, myasthenia gravis, etc.) and the like can be mentioned.
  • the administration subject is a mammal, preferably a human.
  • the dose varies depending on the condition, age, administration route and the like of the administration subject, it may be administered, for example, such that the daily dose converted to double-stranded nucleic acid is 0.1 ⁇ g to 1000 mg. It is preferable to administer to 1 to 100 mg.
  • the invention also relates to nucleic acid complexes for use in the treatment of diseases; pharmaceutical compositions for use in the treatment of diseases; use of nucleic acid complexes for the treatment of diseases; in the manufacture of a medicament for the treatment of diseases
  • a nucleic acid complex for use in the manufacture of a medicament for treating a disease a method for treating or preventing a disease comprising administering an effective amount of the nucleic acid complex to a subject in need thereof; provide.
  • Mobile phase A aqueous solution containing 0.1% formic acid
  • B acetonitrile solution gradient: linear gradient (10 minutes-90%) of mobile phase B (3 minutes)
  • Synthesis step 1 of compound RE1-4 The compound RE1-1 (0.9602 g, 2.1460 mmol) is dissolved in N, N'-dimethylformamide (10 mL), N-Boc-ethylenediamine (manufactured by Sigma-Aldrich, 0.6877 g, 4.292 mmol), diisopropylethylamine (1.90 mL) , 10.87 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (manufactured by Wako Pure Chemical Industries, Ltd., 1.6437 g, 4.3229 mmol) was added and stirred overnight at room temperature.
  • Reference Example 3 Step 7 Reference Example 3 Compound RE 3-7 (180 mg, 0.406 mmol) synthesized in step 6 N ⁇ , N ⁇ -bis (tert-butoxycarbonyl) -L-lysine (manufactured by Nova Biochem, 295 mg, 0.852 mmol), diisopropylethylamine ( Add 0.354 mL, 2.029 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (324 mg, 0.852 mmol) at room temperature And stirred overnight. The reaction mixture was ice-cooled, 10% aqueous citric acid solution was added, and the mixture was extracted with chloroform.
  • Reference Example 4 Step 1 Compound RE4-1 synthesized by the method described in Reference Example 3 (compound RE3-5, 0.5716 g, 0.7582 mmol in Reference Example 3), Bioconjugate Chemistry, Volume 22, pp. 690-699, Dodecanoic acid monobenzyl ester (0.4859 g, 1.5164 mmol), diisopropylethylamine (0.662 mL, 3.79 mmol) synthesized by the method described in 2011, and 2- (1H-benzotriazol-1-yl) -1,1 3,3,3-tetramethyluronium hexafluorophosphate (0.5766 g, 1.516 mmol) was dissolved in N, N dimethylformamide (12 mL) and stirred at room temperature for 1 hour.
  • Reference Example 4 Step 3 Reference Example 4 Compound RE4-3 (0.437 g, 0.7143 mmol) synthesized in step 2, N ⁇ , N ⁇ -bis (tert-butoxycarbonyl) -L-lysine (manufactured by Nova Biochem, 0.5483 g, 1.583 mmol), diisopropylethylamine ( Add 0.624 mL, 3.57 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (0.5703 g, 1.5 mmol) to room temperature. The mixture was stirred for 2 hours.
  • Reference Example 6 Step 2
  • Reference Example 6 Compound RE6-3 was quantitatively obtained in the same manner as in step 4 of reference example 3 using compound RE6-2 (0.9622 g, 1.952 mmol) synthesized in step 1.
  • Reference Example 6 Step 3 Reference Example 6 Compound RE6-3 (0.1146 g, 0.290 mmol) synthesized in step 2 and N-succinimidyl 15-azido-4,7,10,13-tetraoxapentadecanoic acid (N3-PEG4-NHS, Tokyo Chemical Industry Co., Ltd. Compound RE6-4 was quantitatively obtained in the same manner as in step 2 of Reference Example 5, using company-made, 0.0750 g, 0.1931 mmol).
  • Reference Example 6 Step 5
  • Reference Example 6 The process of Reference Example 3 using compound RE6-5 (0.1252 g, 0.193 mmol) synthesized in step 4 and L-glutamic acid di-tert-butyl ester (manufactured by Watanabe Kagaku Co., Ltd., 0.1180 g, 0.399 mmol).
  • Compound RE6-6 (0.0521 g, yield 24%) was obtained in the same manner as 3.
  • Reference Example 7 Step 2
  • Reference Example 7 Compound RE7-2 (0.1524 g, yield 61%) was obtained in the same manner as in step 1 of reference example 5 using compound RE7-2 (0.2653 g, 0.1097 mmol) synthesized in step 1.
  • Reference Example 10 Step 3 Reference Example 10 Compound RE10- was prepared using Compound D1 (0.1091 g, 0.044 mmol) synthesized in Step 2 and Compound RE2-3 (0.0748 g, 0.184 mmol) of Reference Example 2 in the same manner as in Step 3 of Reference Example 3. 3 was obtained as a crude product.
  • Reference Example 10 Step 5 Reference Example 10 Compound RE10-4 synthesized in step 4 (0.0816 g, 0.02734 mmol), O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (0.0221 g, 0.05827 mmol) and diisopropylethylamine (0.02 mL, 0.1094 mmol) are dissolved in N, N-dimethylformamide (4 mL), LCAA-CPG (Chem Gene, 0.4882 g) is added, and the mixture is allowed to reach room temperature. Stir overnight.
  • O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate 0.0221 g, 0.05827 mmol
  • diisopropylethylamine 0.02 mL, 0.1094 mmol
  • the mixture was filtered off, washed successively with dichloromethane, 10% methanol solution in dichloromethane and diethyl ether, and then reacted with acetic anhydride / pyridine solution to obtain compound C1 (49.5 ⁇ mol / g, yield 89%) .
  • the yield was calculated from the introduction ratio to the solid phase carrier which can be calculated from the absorption derived from the DMTr group by adding 1% trifluoroacetic acid / dichloromethane solution to the solid phase carrier.
  • Compound RE 11-2 (1.050) was prepared according to the method described in Compound RE 11-1 (1.200 g, 3.640 mmol), Journal of Medicinal Chemistry, Volume 59, 2718-2733 (2016). g, 50% yield) was synthesized.
  • Compound RE 13-1 (Compound RE 1-1, 898.0 mg, 2.007 mmol in Reference Example 1) was dissolved in dichloromethane (15 mL), 1-hydroxybenzotriazole monohydrate (338.0 mg, 2.208 mmol), 1- (1 Add 3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (343 mg, 2.208 mmol), and N-1-Z-l, 3-diaminopropane hydrochloride (0.4910 mL, 2.208 mmol), and add 3 hours at room temperature. It stirred.
  • Reference Example 15 Dissolve iminodiacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 1.5 g, 6.43 mmol,) in methylene chloride (30 mL), pentafluorotrifluoroacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 2.75 mL, 16.08 mmol), triethylamine (4.48) mL, 32.2 mmol) was added and stirred for 4 hours. To the reaction solution was added 10% aqueous citric acid solution, and the mixture was extracted with chloroform, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate.
  • Reference Example 16 Step 1 Using Compound RE16-1 (1.855 g, 3.19 mmol) synthesized by the method described in Reference Example 11 using N- (t-butoxycarbonyl) -L-glutamic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), Reference Example 15 A crude purified product of compound RE16-2 was obtained in the same manner as in the step 1). ESI-MS m / z: 1105 (M + H) +
  • Reference Example 20 Step 1 The same method as in step 3 of Reference Example 3 using compound RE20-1 (AstaTech, 100 mg, 1.148 mmol) and Fmoc-Ser (tBuMe2Si) -OH (Watanabe Chemical Industry, 532 mg, 1.205 mmol) Thus, compound RE20-2 (410 mg, yield 70%) was obtained.
  • Reference Example 22 step 1 A crude product of compound RE22-2 was obtained in the same manner as in step 1 of Reference Example 2 using compound RE22-1 (manufactured by Tokyo Chemical Industry Co., Ltd., 1.2 g, 4.24 mmol).
  • Reference Example 22 Process 3 Compound RE22- was prepared by the same method as in step 3 of Reference Example 3 using compound RE22-3 (1.15 g, 3.16 mmol) and Fmoc-Ser (tBuMe2Si) -OH (manufactured by Watanabe Chemical Industries, Ltd., 1.677 g, 3.8 mmol). 4 (560 mg, 31% yield) was obtained.
  • 1 H-NMR 400 MHz, CDCl 3 ) ⁇ : 0.00-0.07 (6 H, m), 0.83-0.89 (9 H, m), 3.18-3.26 (2 H, m), 3.39-3. 46 (2 H, m), 3.61- 3.68 (1 H, m), 3. 76 (6 H, s), 3.
  • Reference Example 23 The compounds RE23-1 to RE23-5 listed in Table Y-1 and Fmoc-Ser (tBuMe 2 Si) -OH were listed in Table Y-2 in the same manner as in Reference Example 22. Compounds RE23-6 to RE23-10 were obtained. The compound RE23-11 described in Table Y-2 was obtained in the same manner as in Reference Example 22 using the compound RE22-3 in Reference Example 22 and Fmoc-Thr (tBuMe 2 Si) -OH. The compound RE23-12 described in Table Y-2 is obtained in the same manner as in Reference Example 22 using the compound RE23-1 and Fmoc-Thr (tBuMe 2 Si) -OH described in Table Y-1. The The NMR analysis data of the compound synthesized according to this example is shown in Table Y-3.
  • Compound RE24-2 was obtained in the same manner as in Step 2 of Reference Example 2 using Compound RE24-1 (compound RE22-4 in Reference Example 22; 2.487 g, 3.16 mmol) synthesized by the method described in Reference Example 22. (1.2 g, 67% yield).
  • Reference Example 25 Compounds RE25-1 to RE25-7 described in Table Z-1 were obtained in the same manner as in Reference Example 24 using compounds RE23-6 to RE23-12 described in Table Y-2. The mass spectrometry results of the compound synthesized according to this example are shown in Table Z-2.
  • Reference example 26 process 1 Compound RE26-1 (2.00 g, 9.47 mmol) was dissolved in N, N'-dimethylformamide (40 mL), iminodiacetic acid di-tert-butyl ester (5.11 g, 20.84 mmol) at room temperature, 1- ( 3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.00 g, 20.84 mmol) and 1-hydroxybenzotriazole monohydrate (145 mg, 0.947 mmol) were added and stirred for 2 hours.
  • Reference Example 28 step 1 Compound RE28-1 (Compound RE 26-3, 474 mg, 0.744 mmol in Reference Example 26) synthesized by the method described in Reference Example 26 is dissolved in N, N'-dimethylformamide (10 mL), and a journal is prepared at room temperature. Trans-cyclohexane-1,4-dicarboxylic acid monobenzyl ester (0.234 mg, 0.893) synthesized by the method described in Journal of Medicinal Chemistry, Vol. 54, p. 2433-2446, 2011.
  • Reference Example 31 step 1 Dissolve 4-nitroisophthalic acid RE31-1 (500 mg, 2.37 mmol) and N-Boc-ethylenediamine (808 mg, 5.21 mmol) in N, N'-dimethylformamide (10 mL) and use triethylamine (0.90) at room temperature. Add 1 mL (7.11 mmol), 1-hydroxybenzotriazole monohydrate (703 mg, 5.21 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.36 g, 7.11 mmol) to Stir for hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE31-2 (650 mg, yield 55%).
  • Reference example 32 process 1 Dissolve 3,5-dinitrobenzoic acid RE 32-1 (500 mg, 2.36 mmol) and N-Cbz-ethylenediamine (588 mg, 2.83 mmol) in N, N'-dimethylformamide (5.0 mL) and use triethylamine at room temperature Add (0.65 mL, 4.72 mmol), 1-Hydroxybenzotriazole monohydrate (380 mg, 2.83 mmol) and 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (675 mg, 3.54 mmol) Stir for 16 hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE32-2 (445 mg, yield 48%).
  • Reference example 32 process 2 Compound RE32-2 (200 mg, 0.515 mmol) is dissolved in ethanol (5.0 mL), tin (II) chloride (584 mg, 3.09 mmol) and concentrated hydrochloric acid (0.2 mL) are added at room temperature, and Stir for hours. The reaction mixture was worked up to give compound RE32-3 (180 mg, quantitative). ESI-MS m / z: 329 (M + H) +
  • Reference Example 37 Step 1 Compound RE 37-1 (Compound RE 13-2 in Reference Example 13, 430 mg, 0.674 mmol) synthesized by the method described in Reference Example 13 was dissolved in N, N′-dimethylformamide (6 mL), % Palladium carbon powder (water content, 54.29%; 79 mg) was added and stirred for 4 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE26-5 (105.0 mg, 0.148 mmol) in Reference Example 26, 1-hydroxybenzotriazole monohydrate (11. 31 mg, 0.074 mmol), and 1- (3-dimethylaminopropyl) -3-ethyl were added to the filtrate.
  • Reference Example 39 Step 1 Compound RE 39-1 (Compound RE 13-2, 418 mg, 0.655 mmol in Reference Example 13) synthesized by the method described in Reference Example 13 was dissolved in N, N′-dimethylformamide (6 mL), % Palladium carbon powder (hydrous, 54.29%; 77 mg) was added and stirred for 5 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE27-6 (85 mg, 0.144 mmol), 1-hydroxybenzotriazole monohydrate (121.0 mg, 0.791 mmol), and 1- (3-dimethylaminopropyl) -3 which were synthesized in Reference Example 27 were added to the filtrate.
  • Reference Example 45 Step 1 Compound RE 45-1 (Compound RE 32-3, 75.0 mg, 0.228 mmol in Reference Example 32) synthesized by the method described in Reference Example 32 is dissolved in N, N'-dimethylformamide (3.0 mL), and the reaction is performed at room temperature Compound RE 14-3 of Example 14 (574 mg, 0.571 mmol), diisopropylethylamine (0.199 mL, 1.14 mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetra Methyluronium hexafluorophosphate (217 mg, 0.571 mmol) was added and stirred overnight.
  • Reference Example 47 Step 1 Using compound RE47-1 (compound RE33-4 in Reference Example 33, 0.101 g, 0.282 mmol) synthesized by the method described in Reference Example 33, Compound RE15-2 (0.607 g, 0.613 mmol) of Reference Example 15 was synthesized. The compound RE47-2 (0.25 g, yield 39%) was obtained by the same method as used in Step 3 of Reference Example 3. ESI-MS m / z: 2304 (M + H) +
  • a compound A6 was synthesized by the same method as the synthesis of the compound A1 of Reference Example 8 using the compound RE49-1 (0.048 g, 0.021 mmol) and N-succinimidyl 3-maleimidopropionate (manufactured by Tokyo Chemical Industry Co., Ltd., 0.017 g, 0.064 mmol). (0.040 g, 78% yield) was obtained.
  • Reference Example 51 step 1 Compound RE51-1 (0.122 g, 0.054 mmol) synthesized by the method described in Reference Example 7 and the method described in Bioconjugate Chemistry, Volume 22, pp. 690-699, 2011 A compound RE51-2 (0.076 g, yield 56%) was obtained in the same manner as in Reference Example 4, step 1, using hexanoic acid monobenzyl ester synthesized using a similar method.
  • Reference Example 59 step 1 Method similar to step 3 of Reference Example 3 using compound D10 (74.1 mg, 0.029 mmol) synthesized by the method described in Reference Example 46 and compound RE22-2 (15 mg, 0.027 mmol) of Reference Example 22; Alternatively, a crude organism of compound RE59-1 was obtained by the method described in Bioconjugate Chemistry, Vol. 26, pp. 1451-1455 (2015). ESI-MS m / z: 1392 (M + H) + , detected as de-DMTr body
  • Reference Example 60 Tables P-1 and P-2 in the same manner as in Reference Example 59, Steps 1 and 2, using Compound D1 and Compound RE20-4 of the structure of Reference Example 20 described in Table Z-1 or Reference Example 20 The compound described in was obtained.
  • the mass spectrometry results of the compound synthesized according to this example are shown in Table P-3.
  • Step 1 Reference Example Compound A1 and a terminal thiolated oligonucleotide synthesized by the method described in Molecules, Vol. 17, p. 13825-13843, 2012 were added and allowed to stand at room temperature for 4 hours. Sodium carbonate was added to the reaction mixture and allowed to stand overnight at 4 ° C.
  • Step 2 Compound 1-1 (3′-CFB-ssRNA) synthesized in step 1 was prepared in a mixed buffer (100 mmol / L potassium acetate, 30 mmol / L 2- [4- (2-hydroxyethyl) piperazin-1-yl The concentration was adjusted (50 ⁇ mol / L) with ethanesulfonic acid, HEPES) -KOH (pH 7.4), 2 mmol / L magnesium acetate). Equal amounts of the sense strand and the antisense strand (50 ⁇ mol / L) were mixed and allowed to stand at 80 ° C. for 10 minutes. Antisense strand sequences are as described in Table R-2. The temperature was gradually lowered and allowed to stand at 37 ° C. for 1 hour to obtain a double-stranded compound 1-2.
  • Examples 2 to 11 Synthesis of Compounds 2-1 to 11-1 and Compounds 2-2 to 11-2 Compound 2- was prepared in the same manner as in Example 1 except that oligonucleotides having a different base sequence from Example 1 were used. 1-11-1 and compounds 2-2-11-2 were synthesized.
  • Step 1 Reference Example Compound D5 is used to add an amino-terminal modified oligonucleotide synthesized by the method described in Molecules, Vol. 17, p. 13825-13843, 2012, to obtain bioconjugate chemistry. 22: 1723-1728 (2011) or Bioconjugate Chemistry, 26: 1451-1455 (2015). Purification by the method described in Example 1 gave compound 12-1.
  • Step 2 Compound 12-2 was obtained in the same manner as in step 2 of Example 1.
  • Examples 13 to 15 Synthesis of Compounds 13-1 to 15-1 and Compounds 13-2 to 15-2 In the same manner as in Example 1 except that oligonucleotides having a different base sequence from Example 1 were used, Compound 13 -1 to 15-1 and compounds 13-2 to 15-2 were synthesized.
  • N (M) represents 2'-O-methyl modified RNA
  • N (F) represents 2'-fluorine modified RNA
  • ⁇ represents phosphorothioate.
  • N (M) represents 2'-O-methyl modified RNA
  • N (F) represents 2'-fluorine modified RNA
  • p represents phosphorylation at the 5 'end
  • ⁇ represents phosphorothioate.
  • Reference test example 1 Measurement of knockdown activity of CFB mRNA in human cells Huh 7 cells (National Research and Development Corporation, National Institute of Biomedical Innovation and Health and Nutrition Research Institute, JCRB Cell Bank) ) Were seeded at 10,000 cells / 80 ⁇ L / well.
  • DMEM medium Life Technology, catalog number 08458-16
  • FBS fetal bovine serum
  • n (n 2 to 124) and the antisense strand shown in SEQ ID NO: [n + 123] form a pair).
  • the double-stranded nucleic acid described in Tables 1-1 to 1-3 and RNAiMax transfection reagent (Life Technology, catalog number: 1401251) were prepared using Opti-MEM medium (Life Technology, catalog number 11058-021).
  • the amount of CFB mRNA relative to the amount of CFB mRNA introduced when each double stranded nucleic acid was introduced was calculated, assuming that the amount of CFB mRNA was 1.0 when the Huh7 cells were treated with the transfection reagent alone without addition of siRNA. This experiment was performed multiple times, and the average value of the relative expression amount of CFB mRNA is shown in Tables 1-1 to 1-3.
  • human primary hepatocytes (Bioprediction International, catalog number HEP 187) suspended in plating medium (Bioprediction International, catalog number LV 0304-2), the cell number 10000
  • the cells are seeded to be cells / 80 ⁇ L / well and cultured at 37 ° C., 5% CO 2 for 6 hours, and then the culture supernatant is carefully removed, and the incubation medium (Bioprediction International, catalog number LV0304)
  • Each nucleic acid complex was subjected to human primary hepatocytes by adding -2).
  • cells treated with nothing were seeded.
  • the cells to which each nucleic acid complex has been added are cultured in a 5% CO 2 incubator at 37 ° C.
  • the cDNA was prepared by reverse transcription reaction.
  • the glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase) is a CFB gene and a constitutively expressed gene by PCR reaction according to the method described in the attached instruction manual.
  • the following PCR reaction was performed on the gene (hereinafter referred to as gapdh) to measure the amount of mRNA amplification, and the amount of amplification of gapdh was used as an internal control to calculate a semi-quantitative value of mRNA of CFB.
  • the expression rate of CFB mRNA was determined from the semi-quantitative value of CFB mRNA, where the semi-quantitative value of CFB mRNA in the negative control measured similarly was 1.
  • the results of the expression rate of the obtained CFB mRNA are shown in Table S1. As apparent from Table S1, each nucleic acid complex suppressed the expression of mRNA of CFB gene after addition to human primary hepatocytes.
  • Test Example 2 In Vitro Knockdown Test of Nucleic Acid Complex on Mouse Primary Hepatocytes
  • the in vitro knockdown activity of mouse primary hepatocytes was measured for each of the nucleic acid complexes obtained in Examples 10, 11, 14 and 15. .
  • Williams' E Medium Wood's E Medium
  • Primary Hepatocyte Thawing and Plating Supplements Thermo Fisher Scientific, Catalog No.
  • CM 3000 The CD1 mouse-derived mouse primary hepatocytes (manufactured by Thermo Fisher Scientific, catalog number MSCP 10) suspended in (Thermo Fisher Scientific, catalog number A12176-01) were seeded so that 10000cells / 80 ⁇ L / well, 37 ° C., the After incubation for 6 hours under 5% CO 2, culture Each nucleic acid complex was added to mouse primary hepatocytes by carefully removing the supernatant and adding Williams Emedium containing Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, catalog number CM4000). Provided for. In addition, as a negative control group, cells treated with nothing were seeded.
  • the cells to which each nucleic acid complex has been added are cultured in a 5% CO 2 incubator at 37 ° C. for 18 hours, washed with ice-cold phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque) and superprep cell Total RNA was recovered and the obtained total RNA was templated according to the method described in the instruction attached to the product, using Lysis and T kit ForQ PCR (Toyobo Co., Ltd., catalog number SCQ-201). The cDNA was prepared by reverse transcription reaction.
  • DPBS ice-cold phosphate buffered saline
  • the glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase) is a CFB gene and a constitutively expressed gene by PCR reaction according to the method described in the attached instruction manual.
  • the following PCR reaction was performed on the gene (hereinafter referred to as gapdh) to measure the amount of mRNA amplification, and the amount of amplification of gapdh was used as an internal control to calculate a semi-quantitative value of mRNA of CFB.
  • the expression rate of CFB mRNA was determined from the semi-quantitative value of CFB mRNA, where the semi-quantitative value of CFB mRNA in the negative control measured similarly was 1.
  • the expression rate of the obtained CFB mRNA is shown in Table S2.
  • each nucleic acid complex suppressed the expression of mRNA of CFB gene after addition to mouse primary hepatocytes.
  • Test Example 3 In Vivo Knockdown Test of Nucleic Acid Complex in Mouse
  • DPBS phosphate buffered saline
  • BALB / cA obtained from CLEA Japan, Inc.
  • each nucleic acid complex was subcutaneously administered to mice at 30 mg / kg, 10 mg / kg, 3 mg / kg or 0.3 mg / kg.
  • DPBS phosphate buffered saline
  • N.T. represents that it did not evaluate.
  • nucleic acid complex of the present invention was administered to mice to reduce the expression of the CFB gene in the liver.
  • Test Example 4 In Vitro Knockdown Test of Nucleic Acid Complex on Rat Primary Hepatocytes
  • the in vitro knockdown activity of rat primary hepatocytes was measured for each of the nucleic acid complexes shown in Table S4.
  • 96 well culture plate of each nucleic acid complex diluted with Optim (Opti-MEM) (Thermo Fisher Scientific, Catalog No. 31985) to a final concentration of 30, 10, 3 or 1 nmol / L After dispensing 20 ⁇ l each into the tube, Williams 'E Medium (Primary Hepatocyte Thawing and Plating Supplements) (Thermo Fisher Scientific, Catalog No.
  • CM 3000 containing Williams' E Medium (William's E Medium)
  • the primary rat hepatocytes (Thermo Fisher Scientific, catalog number: RTCP10) suspended in (Thermo Fisher Scientific, Catalog No. A12176-01) become 10000 cells / 80 ⁇ L / well of the cell number. seeded manner, 37 ° C., the After incubation for 6 hours under 5% CO 2, and carefully removing the culture supernatant, Lai Mali HEPA door site maintenance supplements (Primary Hepatocyte Maintenance Supplements) (Thermo Fisher Scientific, Inc., catalog number CM4000) with the addition of Williams E medium, including, each nucleic acid complexes were subjected to rat primary hepatocytes.
  • Lai Mali HEPA door site maintenance supplements Primary Hepatocyte Maintenance Supplements
  • the cDNA was prepared by reverse transcription reaction.
  • the glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase) is a CFB gene and a constitutively expressed gene by PCR reaction according to the method described in the attached instruction manual.
  • the following PCR reaction was performed on the gene (hereinafter referred to as gapdh) to measure the amount of mRNA amplification, and the amount of amplification of gapdh was used as an internal control to calculate a semi-quantitative value of mRNA of CFB. Assuming that the semiquantitative value of CFB mRNA in the negative control similarly measured was 1, the expression rate of CFB mRNA was determined. The results of the expression rate of the obtained CFB mRNA are shown in Table S4. As apparent from Table S4, each nucleic acid complex suppressed the expression of mRNA of CFB gene after addition to rat primary hepatocytes.
  • Test Example 5 In Vivo Activity of Nucleic Acid Complex in Rat
  • a rat in vivo knockdown test was performed by the following method.
  • Each nucleic acid complex was diluted with phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque) according to the test.
  • DPBS phosphate buffered saline
  • DPBS phosphate buffered saline
  • RNA samples were prepared using Trizol® ARE Isolation Reagents (Thermo Fisher Scientific, Catalog No. 1559 026) and Magna Pure 96 (MagNA Pure 96) (Roche Life Sciences).
  • Total RNA was recovered according to the method described in the attached manual.
  • reverse transcription using the obtained total RNA as a template according to the method described in the instruction attached to the product using Transcriptor First Strand CDNA Synthesis Kit (Roche Life Science, catalog number 04897030001)
  • the cDNA was prepared by the reaction.
  • the expression rate of CFB mRNA was determined from the quasi-quantitative value of CFB mRNA, where the quasi-quantitative value of CFB mRNA in the PBS administration group measured in the same manner was 1.
  • the expression rate of the obtained CFB mRNA is shown in Table S5.
  • nucleic acid complex of the present invention is administered to rats to reduce the expression of the CFB gene in the liver.
  • the nucleic acid complex of the present invention can be administered to mammals and used to treat CFB related diseases in vivo.

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Abstract

L'invention fournit un complexe d'acide nucléique qui est représenté par la formule (1), et qui permet d'inhiber l'expression d'un facteur B de complément (CFB). Formule (1) : (Dans la formule (1), X consiste en un acide nucléique à double-brin constitué d'un brin sens et d'un brin antisens, et contenant les régions double-brin d'au moins 11 paires de base, cet acide nucléique à double-brin est complémentaire vis-à-vis de séquences ARNmCFB cibles énumérées dans les tableaux 1-1 à 1-3, dans un brin d'oligonucléotide long de 17 à 30 nucléotides dans le brin antisens, l'extrémité 3' ou l'extrémité 5' du brin sens est liée en S3, L1 et L2 représentent chacun indépendamment un ligand de sucre, et S1, S2 et S3 représentent chacun indépendamment un lieur.)
PCT/JP2018/029113 2017-08-02 2018-08-02 Complexe d'acide nucléique Ceased WO2019027015A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020111280A1 (fr) * 2018-11-30 2020-06-04 協和キリン株式会社 Complexe d'acide nucléique
CN113226332A (zh) * 2018-11-02 2021-08-06 阿布特斯生物制药公司 二价靶向缀合物
WO2021222549A1 (fr) * 2020-04-30 2021-11-04 Alnylam Pharmaceuticals, Inc. Compositions d'arni du facteur b du complément (cfb) et leurs procédés d'utilisation
WO2023031359A1 (fr) 2021-09-02 2023-03-09 Silence Therapeutics Gmbh Acides nucléiques pour inhiber l'expression du facteur b du complément (cfb) dans une cellule
WO2023076451A1 (fr) * 2021-10-29 2023-05-04 Alnylam Pharmaceuticals, Inc. Compositions d'arni du facteur b du complément (cfb) et leurs procédés d'utilisation
WO2023143374A1 (fr) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, son procédé de préparation, et son utilisation
US12258565B2 (en) 2019-10-22 2025-03-25 Alnylam Pharmaceuticals, Inc. Complement component C3 iRNA compositions and methods of use thereof
US12378558B2 (en) 2023-03-21 2025-08-05 Arrowhead Pharmaceuticals, Inc. RNAi agents for inhibiting expression of complement factor B (CFB), pharmaceutical compositions thereof, and methods of use
EP4373940A4 (fr) * 2021-07-17 2025-10-01 Sirnaomics Inc Produits et compositions

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009073809A2 (fr) * 2007-12-04 2009-06-11 Alnylam Pharmaceuticals, Inc. Conjugués glucidiques utilisés en tant qu'agents d'administration pour des oligonucléotides
WO2013075035A1 (fr) * 2011-11-18 2013-05-23 Alnylam Pharmaceuticals Agents arni, compositions et procédés d'utilisation de ceux-ci pour traiter des maladies associées à la transthyrétine (ttr)
WO2014179627A2 (fr) * 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions et méthodes pour moduler l'expression de hbv et de ttr
WO2015006740A2 (fr) * 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Conjugués ligands d'oligonucléotides et procédé pour leur préparation
WO2015038939A2 (fr) * 2013-09-13 2015-03-19 Isis Pharmaceuticals, Inc. Modulateurs du facteur b du complément
WO2015089368A2 (fr) * 2013-12-12 2015-06-18 Alnylam Pharmaceuticals, Inc. Composition d'arni d'élément de complément et procédés pour les utiliser
WO2015105083A1 (fr) * 2014-01-07 2015-07-16 塩野義製薬株式会社 Oligonucléotide double brin contenant un oligonucléotide antisens et un dérivé de sucre
WO2017131236A1 (fr) * 2016-01-29 2017-08-03 協和発酵キリン株式会社 Complexe d'acides nucléiques
WO2017135397A1 (fr) * 2016-02-05 2017-08-10 協和発酵キリン株式会社 Oligonucléotide antisens destiné à supprimer l'expression du facteur du complément b
WO2018004004A1 (fr) * 2016-06-30 2018-01-04 協和発酵キリン株式会社 Complexe d'acides nucléiques

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009073809A2 (fr) * 2007-12-04 2009-06-11 Alnylam Pharmaceuticals, Inc. Conjugués glucidiques utilisés en tant qu'agents d'administration pour des oligonucléotides
WO2013075035A1 (fr) * 2011-11-18 2013-05-23 Alnylam Pharmaceuticals Agents arni, compositions et procédés d'utilisation de ceux-ci pour traiter des maladies associées à la transthyrétine (ttr)
WO2014179627A2 (fr) * 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions et méthodes pour moduler l'expression de hbv et de ttr
WO2014179620A1 (fr) * 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Composés antisens conjugués et leur utilisation
WO2014179629A2 (fr) * 2013-05-01 2014-11-06 Isis Pharmaceuticals, Inc. Compositions et procédés
WO2015006740A2 (fr) * 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Conjugués ligands d'oligonucléotides et procédé pour leur préparation
WO2015038939A2 (fr) * 2013-09-13 2015-03-19 Isis Pharmaceuticals, Inc. Modulateurs du facteur b du complément
WO2015089368A2 (fr) * 2013-12-12 2015-06-18 Alnylam Pharmaceuticals, Inc. Composition d'arni d'élément de complément et procédés pour les utiliser
WO2015105083A1 (fr) * 2014-01-07 2015-07-16 塩野義製薬株式会社 Oligonucléotide double brin contenant un oligonucléotide antisens et un dérivé de sucre
WO2017131236A1 (fr) * 2016-01-29 2017-08-03 協和発酵キリン株式会社 Complexe d'acides nucléiques
WO2017135397A1 (fr) * 2016-02-05 2017-08-10 協和発酵キリン株式会社 Oligonucléotide antisens destiné à supprimer l'expression du facteur du complément b
WO2018004004A1 (fr) * 2016-06-30 2018-01-04 協和発酵キリン株式会社 Complexe d'acides nucléiques

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113226332B (zh) * 2018-11-02 2025-05-09 阿布特斯生物制药公司 二价靶向缀合物
JP2022506517A (ja) * 2018-11-02 2022-01-17 アルブータス・バイオファーマー・コーポレイション 標的指向化二価結合体
EP3873486A4 (fr) * 2018-11-02 2022-10-19 Arbutus Biopharma Corporation Conjugués ciblés bivalents
CN113226332A (zh) * 2018-11-02 2021-08-06 阿布特斯生物制药公司 二价靶向缀合物
WO2020111280A1 (fr) * 2018-11-30 2020-06-04 協和キリン株式会社 Complexe d'acide nucléique
US12365896B2 (en) 2019-10-22 2025-07-22 Alnylam Pharmaceuticals, Inc. Complement component C3 iRNA compositions and methods of use thereof
US12258565B2 (en) 2019-10-22 2025-03-25 Alnylam Pharmaceuticals, Inc. Complement component C3 iRNA compositions and methods of use thereof
WO2021222549A1 (fr) * 2020-04-30 2021-11-04 Alnylam Pharmaceuticals, Inc. Compositions d'arni du facteur b du complément (cfb) et leurs procédés d'utilisation
JP2023523790A (ja) * 2020-04-30 2023-06-07 アルナイラム ファーマシューティカルズ， インコーポレイテッド 補体因子Ｂ（ＣＦＢ）ｉＲＮＡ組成物およびその使用方法
CN116096381A (zh) * 2020-04-30 2023-05-09 阿尔尼拉姆医药品有限公司 补体因子B（CFB）iRNA组合物及其使用方法
EP4373940A4 (fr) * 2021-07-17 2025-10-01 Sirnaomics Inc Produits et compositions
WO2023031359A1 (fr) 2021-09-02 2023-03-09 Silence Therapeutics Gmbh Acides nucléiques pour inhiber l'expression du facteur b du complément (cfb) dans une cellule
US11965166B2 (en) 2021-10-29 2024-04-23 Alnylam Pharmaceuticals, Inc. Complement factor B (CFB) iRNA compositions and methods of use thereof
WO2023076451A1 (fr) * 2021-10-29 2023-05-04 Alnylam Pharmaceuticals, Inc. Compositions d'arni du facteur b du complément (cfb) et leurs procédés d'utilisation
WO2023143374A1 (fr) * 2022-01-30 2023-08-03 成都凌泰氪生物技术有限公司 Ligand, son procédé de préparation, et son utilisation
US12378558B2 (en) 2023-03-21 2025-08-05 Arrowhead Pharmaceuticals, Inc. RNAi agents for inhibiting expression of complement factor B (CFB), pharmaceutical compositions thereof, and methods of use

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