WO2017135397A1 - Oligonucléotide antisens destiné à supprimer l'expression du facteur du complément b - Google Patents

Oligonucléotide antisens destiné à supprimer l'expression du facteur du complément b Download PDF

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WO2017135397A1
WO2017135397A1 PCT/JP2017/003904 JP2017003904W WO2017135397A1 WO 2017135397 A1 WO2017135397 A1 WO 2017135397A1 JP 2017003904 W JP2017003904 W JP 2017003904W WO 2017135397 A1 WO2017135397 A1 WO 2017135397A1
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antisense oligonucleotide
seq
nos
base sequence
bases
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PCT/JP2017/003904
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陽史 山田
清 清水
宏徒 岩井
麻奈 牧野
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協和発酵キリン株式会社
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to an antisense oligonucleotide for use in suppressing the expression of complement factor B or a pharmaceutical composition containing the antisense oligonucleotide.
  • Complements are a group of proteins in the blood that mediate immune responses. Complement effects include phagocytosis of pathogenic bacteria by phagocytic cells, damage to viruses with outer membranes, and loss of infectivity. Is mentioned. Among complements, C3 is the most abundant in serum, and its action is controlled by being activated by complement factor B (CFB) in the alternative pathway (non-patented) Reference 1).
  • CFB complement factor B
  • C3 continues to be activated due to abnormalities in regulatory factors related to the alternative pathway of the complement or stabilization with autoantibodies to C3 convertase, atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria Disease (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), It is known to develop diseases such as systemic lupus erythematosus (SLE), asthma, psoriasis, neuromyelitis optica, myasthenia gravis and autoimmune disease (Non-patent Document 2).
  • aHUS atypical hemolytic uremic syndrome
  • PNH paroxysmal nocturnal hemoglobinuria Disease
  • AMD age-related macular degeneration
  • MPGN membran
  • complement factor B is present in the blood at a relatively high concentration of 300 ⁇ g / mL ( Non-patent document 3), it is not easy to continue to inhibit all these complement factor B by, for example, a general antibody drug.
  • an antisense method is known as a method for suppressing gene expression itself (Patent Document 1).
  • oligonucleotides complementary to the target gene mRNA or mRNA precursor are introduced into the target gene mRNA or mRNA precursor in two. It forms a chain and can specifically suppress the expression of the target gene.
  • RNAi RNA interference
  • the expression of the target gene can be specifically suppressed by introducing a double-stranded RNA (siRNA) having the same sequence as the target gene (Patent Document 2).
  • Patent Document 3 A part of siRNA sequence targeting human complement factor B gene has been disclosed (Patent Document 3). Moreover, although some antisense sequences targeting the gene have been disclosed (Patent Document 4), the present invention is a sequence different from these sequences.
  • the present invention relates to the following (1) to (13).
  • An antisense oligonucleotide that suppresses the expression of complement factor B and is capable of hybridizing under stringent conditions to a nucleic acid comprising the target base sequence represented by any of SEQ ID NOs: 53 to 103. 8 Antisense oligonucleotide of ⁇ 80 bases length.
  • An antisense oligonucleotide having a length of 8 to 80 bases comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 53 to 103.
  • the antisense oligonucleotide according to (3) comprising the base sequence represented by any of SEQ ID NOs: 2-52.
  • the antisense oligonucleotide according to (4) comprising a sequence in which one to several bases are deleted, substituted, inserted or added in the base sequence represented by any of SEQ ID NOs: 2 to 52 .
  • An antisense oligonucleotide comprising the base sequence represented by any of SEQ ID NOs: 2-52.
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (10).
  • (12) A method for treating a disorder mediated by an abnormality in the alternative complement pathway, comprising a therapeutically effective amount of the antisense oligonucleotide according to any one of (1) to (10) or (11 ) Comprising administering to a human in need of such treatment.
  • the disorder is atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma or autoimmune disease, the method according to (12).
  • antisense oligonucleotide of the present invention or the pharmaceutical composition containing the antisense oligonucleotide.
  • the antisense oligonucleotide of the present invention or the pharmaceutical composition containing the antisense oligonucleotide is useful for the treatment / prevention of a disorder mediated by an abnormality in the alternative complement pathway.
  • FIG. 1 shows the results of in vivo knockdown in mice administered with an antisense oligonucleotide represented by any of SEQ ID NOs: 118, 125 and 150.
  • the vertical axis shows the relative expression rate of CFB mRNA in each antisense oligonucleotide administration group when the semi-quantitative value of complement factor B (CFB) mRNA in the control group is 1.
  • FIG. 2 shows the results of in vivo knockdown in mice administered with the antisense oligonucleotide represented by SEQ ID NO: 155.
  • the vertical axis shows the relative expression rate of CFB mRNA in the antisense oligonucleotide-administered group when the semi-quantitative value of CFB mRNA in the control group is 1.
  • Antisense oligonucleotide of the present invention provides an antisense oligonucleotide that suppresses the expression of complement factor B (CFB) (also referred to herein as the “antisense oligonucleotide of the present invention”). .
  • CFB complement factor B
  • antisense oligonucleotide refers to a DNA constituting a target gene, an mRNA precursor transcribed from such DNA, and an oligonucleotide complementary to the mRNA. Suppresses the action of DNA, mRNA precursor or mRNA (transcription, post-transcriptional editing, translation, etc.) by forming double-stranded or triple-stranded with target DNA, mRNA precursor or mRNA.
  • CFB gene As a gene encoding CFB targeted by the antisense oligonucleotide of the present invention (hereinafter also referred to as CFB gene), a cDNA base sequence corresponding to human CFB full-length mRNA registered as Genbank Accession No. NM_001710 (SEQ ID NO: 1), and naturally occurring variants thereof (see, for example, Hum. Mutat. 31: E1445-E1460 (2010), refsnp No. rs12614, rs641153, etc.) are also antisense of the present invention. It goes without saying that it can be a target gene for oligonucleotides.
  • complementary refers to, for example, base pairing between two bases via a slow hydrogen bond, such as the relationship between adenine and thymine or uracil, and the relationship between guanine and cytosine. Means a relationship.
  • “complementary” means not only when two nucleotide sequences are completely complementary in a double-stranded region (a region overlapping when both sequences are aligned), but also the entire double-stranded region. As long as it can have a double helix structure, that is, as long as both sequences can hybridize under stringent conditions, a case where 1 to several mismatches exist is also included.
  • the antisense oligonucleotide of the present invention is a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid comprising DNA encoding CFB, an mRNA precursor, or a target base sequence in mRNA.
  • stringent conditions means that the antisense oligonucleotide of the present invention hybridizes to the target base sequence of the CFB gene, but does not hybridize to other sequences or even if it is a target. It means a condition that is significantly less than the amount hybridized to the base sequence and hybridizes only in a negligible amount that is relatively negligible. Such conditions can be easily selected by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like.
  • 6xSSC 0.09M trisodium citrate
  • 6xSSPE 3M NaCl, 0.2M NaH 2 PO 4 , 20mM EDTA ⁇ 2Na, pH7.4
  • the conditions for washing with 0.5xSSC are examples of stringent conditions, but are not limited thereto.
  • a hybridization method for example, Southern blot hybridization method or the like can be used. Specifically, it should be performed according to the method described in Molecular Cloning: A Laboratory Mannual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), etc. Can do.
  • the antisense oligonucleotide of the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to those of the nucleotides.
  • DNA that is a polymer of deoxyribonucleotides, and DNA ribonucleotides.
  • examples thereof include RNA as a combination, chimeric nucleic acids composed of DNA and RNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide.
  • uracil (U) in RNA can be uniquely read as thymine (T) in DNA.
  • nucleotide derivatives examples include nucleotide derivatives.
  • the nucleotide derivative may be any molecule as long as it is a molecule in which nucleotides have been modified.
  • deoxyribonucleotides or molecules modified with ribonucleotides are preferably used.
  • nucleotide examples include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, and a nucleotide in which two or more of the sugar moiety, phosphodiester bond and base are modified.
  • any or all of the chemical structure of the sugar of the nucleotide may be modified or substituted with any substituent, or may be substituted with any atom.
  • '-Modified nucleotides are preferably used.
  • 2′-modified nucleotides include ribose 2′-OH groups from OR, R, R′OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br, and I.
  • Examples include modified nucleotides. More preferred examples include 2′-modified nucleotides substituted with a substituent selected from the group consisting of a methoxy group and a 2- (methoxy) ethoxy group.
  • a crosslinked structure-type artificial nucleic acid having two circular structures by introducing a crosslinked structure into the sugar moiety is also preferably used.
  • LNA locked artificial nucleic acid
  • Ethylene bridged nucleic acid ENA
  • cEt Constrained Ethyl
  • AmNA AmNA
  • scpBNA 2'-O, 4'-C-Spirocyclopropylene bridged nucleic acid
  • peptide nucleic acid [Acc. Chem. Res., 32, 624 (1999)]
  • oxypeptide nucleic acid OPNA
  • peptide ribonucleic acid 2001
  • PRNA peptide ribonucleic acid
  • any or all of the chemical structure of the nucleotide phosphodiester bond modified or substituted with any substituent or any atom may be used.
  • a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate
  • examples include nucleotides in which a diester bond is substituted with a phosphoramidate bond, and preferably nucleotides in which a phosphodiester bond is substituted with a phosphorothioate bond.
  • the base-modified nucleotide may be any nucleotide as long as it is part or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent, or substituted with an arbitrary atom.
  • oxygen atoms are substituted with sulfur atoms
  • hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms
  • halogen groups methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms
  • an amino group is substituted with 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 derivatives include nucleotide derivatives modified with at least one of nucleotide or sugar moiety, phosphodiester bond or base, ligands such as lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc) , Sugars such as galactose (Gal) and mannose (Man), full antibodies, fragment antibodies such as Fab, scFv, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, RGD, NGR, R9, CPP, etc.
  • ligands such as lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc) , Sugars such as galactose (Gal) and mannose (Man), full antibodies, fragment antibodies such as Fab, scFv, VHH, proteins such as low density lipoprotein (LDL),
  • Peptides small molecules such as phenazine, phenanthridine, anthraquinone, folic acid, synthetic polymers such as synthetic polyamino acids, dyes such as nucleic acid aptamer, acridine, fluorescein, rhodamine, coumarin, Cy3 series, Alexa series, black hole quencher Such as fluorescence There may be mentioned those obtained by adding another chemical substance such as a group directly or via a linker.
  • polyamine addition nucleotide derivatives examples include 6-FAM-added nucleotide derivatives and biotin-added nucleotide derivatives, and preferably include GalNAc-added nucleotide derivatives.
  • modifying agent capable of reacting on the solid phase during the extension reaction on the solid phase. It can also be obtained by previously synthesizing and purifying nucleic acids into which a functional group such as an amino group, mercapto group, azide group or triple bond has been introduced, and allowing a modifier to act on them.
  • the nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, or a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.
  • the antisense oligonucleotide of the present invention includes those in which some or all atoms in the molecule are substituted with atoms (isotopes) having different mass numbers.
  • the antisense oligonucleotide of the present invention may be introduced into the form of a hairpin oligomer or circular oligomer as long as it is a nucleic acid that hybridizes under stringent conditions to the target base sequence of the CFB gene. It may contain structural elements such as loops.
  • the length of the antisense oligonucleotide of the present invention is 8 to 80 bases, preferably 8 to 30 bases.
  • it can be 8-20 bases, 10-20 bases, 13-20 bases, 13-16 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases.
  • the target base sequence of the antisense oligonucleotide of the present invention include a partial base sequence of CFB mRNA (cDNA) represented by SEQ ID NOs: 53 to 103 described in Table 1. Therefore, the antisense oligonucleotide of the present invention is an oligonucleotide capable of hybridizing under stringent conditions to the target base sequence represented by any of SEQ ID NOs: 53 to 103. As long as the antisense oligonucleotide of the present invention can hybridize to any one of the above target base sequences under stringent conditions, not only the sequence completely complementary to the target base sequence but also 1 to the target base sequence.
  • the antisense oligonucleotide of the present invention includes a base sequence complementary to the target base sequence represented by any of SEQ ID NOs: 53 to 103 and a sequence completely complementary to the target base sequence. , 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base deleted, substituted, inserted or added may be used.
  • Antisense oligonucleotides having a length of 8 to 80 bases containing a series of bases are also preferred antisense oligonucleotides. More preferably 9 or more, still more preferably 10 or more, still more preferably 11 or more, particularly preferably 12 or more, most preferably selected from each antisense base sequence described in Table 1
  • Antisense oligonucleotides containing 13 consecutive bases and having a length of 8 to 80 bases, preferably 8 to 30 bases are also preferred antisense oligonucleotides.
  • Typical preferred antisense oligonucleotides include at least 8, more preferably 9 or more, even more preferably 10 or more, and even more preferably 11 from the 5 ′ end of each antisense base sequence listed in Table 1. Oligonucleotides comprising one or more, particularly preferably 12 or more consecutive nucleobases are included. Similarly, preferable antisense oligonucleotides include at least 8 or more, more preferably 9 or more, still more preferably 10 or more, and still more preferably 11 from the 3 ′ end of each antisense base sequence described in Table 1. Oligonucleotides comprising one or more, particularly preferably 12 or more consecutive nucleobases are included. Most preferably, it is an oligonucleotide containing each antisense base sequence described in Table 1.
  • the antisense oligonucleotide of the present invention includes each antisense base sequence described in Table 1, and lacks 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the antisense base sequence. A deletion, substitution, insertion or addition may be used.
  • an antisense oligonucleotide consisting of the nucleotide sequence represented by any of SEQ ID NOs: 104 to 154 and 155 described in Table 2 is preferably used.
  • Antisense oligonucleotides of the present invention include antisense oligonucleotides described in Table 2 and Table 3, each having an antisense oligonucleotide sequence having a CFB relative expression level of 0.5 or less. It is more preferable to use nucleotides, more preferably antisense oligonucleotides having a CFB relative expression level of 0.3 or less, and particularly preferably antisense oligonucleotides having a CFB relative expression level of 0.1 or less.
  • the antisense oligonucleotide of the present invention When the antisense oligonucleotide of the present invention is introduced into a cell, it binds to complementary mRNA and mRNA precursor, and sterically inhibits translation of the mRNA and mRNA precursor into a protein, thereby inhibiting the CFB gene. Can be suppressed.
  • the antisense oligonucleotide of the present invention introduced into a cell may bind to complementary mRNA and mRNA precursor in the cell and cleave the mRNA and mRNA precursor.
  • RNaseH an action through RNaseH, which is an endonuclease that cleaves RNA strands of RNA and DNA double strands.
  • RNaseH an endonuclease that cleaves RNA strands of RNA and DNA double strands
  • an antisense oligonucleotide having 4 to 80 continuous DNA regions is preferable.
  • the antisense oligonucleotide preferably has 0 to 80% sugar-modified nucleotides, more preferably 10 to 60%, and even more preferably 20 to 50%.
  • the number of continuous DNA regions having a sugar-modified nucleotide is more preferably 4 to 20, more preferably 4 to 15, and most preferably 5 to 10.
  • the position of the sugar-modified nucleotide in the antisense oligonucleotide is preferably arranged in the vicinity of the 5 ′ end and / or in the vicinity of the 3 ′ end, and the position within 30% of the total length from the 5 ′ end and / or More preferably, it is arranged at a position within 30% of the total length from the 3 ′ end, and the sugar-modified nucleotide is located at a position within 25% of the total length from the 5 ′ end of the antisense oligonucleotide and / or More preferably, it is arranged at a position within 25% of the total length from the 3 ′ end.
  • the antisense oligonucleotide of the present invention When introduced into a cell, the antisense oligonucleotide of the present invention also binds to genomic DNA encoding a complementary CFB, inhibits transcription of the DNA to an mRNA precursor, and inhibits expression of the CFB gene. Can be suppressed.
  • the vicinity of the 5 'end and / or the vicinity of the 3' end is a sugar-modified nucleotide.
  • the vicinity of the 5 ′ end is a sugar moiety-modified nucleotide means that 1 to 4, preferably 2 to 4 consecutive nucleotides from the 5 ′ end are sugar part-modified nucleotides
  • the vicinity of the 3 ′ end is a sugar part modified nucleotide means that 1 to 4, preferably 2 to 4 consecutive nucleotides from the 3 ′ end are sugar part modified nucleotides.
  • the antisense oligonucleotide of the present invention is preferably an antisense oligonucleotide in which 1 to 4 nucleotides from the 5 ′ end are sugar-modified nucleotides, more preferably 2 to 4 from the 5 ′ end.
  • the nucleotide is a sugar-modified nucleotide.
  • the antisense oligonucleotide of the present invention is preferably an antisense oligonucleotide in which 1 to 4 nucleotides from the 3 ′ end are sugar-modified nucleotides, more preferably 2 to 4 nucleotides from the 3 ′ end. Can be used in which is a sugar-modified nucleotide.
  • the method for producing the antisense oligonucleotide of the present invention is not particularly limited, and includes a method using a known chemical synthesis, an enzymatic transcription method, or the like.
  • known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic® Acid® Research, 35, 3287] (2007)].
  • ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed.
  • an antisense oligonucleotide having a purity of 90% or more, preferably 95% or more by purification is desirable to obtain an antisense oligonucleotide having a purity of 90% or more, preferably 95% or more by purification.
  • a transcription method using a phage RNA polymerase for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template.
  • the antisense oligonucleotide of the present invention can be introduced into cells using a transfection carrier, preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.
  • the antisense oligonucleotide of the present invention can also induce suppression of target gene expression by forming a double strand with a complementary oligonucleic acid and introducing it into a cell as a double-stranded nucleic acid (International Publication No. 2005). / 113571).
  • the position at which the double-stranded nucleic acid is modified with a ligand is preferably the 5 'end or 3' end of a complementary oligonucleic acid.
  • the antisense oligonucleotide of the present invention has a cDNA base sequence (SEQ ID NO: 1) corresponding to the full-length mRNA of human CFB or a genomic sequence corresponding to the mRNA precursor. Can be designed based on.
  • the cDNA of human CFB full-length mRNA is registered, for example, as Genbank Accession No. NM_001710, and the genomic sequence containing the human CFB mRNA precursor is, for example, Genbank Accession No. It is registered.
  • the antisense oligonucleotide having CFB expression suppression activity is selected from the group described in Table 1 or Table 2, for example.
  • antisense oligonucleotides composed of antisense base sequences is 8 to 80 bases, preferably 8 to 30 bases.
  • it can be 8-20 bases, 10-20 bases, 13-20 bases, 13-16 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases.
  • CFB expression can be suppressed by introducing these antisense oligonucleotides into cells.
  • the antisense oligonucleotide of the present invention can suppress the expression of CFB mRNA after being introduced into cells at a concentration of several nM to several ⁇ M and then cultured for 24 hours or more, for example 48 hours.
  • the evaluation of the CFB mRNA expression inhibitory activity of the antisense oligonucleotide of the present invention can be carried out using the antisense oligonucleotide as a cationic liposome or the antisense oligonucleotide as it is, or the antisense oligonucleotide.
  • the antisense oligonucleotide of the present invention may contain a ligand.
  • the ligand may be one that directly modifies the 5 ′ end, 3 ′ end and / or the sequence interior of the antisense oligonucleotide of the present invention.
  • the ligand contained in the antisense oligonucleotide of the present invention may be any molecule that has affinity for biomolecules.
  • lipids such as cholesterol, fatty acids, tocopherols, retinoids, N-acetylgalactosamine (GalNAc), Sugars such as galactose (Gal) and mannose (Man), full antibodies, fragment antibodies such as Fab, scFV, VHH, proteins such as low density lipoprotein (LDL), human serum albumin, RGD, NGR, R9, CPP, etc.
  • Peptides, low molecular weight compounds such as folic acid, synthetic polymers such as synthetic polyamino acids, nucleic acid aptamers, and the like can be mentioned, but the invention is not limited thereto, and these can be used in appropriate combinations.
  • a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifier to act on them.
  • composition of the present invention relates to a pharmaceutical composition containing the antisense oligonucleotide of the present invention as an active ingredient (hereinafter also referred to as the pharmaceutical composition of the present invention).
  • the pharmaceutical composition can further contain a carrier effective for transferring the antisense oligonucleotide into the cell.
  • the pharmaceutical composition of the present invention comprises atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN), C3 Nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) ) And the like.
  • aHUS atypical hemolytic uremic syndrome
  • PNH paroxysmal nocturnal hemoglobinuria
  • AMD age-related macular degeneration
  • MPGN membranoproliferative glomerulonephriti
  • Examples of the carrier effective for transferring the antisense oligonucleotide into the cell include a cationic carrier.
  • Examples of the cationic carrier include cationic liposomes and cationic polymers.
  • a carrier utilizing a viral envelope may be used as a carrier effective for transferring the antisense oligonucleotide into the cell.
  • cationic liposomes include liposomes containing 2-O- (2-diethylaminoethyl) carbamoyl-1,3-O-dioleoylglycerol (hereinafter also referred to as liposome A), oligofectamine (Invitrogen), lipofectin ( Invitrogen), Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene Therapy Systems), TransMessenger (QIAGEN), TransIT TKO (Mirus), etc. Is preferably used.
  • JetSI Qbiogene
  • Jet-PEI polyethyleneimine; Qbiogene
  • GenomeOne HVJ-E liposome; Ishihara Sangyo Co., Ltd.
  • the pharmaceutical composition of the present invention comprising the above-described carrier in the antisense oligonucleotide of the present invention can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion having an appropriate concentration and an antisense oligonucleotide solution.
  • a carrier dispersion having an appropriate concentration
  • an antisense oligonucleotide solution When a cationic carrier is used, the antisense oligonucleotide is negatively charged in an aqueous solution, and therefore can be easily prepared by mixing in an aqueous solution by a conventional method.
  • the aqueous solvent used for preparing the composition include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution.
  • conditions such as pH and temperature when preparing the composition can be appropriately selected by those skilled in the art.
  • liposome A prepared by gradually adding an antisense oligonucleotide in 10% maltose aqueous solution to 16 mg / ml liposome dispersion in 10% maltose aqueous solution with stirring at pH 7.4 and 25 ° C. can do.
  • the composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersion device or a high-pressure emulsification device if necessary.
  • the optimum method and conditions for the preparation of the composition comprising the carrier and the antisense oligonucleotide depend on the carrier to be used, and those skilled in the art can select the optimum method for the carrier to be used without being bound by the above method. it can.
  • the pharmaceutical composition of the present invention comprises a composite particle comprising an antisense oligonucleotide and a lead particle as constituents and a lipid membrane covering the composite particle, wherein the constituent of the lipid membrane is soluble
  • a liposome containing a liquid containing the polar organic solvent at a concentration capable of dispersing the constituent components of the lipid membrane and the composite particles can also be suitably used.
  • the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, and fine particle preparations, and liposomes are preferably used.
  • the lead particles in the present invention may be composed of a complex obtained by combining two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, A complex formed by combining a polymer, a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.
  • another compound eg, sugar, lipid, inorganic compound, etc.
  • lipid membrane that coats the composite particles examples include neutral lipids and polyethylene glycol-phosphatidylethanolamine as constituent components.
  • the liposome can be prepared, for example, according to the method described in WO2006 / 080118.
  • the mixing ratio of the antisense oligonucleotide and the carrier contained in the pharmaceutical composition of the present invention is suitably 1 to 200 parts by weight of the carrier with respect to 1 part by weight of the antisense oligonucleotide.
  • the amount is 2.5 to 100 parts by weight, more preferably 10 to 20 parts by weight, based on 1 part by weight of the antisense oligonucleotide.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent in addition to the above carrier.
  • a pharmaceutically acceptable carrier or diluent or the like is an essentially chemically inert and harmless composition that does not affect the biological activity of the pharmaceutical composition of the present invention at all. Examples of such carriers or diluents include but are not limited to salt solutions, sugar solutions, glycerol solutions, ethanol and the like.
  • disorders mediated by abnormalities in the alternative complement pathway include atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), Membranoproliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE)) Psoriasis, optic neuritis, myasthenia gravis, etc.).
  • aHUS atypical hemolytic uremic syndrome
  • PNH paroxysmal nocturnal hemoglobinuria
  • AMD age-related macular degeneration
  • MPGN Membranoproliferative glomerulonephritis
  • C3 nephritis C3 nephritis
  • the pharmaceutical composition of the present invention comprises atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN) , C3 nephritis, membranous nephropathy, rapidly progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis It can be used as a therapeutic or prophylactic agent such as atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranoproliferative glomerulonephritis (MPGN) , C3 nephritis, membranous n
  • the pharmaceutical composition of the present invention can be provided in a form that contains an amount of the complex effective for treating or preventing a disease and can be appropriately administered to a patient.
  • the preparation form of the pharmaceutical composition of the present invention may be, for example, injections, eye drops, liquids for inhalation, etc., for example, external preparations such as ointments, lotions and the like.
  • the concentration range of the pharmaceutical composition of the present invention is usually 0.001 to 25% (w / v), preferably 0.01 to 5% (w / v), more preferably 0.1 to 2% ( w / v).
  • the pharmaceutical composition of the present invention may contain an appropriate amount of any pharmaceutically acceptable additive, for example, an emulsification aid, a stabilizer, an isotonic agent, a pH adjuster and the like. Any pharmaceutically acceptable additive can be added in an appropriate step before or after dispersion of the complex.
  • the pharmaceutical composition of the present invention can also be provided as a lyophilized preparation.
  • the lyophilized preparation can be prepared by dispersing the antisense oligonucleotide and the carrier and then lyophilizing.
  • the lyophilization treatment can be performed by a conventional method. For example, a predetermined amount of the complex solution after the above dispersion treatment is aseptically dispensed into a vial, preliminarily dried for about 2 hours under a condition of about -40 to -20 ° C, and about 0 to 10 ° C. Primary drying under reduced pressure, followed by secondary drying under reduced pressure at about 15-25 ° C. and lyophilization. Then, for example, by replacing the inside of the vial with nitrogen gas and stoppering, a freeze-dried preparation of the pharmaceutical composition of the present invention can be obtained.
  • the pharmaceutical composition of the present invention can be redissolved and used by adding any appropriate solution.
  • a solution include electrolytes such as water for injection and physiological saline, glucose solution, and other general infusion solutions.
  • the amount of this solution varies depending on the application and is not particularly limited, but it is preferably 0.5 to 2 times the amount before lyophilization, or 500 ml or less.
  • the pharmaceutical composition of the present invention can be administered to animals including humans, for example, intravenous administration, intraarterial administration, oral administration, tissue administration, transdermal administration, transmucosal administration, or rectal administration. It is preferable to administer by an appropriate method according to the symptoms. In particular, intravenous administration, transdermal administration, and transmucosal administration are preferably used. Moreover, local administration, such as local administration in cancer, can also be performed. Examples of the dosage form suitable for these administration methods include various injections, oral preparations, drops, absorbents, eye drops, ointments, lotions, suppositories and the like.
  • the dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the drug, dosage form, patient condition such as age and weight, administration route, nature and degree of disease, etc.
  • the mass of nucleotide is 0.1 mg to 10 g / day, preferably 1 mg to 500 mg / day per day for an adult. In some cases, this may be sufficient, or vice versa. It can also be administered once to several times a day, and can be administered at intervals of 1 to several days.
  • the present invention is further a method of treating a disorder mediated by an alternative pathway alternative comprising a therapeutically effective amount of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention.
  • a method comprising the step of administering to a human in need of such treatment (the treatment method of the invention).
  • the treatment method of the present invention is preferably an atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 nephritis, membranous nephropathy, rapidly progressive It is a method of treating diseases such as glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis)
  • RPGN glomerulonephritis
  • AKI acute kidney injury
  • SLE systemic lupus erythematosus
  • psoriasis optic neuromyelitis
  • myasthenia gravis a therapeutically effective amount of the antisense oligonucleotide of the present invention or the pharmaceutical composition of the present invention is administered to
  • the administration method, dose, preparation method and the like of the pharmaceutical composition of the present invention can be used.
  • each nucleotide has a phosphodiester bond substituted with a phosphorothioate bond.
  • the antisense oligonucleotide and Lipofectamine LTX & Plus reagent are diluted with Opti-MEM medium (Life Technology, catalog number 11058-021) to terminate the antisense oligonucleotide.
  • 20 ⁇ L of antisense oligonucleotide / Lipofectamine LTX mixed solution was added to each 96-well culture plate so that the concentration was 30 nM, and cultured at 37 ° C. under 5% CO 2 condition for 24 hours.
  • CDNA was synthesized according to the method. Add 5 ⁇ L of this cDNA to a MicroAmpOptical 96-well plate (Applied Biosystems, catalog number 4326659), then add 10 ⁇ L TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016), 3 ⁇ L of UltraPure Distilled Water (Life Technologies) (Cat. No. 10977-015), 1 ⁇ L human CFB probe, and 1 ⁇ L human ⁇ -actin probe were added.
  • PBS Phosphate buffered saline
  • ⁇ -actin is a constitutively expressed gene, measured as an internal control, and corrected for CFB expression.
  • the relative expression level of CFB mRNA when each antisense oligonucleotide was introduced was calculated with 1.0 as the amount of CFB mRNA when Huh7 cells were treated with only the transfection reagent without adding the antisense oligonucleotide. This experiment was performed a plurality of times, and the average value of the relative expression level of CFB mRNA is shown in Table 2.
  • the CFB mRNA expression rate was determined from the CFB mRNA quasi-quantitative value, with the CFB mRNA quasi-quantitative value in the control group measured similarly.
  • the relative expression rate of the obtained CFB mRNA is shown in FIG.
  • the in vitro knockdown activity was adjusted 3 times in the same manner as in Example 1 by adjusting the final concentration of the antisense oligonucleotide (SEQ ID NO: 155) to 30 nM, 10 nM, 3 nM, 1 nM, and 0.3 nM, respectively.
  • the average value of the results is shown in Table 3.
  • In vivo knockdown activity is shown in FIG. 2, in which antisense oligonucleotide (SEQ ID NO: 155) was subcutaneously administered to mice at 10 mg / kg and measured in the same manner as in Example 2.
  • the present invention provides an antisense oligonucleotide having CFB expression-inhibiting activity, a pharmaceutical composition containing the antisense oligonucleotide as an active ingredient, and the like.
  • the antisense oligonucleotide and the pharmaceutical composition of the present invention suppress the expression of CFB, atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranoproliferative glomerulonephritis, C3 Nephritis, membranous nephropathy, rapid progressive glomerulonephritis (RPGN), acute kidney injury (AKI), asthma, autoimmune diseases (eg systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis) It is useful in the treatment and prevention of disorders mediated by abnormalities in the alternative pathway of the complement such as).

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Abstract

La présente invention décrit : un oligonucléotide antisens présentant une activité d'expression-suppression de CFB ; une composition pharmaceutique comprenant l'oligonucléotide antisens ; et un médicament qui supprime l'expression de CFB incluant l'oligonucléotide antisens, le médicament prévenant ou traitant les troubles médiés par les anomalies de la voie alternative du complément telles que le syndrome urémique hémolytique atypique, l'hémoglobinurie nocturne paroxysmique, la dégénérescence maculaire liée à l'âge, la glomérulonéphrite membrano-proliférative, la néphrite C3, la néphropathie membraneuse, la glomérulonéphrite à progression rapide (RPGN), la lésion rénale aiguë (AKI), l'asthme, ou les maladies auto-immunes.
PCT/JP2017/003904 2016-02-05 2017-02-03 Oligonucléotide antisens destiné à supprimer l'expression du facteur du complément b WO2017135397A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2018117253A1 (fr) * 2016-12-23 2018-06-28 協和発酵キリン株式会社 Acide nucléique inhibant l'expression du facteur b du complément
WO2019027015A1 (fr) * 2017-08-02 2019-02-07 協和発酵キリン株式会社 Complexe d'acide nucléique
CN110951915A (zh) * 2019-11-21 2020-04-03 南方医科大学南方医院 一种慢性乙型肝炎抗病毒治疗应答疗效预测的引物以及试剂盒
CN114144525A (zh) * 2019-07-24 2022-03-04 国立大学法人东北大学 嵌合分子、药物组合物、靶核酸的切割方法以及靶核酸切割用或诊断用试剂盒
WO2022124345A1 (fr) * 2020-12-08 2022-06-16 学校法人福岡大学 Arn guide stable d'édition cible dans lequel un acide nucléique chimiquement modifié a été introduit

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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
WO2015188194A1 (fr) * 2014-06-06 2015-12-10 Isis Pharmaceuticals, Inc. Compositions et méthodes assurant une meilleure absorption intestinale de composés oligomères conjugués

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2015188194A1 (fr) * 2014-06-06 2015-12-10 Isis Pharmaceuticals, Inc. Compositions et méthodes assurant une meilleure absorption intestinale de composés oligomères conjugués

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018117253A1 (fr) * 2016-12-23 2018-06-28 協和発酵キリン株式会社 Acide nucléique inhibant l'expression du facteur b du complément
WO2019027015A1 (fr) * 2017-08-02 2019-02-07 協和発酵キリン株式会社 Complexe d'acide nucléique
CN114144525A (zh) * 2019-07-24 2022-03-04 国立大学法人东北大学 嵌合分子、药物组合物、靶核酸的切割方法以及靶核酸切割用或诊断用试剂盒
CN110951915A (zh) * 2019-11-21 2020-04-03 南方医科大学南方医院 一种慢性乙型肝炎抗病毒治疗应答疗效预测的引物以及试剂盒
WO2022124345A1 (fr) * 2020-12-08 2022-06-16 学校法人福岡大学 Arn guide stable d'édition cible dans lequel un acide nucléique chimiquement modifié a été introduit

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